Proteins Associated with the Intracellular Signalling Tail of the ...€¦ · Proteins Associated with the Intracellular Signalling Tail of the Calcium-Sensing Receptor and Their

Proteins Associated with the Intracellular Signalling Tail of the

Calcium-Sensing Receptor and Their Impact on Receptor Function

By

Aaron Magno, B.Sc (Honours)

This thesis is presented for the Degree of Doctor of Philosophy of the University of Western Australia

School of Medicine and Pharmacology

2008

Preface

The experimental work contained within this thesis was conducted in the Department of

Endocrinology and Diabetes, Sir Charles Gairdner Hospital and the Western Australian

Institute for Medical Research, University of Western Australia, under the supervision

of Associate Professor Thomas Ratajczak and Doctor Bryan Ward. All experimental

work presented in this thesis was performed by myself, except for where expressly

stated.

Aaron Magno, B.Sc (Honours)

i

Acknowledgements

I must first express my gratitude to my supervisor Assoc Prof Thomas Ratajczak for

providing me the opportunity to undertake my PhD.

My thanks must also go to my co-supervisor, Dr Bryan Ward, for his guidance and

support throughout the years.

I’d like to express my appreciation to both the members of the Ratajczak lab who have

been a part of my journey since the beginning Dr Rudi Allan, Dr Carmel Cluning and

Dr Danny Mok and the PhD students who have joined more recently, Ajanthy

Arulpragasam and Sarah Rea.

To the Honours students, Bernadette Pederson and Shelby Chew, who have come

through and furthered the CaR studies, I say thankyou.

I must acknowledge the individuals who have provided their expertise, materials and

insight to assist me with my project. Dr Evan Ingley, who provided the yeast two-hybrid

library and advice on examining the identified clones. Assoc Prof Arthur Conigrave, Ed

Nemeth and Donald Ward for supplying the HEK293-CaR stables. The team from

CMCA, Dr Paul Rigby, Kathy Heel-Miller and Tracey Lee-Pullen, for their assistance

with microscopy and cell sorting. Dr Fiona Pixley for her guidance regarding the

cytosketal studies. Dr Kendall Walker for her assistance with the baculoviral

expression. Dr Michael Way for his gift of the testin antibody and Suszanne Brown for

her help with statistical analysis.

I also recognise the financial support provided by Kidney Health Australia and the

National Health and Medical Research Council throughout my time as a student.

Finally, I would like to thank my Mother for eternal support and patience.

ii

Abstract The calcium-sensing receptor (CaR) is a G protein-coupled receptor that can respond to

changes in extracellular calcium and plays an integral role in calcium homeostasis.

Later studies revealed that the CaR was stimulated by not just calcium, but a diverse

range of stimuli and that activation of the receptor regulated a host of different

biological processes. The CaR is linked to these cellular responses via the various

signalling pathways initiated by the receptor. Recent yeast two-hybrid studies have

identified a number of accessory proteins that, through their interaction with the

intracellular tail of the CaR, are able to regulate important functional aspects of the

receptor, including its signalling and degradation. We hypothesised that many more

proteins that bind to the CaR-tail await identification, especially since most of the

previous studies used the yeast two-hybrid system to screen cDNA libraries generated

from tissues that are important to whole body calcium homeostasis, such as the

parathyroid gland and kidney. In order to identify novel binding partners of the CaR,

which may affect its function, particularly in biological processes that might be

unrelated to calcium homeostasis, our laboratory performed a yeast two-hybrid screen

of an EMLC.1 mouse pluripotent haemopoietic cell line library using the intracellular

tail of the human CaR as bait. This screen revealed a large number of “potentially

interacting” clones when plated on selective medium, 130 of which were confirmed as

such using a Lac Z reporter assay.

The aims of this thesis were:

(i) to examine 60 of these “potentially interacting” clones to determine that they were

“true positives” and once confirmed to establish the identity of the interacting proteins

by sequence analysis. Following this, a secondary aim was to establish the region of the

CaR-tail to which these partner proteins bind, using yeast two-hybrid CaR-tail deletion

mapping studies.

(ii) The second aim was to examine in greater detail two of the proteins, filamin A, a

cytoskeletal protein shown previously to interact with the CaR and influence CaR-

mediated cell signalling, and testin, a LIM domain containing, focal adhesion protein

also known to have effects on the cytoskeleton.

This screen revealed a total of seven CaR interacting proteins, namely filamin A,

filamin B, testin, 14-3-3 θ, OS-9, Ubc9 and MPc2. This included six novel CaR binding

iii

partners and an interacting clone of filamin A that was different to that previously

published. This diverse collection of proteins is associated with a variety of functions

that range from the regulation of intracellular signalling, cytoskeletal organisation,

trafficking, degradation, posttranslational modification and transcriptional repression, to

acting as scaffolding proteins. In addition to demonstrating an interaction between these

interacting proteins and the CaR, their binding sites within the CaR-tail were also

mapped using the yeast two-hybrid system. Data from these studies and the known

interaction domains of previously identified CaR binding partners have revealed two

regions of the CaR intracellular tail, 865-922 and 965-986, which appear to be essential

for the interaction of accessory proteins.

The scaffolding protein, filamin A, was previously shown to bind to the CaR-tail and

influence receptor signalling and degradation. However, two distinct library clones

corresponding to filamin A (one isolated directly from the yeast two-hybrid library

screen and one based on the filamin B clone) did not overlap with the previously

identified site of interaction with the CaR. Direct interaction studies performed in vitro

using pulldown assays confirmed that these two regions of filamin A contained novel

sites of direct interaction with the CaR-tail. Sequence alignment between the two CaR

binding domains identified in this study and the previously defined binding domain

revealed a 40 amino acid region that was highly homologous in all three.

The CaR is the first receptor that has been found to interact with testin. Although direct

interaction of CaR and testin was unable to be confirmed due to insolubility of testin

fusion proteins, coimmunoprecipitation and confocal microscopy experiments

demonstrated that the CaR and testin could interact and colocalise in mammalian cells.

Furthermore, the binding of testin was found to occur at the membrane proximal region

of the CaR-tail, a region known to be important for signalling. Mapping studies

indicated that the interaction between the CaR and testin required key residues essential

in maintaining the integrity of the second zinc finger of LIM domain 1, as well as

additional residues of the zinc finger which may also be critical in maintaining its

structure. The overexpression of testin did not alter the level of CaR-mediated ERK

phosphorylation, but was found to enhance the level of CaR-induced Rho kinase

activity. The work of previous studies showing that CaR agonist stimulation of

HEK293-CaR cells caused changes in cell morphology and actin stress fibre assembly,

was replicated, with the additional finding that focal adhesion formation was also

iv

increased upon CaR activation. Surprisingly, unstimulated HEK293-CaR stable cells in

which testin had been knocked down using shRNA technology exhibited the same cell

morphology, actin stress fibre formation and focal adhesion formation as seen in

stimulated wild-type HEK293-CaR cells.

These studies have shown that the CaR is capable of interacting with a much larger

number of accessory proteins than previously known and highlighted two regions of the

receptor’s intracellular tail that contain elements important to partner protein binding.

Further investigations of the interaction between filamin A and the CaR have revealed

multiple sites of interaction suggesting mechanisms by which filamin A may act as a

more versatile scaffolding protein or perhaps a more efficient clamp for the CaR.

Finally, the novel CaR interacting protein, testin, was shown to enhance CaR-induced

Rho signalling and may potentially be involved in CaR-mediated changes to cell

morphology and cytoskeletal reorganisation.

v

Abbreviations

Associated Molecule with SH3 Domain AMSH

Bicinchoninic Acid BCA

Bovine Serum Albumin BSA

Calcium-Sensing Receptor CaR

Calmodulin-Dependent Protein Kinase CaMK

C-Jun NH2 Terminal Kinase JNK

C-Jun NH2 Terminal Kinase Kinase JNKK

Cyclic adenosine monophosphate cAMP

Dimethyl sulphoxide DMSO

Diacylglycerol DAG

Dithiothreitol DTT

Dulbecco’s Modified Eagle Medium DMEM

Endoplasmic Reticulum ER

Endoplasmic Reticulum-Associated Degradation ERAD

Enhanced Green Fluorescent Protein EGFP

Epidermal Growth Factor EGF

EGF Receptor EGFR

Ethylenediaminetetra-acetic acid EDTA

Extracellular Signal Regulated Kinase ERK

Fetal Calf Serum FCS

Familial Hypocalciuric Hypercalcaemia FHH

G-Protein Coupled Receptor GPCR

G Protein Receptor Kinase GRK

Glutathione S-Transferase GST

γ-Aminobutyric AcidB GABAB

Heparin-Binding Epidemral Growth Factor HB-EGF

Hours hr

Horseradish Peroxidase HRP

Inositol Phosphate IP3

Isopropanol β-thiogalactopyranoside IPTG

Lin-11, Isl-1, Mec-3 LIM

Lymphoid Blast Crisis Lbc

Matrix Metalloprotease MMP

Metabotropic Glutamate Receptors mGluR

vi

Minutes min

Mitogen Activated Protein Kinase MAPK

Mouse Tonicity Phosphate Buffered Saline MT-PBS

Neonatal Severe Hyperparathyroidism NSHPT

Nickel-Nitriloacetic acid Ni-NTA

Optical Density OD

Parathyroid Hormone PTH

Parathyroid Hormone-Related Protein PTHrP

Phenylmethylsulphonylflouride PMSF

Phorbol Myrisate Acetate PMA

Phosphate Buffered Saline PBS

Phosphatidyl Inositol 3 Kinase PI3K

Phosphatidyl inositol 4,5-bisphosphonate PIP2

Phospholipase A2 PLA2

Phospholipase C PLC

Phospholipase D PLD

Polymerase Chain Reaction PCR

Prickle, Espinas, Testin PET

Proheparin-Binding Epidemral Growth Factor ProHB-EGF

Protein Kinase C PKC

Protein Kinase A PKA

Receptor-Activity-Modifying Protein RAMP

Reverse Transcriptase Polymerase Chain Reaction RT-PCR

Rho-Guanine Nucleotide Exchange Factor Rho-GEF

Seconds sec

Serum Response Element SRE

Sodium Dodecyl Sulphate SDS

Sodium Dodecyl Sulphate Polyacrylamide Gel Electrophoresis SDS-PAGE

Transient Receptor Potential Vailloid Group 4 TRPV4

vii

TTaabbllee ooff CCoonntteennttss

Preface i

Acknowledgements ii

Abstract iii

Abbreviations vi

Table of Contents viii

List of Figures xvii

List of Tables xviii

Chapter 1: Introduction

1.1 Discovering the Calcium-Sensing Receptor 1

1.2 The Calcium-Sensing Receptor Gene 2

1.3 The Calcium-Sensing Receptor is a G Protein-Coupled Receptor 2

1.4 Properties of the Calcium-Sensing Receptor 6

1.4.1 Calcium-Sensing Receptor Dimerisation 6

1.5 Calcium-Sensing Receptor Structure 7

1.5.1 The Extracellular Domain 7

1.5.1.1 Bilobed Venus-Flytrap 7

1.5.1.2 Ca2+-Binding Pocket 8

1.5.1.3 Signal Peptide Cleavage Site 9

1.5.1.4 Cysteines 9

1.5.1.5 Peptide Linker 10

15.1.6 N-Linked Glycosylation Sites 10

1.5.2 The Transmembrane Domain 11

1.5.2.1 Membrane Spanning Region 11

1.5.2.2 Intracellular Loops 12

1.5.2.3 Extracellular Loops 13

1.5.2.4 Binding of Allosteric Modulators 13

1.5.3 The Intracellular Tail 14

1.5.3.1 Membrane Proximal Region 14

1.5.3.2 Phosphorylation Sites 16

1.6 Calcium-Sensing Receptor Signalling 17

viii

1.6.1 Calcium-Sensing Receptor Stimuli 17

1.6.1.1 Cations 17

1.6.1.2 Amino Acids 18

1.6.1.3 Pharmacological Agents 18

1.6.1.4 Polyamines 18

1.6.1.5 Polypeptides 19

1.6.1.6 Aminoglycoside Antibiotics 19

1.6.1.7 Ionic Strength 19

1.6.1.8 pH 19

1.6.2 Intracellular Signalling Pathways Regulated by the Calcium-Sensing

Receptor 20

1.6.2.1 Phospholipase Signalling 20

1.6.2.2 Mitogen Activated Protein Kinase Signalling 22

1.6.2.2.1 Extracellular Signal Regulated Kinase 22

1.6.2.2.2 c-Jun NH2 Terminal Kinase 24

1.6.2.2.3 p38 Mitogen Activated Protein Kinase 24

1.6.2.3 Inhibition of Cyclic AMP 25

1.6.2.4 Rho Signalling 25

1.7 The Biological Roles of the Calcium-Sensing Receptor 26

1.7.1 Calcium-Sensing Receptor in the Parathyroid 28

1.7.2 Calcium-Sensing Receptor in the Kidney 28

1.7.3 Calcium-Sensing Receptor in the Gastrointestinal Tract 29

1.7.4 Calcium-Sensing Receptor in Bone 30

1.7.5 Calcium-Sensing Receptor in the Nervous System 31

1.7.6 Calcium-Sensing Receptor in Breast 32

1.7.7 Calcium-Sensing Receptor in Epidermal Cells 33

1.8 Interacting Protein Partners of the Calcium-Sensing Receptor 33

1.8.1 Filamin 33

1.8.2 Potassium Channels 34

1.8.3 Dorfin 35

1.8.4 Associated Molecule with SH3 Domain of STAM (AMSH) 36

1.8.5 Receptor-Activity-Modifying Proteins 36

1.8.6 β-Arrestins 37

1.9 Statement of Aims 38

ix

Chapter 2: Materials and Methods

2.1 Materials 40

2.1.1 Reagents 40

2.1.2 Plasmids 43

2.1.3 Enzymes 43

2.1.4 Cell lines 44

2.1.5 Antibodies 44

2.1.6 Equipment 44

2.1.7 Commercial Suppliers 45

2.2 Methods 46

2.2.1 General Methods 46

2.2.1.1 Tissue Culture Methodology 46

2.2.1.1.1 Maintenance of Cell Lines 46

2.2.1.1.2 Transfection 47

2.2.1.1.3 Lysis of Cultured Mammalian Cells 47

2.2.1.2 Transformation of Competent cells 47

2.2.1.3 Plasmid DNA Preparation 48

2.2.1.4 Quantitation of DNA 49

2.2.1.5 Agarose Gel Electrophoresis 49

2.2.1.6 Purification of DNA 49

2.2.1.6.1 Purification of DNA from Agarose Gels 49

2.2.1.6.2 Purification of DNA Using the QIAquick PCR

Purification Kit 49

2.2.1.7 Ethanol Precipitation of DNA 50

2.2.1.8 Restriction Enzyme Digestion 50

2.2.1.9 Dephosphorylation of 5’-Ends 50

2.2.1.10 Ligations 50

2.2.1.11 Reverse Transcriptase-PCR 51

2.2.1.12 PCRs Using a Proofreading Enzyme 51

2.2.1.13 Site-Directed Mutagenesis 52

2.2.1.14 DNA Sequencing 52

2.2.1.15 Quantification of Protein Concentration Using a BCA

Assay Kit 53

2.2.1.16 Quantification of Protein Concentration Using a Bradford

Assay 53

x

2.2.1.17 Preparation of Gels and Electrophoresis 53

2.2.1.18 Western Blotting 54

2.2.1.19 Densitometry 54

2.2.1.20 Statistical Analysis 55

2.2.2 Identification of Positive Clones from a Yeast Two-Hybrid Library

Screen 55

2.2.2.1 DNA Extraction from Yeast 55

2.2.2.2 Profiling of Plasmids by Restriction Enzyme Digestion 55

2.2.2.3 Plasmid Recovery of Library Clones 56

2.2.2.4 Cotransformation of Bait and Library Plasmids with Yeast

L40 56

2.2.2.5 β-galactosidase Colony Lift Assays 58

2.2.3 Protein Interaction Studies 58

2.2.3.1 Baculoviral Expression and Purification of His-Tagged

CaR-Tail 58

2.2.3.2 Bacterial Expression and Purification of His-Tagged

CaR-Tail 59

2.2.3.3 Bacterial Expression and Purification of Glutathione S- 60

Transferase (GST)-Fusion Proteins

2.2.3.4 Alternate Purification Method for GST-Testin 61

2.2.3.5 Pulldown Assay with His-Tagged CaR-Tail 61

2.2.3.6 Staining of Polyacrylamide Gels 62

2.2.3.7 Coimmunoprecipitation 62

2.2.4 Confocal Microscopy 63

2.2.4.1 Detection of CaR-FLAG by Confocal Microscopy 63

2.2.5 Detection of Signalling Pathway Activity 63

2.2.5.1 ERK Assay 63

2.2.5.2 SRE-Luciferase Assay 64

2.2.6 Generation of the Testin Knockdown HEK293-CaR Stable Cell Line 65

2.2.6.1 Cloning of Knockdown Target Sequence 65

2.2.6.2 Generating the Stable Packaging Cell Line 65

2.2.6.3 Retroviral Infection of HEK293-CaR Stable Cell Lines 65

2.2.6.4 Enrichment of EGFP-positive Cells and Verification of

Testin Knockdown 66

2.2.7 Studies of Morphological and Cytoskeletal Changes 67

xi

Chapter 3: Identification of Proteins that Interact with the Intracellular Tail of the

Chapter 3: Calcium-Sensing Receptor in a Yeast Two-Hybrid Library Screen

3.1 Introduction 70

3.2 Results 72

3.2.1 Verification of Clones 72

3.2.2 Mapping of Verified Interacting Proteins of the CaR 73

3.2.2.1 Filamin A 76

3.2.2.2 Filamin B 76

3.2.2.3 Testin 76

3.2.2.4 14-3-3 θ 80

3.2.2.5 OS-9 80

3.2.2.5 Ubc9 80

3.2.2.6 MPc2 80

3.3 Discussion 85

3.3.1 Filamins 85

3.3.2 Testin 87

3.3.3 14-3-3 θ 92

3.3.4 OS-9 93

3.3.5 Ubc9 95

3.3.6 MPc2 97

Chapter 4: Investigating the Interaction Between the Intracellular Tail of the

Chapter 4: Calcium-Sensing Receptor and Filamin

4.1 Introduction 98

4.2 Results 99

4.2.1 Construction of Filamin A GST-Fusion Proteins for Pulldown

Studies 99

4.2.2 Purification of His-tagged CaR-tail from Insect Cells 101

4.2.3 Pulldown Assays Performed Using His-tagged CaR-tail Purified

from Insect cells 101

4.2.4 Pulldown Assays Performed Using His-tagged CaR-tail Purified

from Bacteria 104

4.3 Discussion 104

xii

Chapter 5: Investigation of the Interaction Between the Intracellular Tail of the

Calcium-Sensing Receptor and Testin and the Implications for Cell Function

5.1 Introduction 110

5.2 Results 111

5.2.1 Calcium-Sensing Receptor and Testin Interaction Studies 111

5.2.1.1 Yeast Two-Hybrid Mapping 111

5.2.1.2 Cloning of Full-Length Human Testin 113

5.2.1.3 Direct Interaction Studies 114

5.2.1.4 Coimmunoprecipitation Studies 114

5.2.2 Colocalisation of Testin and the Calcium-Sensing Receptor 117

5.2.3 The Effects of Testin on Calcium-Sensing Receptor Activated 117

ERK Signalling

5.2.4 The Effects of Testin on Calcium-Sensing Receptor-Mediated

Rho Signalling 120

5.2.5 The Calcium-Sensing Receptor Regulates Changes in Cell

Morphology 122

5.2.6 The Impact of Testin Knockdown on HEK293 Cells Stably

Expressing the Calcium-Sensing Receptor 126

5.3 Discussion 133

5.3.1 The Calcium-Sensing Receptor and Testin Interaction 133

5.3.2 Sites of Interaction Between the Calcium-Sensing Receptor and

Testin Identified in the Yeast Two-Hybrid System 133

5.3.3 Calcium-Sensing Receptor and Testin Interaction Studies 134

5.3.4 The Effects of Testin Binding on Calcium-Sensing Receptor

Regulated Signalling 135

5.3.4.1 Calcium-Sensing Receptor-Mediated ERK Phosphorylation

is Unaffected by Testin Overexpression 135

5.3.4.2 Testin Accentuates Calcium-Sensing Receptor-Mediated

Rho Kinase Activity 136

5.3.5 The Relationship Between Cell Morphology and the Calcium-

Sensing Receptor’s Interaction with Testin 136

xiii

Chapter 6: General Discussion

6.1 The Calcium-Sensing Receptor 139

6.2 Interacting Protein Partners of the Calcium-Sensing Receptor 141

6.3 Interacting Protein Partners of the Calcium-Sensing Receptor Regulate its

Function 141

6.3.1 The Effect of Interacting protein partners on Calcium-Sensing

Receptor Dimerisation 142

6.3.2 The Regulation of Calcium-Sensing Receptor Trafficking by

Interacting Proteins 143

6.3.3 The Regulation of Calcium-Sensing Receptor Degradation by

Interacting Proteins 143

6.3.4 Calcium-Sensing Receptor-Mediated Intracellular Signalling is 144

Directed by Interacting Proteins

6.2.5 The Role of the Calcium-Sensing Receptor and its Binding Partners

in Cell Morphology and Organisation of the Cytoskeleton 146

6.4 Future Studies

6.4.1 Filamin A 147

6.4.2 Filamin B 147

6.4.3 Testin 148

6.5 Conclusions 149

Chapter 7: References 150

Appendices

Appendix 1: Oligonucleotides 172

Appendix 2: Anitbodies and Western Blotting Conditions 174

xiv

LLiisstt ooff FFiigguurreess

Chapter 1: Introduction

Figure 1.1: A comparison of the amino acid sequences of mammalian CaRs 3

Figure 1.2: CaR-mediated signalling pathways 21

Chapter 3: Identification of Proteins that Interact with the Intracellular Tail of the


Figure 3.1 A schematic representation of the yeast two-hybrid screen using the

LexA system using the intracellular tail of the CaR as bait. 71

Figure 3.2 Profiling of library screen clones by PCR amplification and restriction

enzyme analysis. 74

Figure 3.3 Sites of interaction for the CaR-tail and filamin A identified in the

yeast two-hybrid system. 77

Figure 3.4 Sites of interaction for the CaR-tail and filamin B identified in the


Figure 3.5 Sites of interaction between the CaR and testin identified with the


Figure 3.6 Sites of interaction between the CaR-tail and 14-3-3q identified in a

yeast two-hybrid library screen. 81

Figure 3.7 Sites of interaction between the CaR and OS-9 identified with the


Figure 3.8 Sites of interaction between the CaR-tail and Ubc9 identified in a


Figure 3.9 Sites of interaction between the CaR-tail and MPc2 identified in a


Figure 3.10: A comparison of the amino acid sequence of testin from different

mammalian species. 89

Figure 3.11: Comparison of LIM domains. 90

Figure 3.12 The SUMOylation and ubiquitination pathways. 96

xv

Chapter 4: Investigating the Interaction Between the Intracellular Tail of the

Chapter 4: Calcium-Sensing Receptor and Filamin

Figure 4.1: Alignment of the filamin B fragment that binds to the CaR-tail with

its filamin A counterpart. 100

Figure 4.2: Purification of baculoviral His-tagged CaR-tail from Sf21 insect cells. 102

Figure 4.3: In vitro interaction studies between GST-tagged filamin A fragments

and His-tagged CaR-tail purified from insect cells. 103

Figure 4.4: In vitro interaction studies between GST-tagged filamin A fragments

and His-tagged CaR-tail purified from bacteria. 105

Figure 4.5: A comparison of the amino acid sequence of the identified

CaR-binding sites within human filamin A. 107

Figure 4.6 Schematic representations of proposed roles of multiple CaR binding

sites within filamin A. 109

Chapter 5: Investigation of the Interaction Between the Intracellular Tail of the

Chapter 5: Calcium-Sensing Receptor and Testin and the Implications for Cell

Chapter 5: Function

Figure 5.1: An alanine scan of the second zinc-finger of LIM domain 1 of testin

using the yeast two-hybrid system. 112

Figure 5.2 Examination of the expression and solubility of testin fusion proteins. 115

Figure 5.3 Coimmunoprecipitation of CaR-FLAG and EGFP-testin. 116

Figure 5.4: Colocalisation of CaR-FLAG and EGFP-testin in HEK293 cells. 118

Figure 5.5: The effect of testin on ERK phosphorylation in HEK293 cells stably

expressing the CaR following stimulation with extracellular calcium. 119

Figure 5.6: The effect of testin on ERK phosphorylation in HEK293 cells stably

expressing the CaR following stimulation with extracellular calcium in the

presence of an allosteric modulator. 121

Figure 5.7: The effect of testin on Rho kinase activity measured in either wild-type

HEK293 cells or HEK293 cells stably expressing the CaR. 123

Figure 5.8 Effects of magnesium stimulation on the morphology of HEK293 cells

stably expressing the CaR. 124

Figure 5.9: Detection of actin stress fibres and focal adhesions in HEK293 cells

stably expressing the CaR when exposed to different concentrations of

magnesium. 125

Figure 5.10: Detection of testin in lysates from normal HEK293-CaR stable cells

xvi

or those expressing testin knockdown shRNA by Western analysis. 127

Figure 5.11: Rho kinase activity measured in either wild-type HEK293-CaR stable

cells or testin knockdown HEK293-CaR cells. 127

Figure 5.12: Comparative cellular morphology of wild-type and testin knockdown

HEK293 cells stably expressing the CaR. 129

Figure 5.13: Detection of actin stress fibres in wild-type and testin knockdown

HEK293 cells stably expressing the CaR. 130

Figure 5.14: Detection of focal adhesions in wild-type and testin knockdown 131

HEK293 cells stably expressing the CaR.

Chapter 6: General Discussion

Figure 6.1: A simplistic overview of the translation of extracellular stimuli into an

intracellular response by the CaR. 140

xvii

LLiisstt ooff TTaabblleess Chapter 3: Identification of Proteins that Interact with the Intracellular Tail of the


Table 3.1 Protein interacting partners of the CaR-tail identified in a yeast two-hybrid screen of a haemopoietic cell line library with their comparative binding strengths to the CaR-tail and various CaR-tail truncations. 75 Chapter 5: Investigation of the Interaction Between the Intracellular Tail of the

Chapter 5: Calcium-Sensing Receptor and Testin and the Implications for Cell

Chapter 5: Function

Table 5.1: Observed effects of the knockdown of testin in HEK293 cells stably expressing the CaR. 132

xviii

CChhaapptteerr 11 Introduction

1.1 Discovering the Calcium-Sensing Receptor

The importance of calcium ions in the regulation of physiological functions has been

known since the 19th century, when Sydney Ringer serendipitously discovered that

calcium was essential for the contraction of isolated hearts (Ringer 1883). From that

early discovery, the importance of calcium in biological systems and the necessity for

organisms to tightly regulate calcium homeostasis has been firmly established (Carafoli

2003; Chang and Shoback 2004). Systemic calcium homeostasis is maintained through

the secretion of hormones in response to extracellular calcium and the consequent

actions of these hormones on various tissues to normalise extracellular calcium by

altering the levels of calcium released or absorbed by the affected tissues (Brown 1999).

One such hormone involved in regulating serum calcium levels is the parathyroid

hormone (PTH). Experiments using a radioimmunoassay to detect PTH levels in whole

animals revealed that an increase in serum calcium resulted in a decrease in PTH and

that a decrease in serum calcium caused an increase in PTH levels (Sherwood et al.

1966). The same radioimmunoassay was subsequently used to demonstrate that the

inverse relationship between extracellular calcium and PTH was also observed in

isolated parathyroid glands (Care et al. 1966). It was observed in an

electrophysiological study that extracellular calcium, even in the presence of a calcium

channel blocker, had a depolarising effect on parathyroid cells in a similar manner as

the inverse relationship between extracellular calcium and PTH (Lopez-Barneo and

Armstrong 1983). When dispersed parathyroid cells were exposed to increasing levels

of extracellular calcium, a relative increase in the levels of cytosolic calcium was

detected, which correlated with the suppression of PTH secretion from the treated cells

(Shoback et al. 1983). Further studies revealed that increases in extracellular calcium

increased the production of both inositol phosphate (IP3) and diacylglycerol (DAG),

which are recognised as components of general mechanisms of receptor mediated

intracellular calcium mobilisation (Brown et al. 1987; Kifor and Brown 1988; Shoback

et al. 1988). The data from these studies provided compelling evidence that there was a

receptor at the cell surface of parathyroid cells sensitive to extracellular calcium that

regulated PTH secretion through the mobilisation of intracellular calcium (Nemeth and

Carafoli 1990). In 1993, a receptor exhibiting these traits, the calcium-sensing receptor,

1

was cloned (Brown et al. 1993). The calcium-sensing receptor has two widely used

abbreviations, CaR and CaSR. Throughout this thesis the former will be used.

1.2 The Calcium-Sensing Receptor Gene

The first CaR identified was cloned from a bovine parathyroid gland cDNA library

(Brown et al. 1993). The isolated clone was 5,276 bp long, with an open reading frame

of 3,255 bp that encoded a protein of 1,085 amino acids and when expressed in Xenopus

laevis oocytes displayed a pharmacological profile similar to that observed in

parathyroid cells (Brown et al. 1993). The human CaR equivalent was cloned from

adenomatous parathyroid gland in 1995 and as with the bovine CaR, pharmacological

characteristics resembling those detected in parathyroid cells were observed in Xenopus

laevis oocytes expressing the human CaR (Garrett et al. 1995). The isolated human CaR

cDNA consists of seven exons, the first of which is a 5’-untranslated region while the

other six encode for a protein of 1078 amino acids with 93% identity to the bovine CaR

(Garrett et al. 1995). The identification of a promoter containing TATA and CAAT

boxes and a second GC-rich promoter without a TATA box in the human CaR suggest

that multiple CaR mRNAs may be produced through tissue specific regulation of the

two promoters (Chikatsu et al. 2000). With a known sequence for the human CaR it was

possible to map the CaR gene to human Chromosome 3q13.3-21 using fluorescence in

situ hybridisation (Janicic et al. 1995b). Besides the bovine and human, the CaR has

been found in a wide variety of vertebrates including rat (Riccardi et al. 1995), rabbit

(Butters et al. 1997), mouse (Oda et al. 2000), dog (Skelly and Franklin 2007), chicken

(Diaz et al. 1997), salamander (Cima et al. 1997) and several types of fish (Loretz

2008). Figure 1.1 contains a comparison of the protein sequences of selected

mammalian CaRs.

1.3 The Calcium-Sensing Receptor is a G Protein-Coupled Receptor

After extrapolating the amino acid sequence of the bovine CaR from the isolated cDNA

clone it was found that the CaR shared significant homology to the metabotropic

glutamate receptors (mGluR), which are part of the G protein-coupled receptor (GPCR)

superfamily (Brown et al. 1993). The GPCR superfamily is a diverse group of

membrane bound receptors involved in signal transduction containing 1000 to 2000

members in vertebrates that constitute over 1% of the genome (Bockaert and Pin 1999).

Phylogenetic analysis has been used to classify this superfamily of distinct proteins into

five families; Rhodopsin, Secretin, Adhesion, Glutamate and Frizzled/Taste2

2

Human 1 MAFYSCCWVLLALT-WHTSAYGPDQRAQKKGDIILGGLFPIHFGV 44 Bovine 1 MALYSCCWILLAFSTWCTSAYGPDQRAQKKGDIILGGLFPIHFGV 45 Rat 1 MASYSCCLALLALA-WHSSAYGPDQRAQKKGDIILGGLFPIHFGV 44 Dog 1 MAFHSCSLILLAIT-WCTSAYGPDQRAQKKGDIILGGLFPIHFGV 44 Mouse 1 MAWFGYCLALLALT-WHSSAYGPDQRAQKKGDIILGGLFPIHFGV 44

Human 45 AAKDQDLKSRPESVECIRYNFRGFRWLQAMIFAIEEINSSPALLP 89 Bovine 46 AVKDQDLKSRPESVECIRYNFRGFRWLQAMIFAIEEINSSPALLP 90 Rat 45 AAKDQDLKSRPESVECIRYNFRGFRWLQAMIFAIEEINSSPSLLP 89 Dog 45 AAKDQDLKSRPESVECIRYNFRGFRWLQAMIFAIEEINSSPALLP 89 Mouse 45 AAKDQDLKSRPESVECIRYNFRGFRWLQAMIFAIEEINSSPALLP 89

Loop I

Human 90 NLTLGYRIFDTCNTVSKALEATLSFVAQNKIDSLNLDEFCNCSEH 134 Bovine 91 NMTLGYRIFDTCNTVSKALEATLSFVAQNKIDSLNLDEFCNCSEH 135 Rat 90 NMTLGYRIFDTCNTVSKALEATLSFVAQNKIDSLNLDEFCNCSEH 134 Dog 90 NMTLGYRIFDTCNTVSKALEATLSFVAQNKIDSLNLDEFCNCSEH 134 Mouse 90 NMTLGYRIFDTCNTVSKALEATLSFVAQNKIDSLNLDEFCNCSEH 134

Loop II

Human 135 IPSTIAVVGATGSGVSTAVANLLGLFYIPQVSYASSSRLLSNKNQ 179 Bovine 136 IPSTIAVVGATGSGISTAVANLLGLFYIPQVSYASSSRLLSNKNQ 180 Rat 135 IPSTIAVVGATGSGVSTAVANLLGLFYIPQVSYASSSRLLSNKNQ 179 Dog 135 IPSTIAVVGATGSGISTAVANLLGLFYIPQVSYASSSRLLSNKNQ 179 Mouse 135 IPSTIAVVGATGSGVSTAVANLLGLFYIPQVSYASSSRLLSNKNQ 179 Human 180 FKSFLRTIPNDEHQATAMADIIEYFRWNWVGTIAADDDYGRPGIE 224 Bovine 181 FKSFLRTIPNDEHQATAMADIIEYFRWNWVGTIAADDDYGRPGIE 225 Rat 180 YKSFLRTIPNDEHQATAMADIIEYFRWNWVGTIAADDDYGRPGIE 224 Dog 180 FKSFLRTIPNDEHQATAMADIIEYFRWNWVGTIAADDDYGRPGIE 224 Mouse 180 FKSFLRTIPNDEHQATAMADIIEYFRWNWVGTIAADDDYGRPGIE 224 Human 225 KFREEAEERDICIDFSELISQYSDEEEIQHVVEVIQNSTAKVIVV 269 Bovine 226 KFREEAEERDICIDFSELISQYSDEEKIQQVVEVIQNSTAKVIVV 270 Rat 225 KFREEAEERDICIDFSELISQYSDEEEIQQVVEVIQNSTAKVIVV 269 Dog 225 KFREEAEERDICIDFSELISQYSDEEEIQQVVEVIQNSTAKVIVV 269 Mouse 225 KFREEAEERDICIDFSELISQYSDEEEIQQVVEVIQNSTAKVIVV 269 Human 270 FSSGPDLEPLIKEIVRRNITGKIWLASEAWASSSLIAMPQYFHVV 314 Bovine 271 FSSGPDLEPLIKEIVRRNITGRIWLASEAWASSSLIAMPEYFHVV 315 Rat 270 FSSGPDLEPLIKEIVRRNITGRIWLASEAWASSSLIAMPEYFHVV 314 Dog 270 FSSGPDLEPLIKEIVRRNITGRIWLASEAWASSSLIAMPEYFHVV 314 Mouse 270 FSSGPDLEPLIKEIVRRNITGRIWLASEAWASSSLIAMPEYFHVV 314 Human 315 GGTIGFALKAGQIPGFREFLKKVHPRKSVHNGFAKEFWEETFNCH 359 Bovine 316 GGTIGFGLKAGQIPGFREFLQKVHPRKSVHNGFAKEFWEETFNCH 360 Rat 315 GGTIGFGLKAGQIPGFREFLQKVHPRKSVHNGFAKEFWEETFNCH 359 Dog 315 GGTIGFALKAGQIPGFREFLQKVHPRKSVHNGFAKEFWEETFNCH 359 Mouse 315 GGTIGFGLKAGQIPGFREFLQKVHPRKSVHNGFAKEFWEETFNCH 359

Human 360 LQEGAKGPLPVDTFLRGHEESGDRFSNSSTAFRPLCTGDENISSV 404 Bovine 361 LQEGAKGPLPVDTFLRGHEEGGARLSNSPTAFRPLCTGEENISSV 405 Rat 360 LQEGAKGPLPVDTFVRSHEEGGNRLLNSSTAFRPLCTGDENINSV 404 Dog 360 LQEGAKGPLSMDTFLRGHEEGGGRISNSSTAFRPLCTGDENISSV 404 Mouse 360 LQDGAKGPLPVDTFVRSHEEGGNRLLNSSTAFRPLCTGDENINSV 404

Loop III Human 405 ETPYIDYTHLRISYNVYLAVYSIAHALQDIYTCLPGRGLFTNGSC 449 Bovine 406 ETPYMDYTHLRISYNVYLAVYSIAHALQDIYTCIPGRGLFTNGSC 450 Rat 405 ETPYMDYEHLRISYNVYLAVYSIAHALQDIYTCLPGRGLFTNGSC 449 Dog 405 ETPYMDYTHLRISYNVYLAVYSIAHALQDIYTCLPGRGLFTNGSC 449 Mouse 405 ETPYMGYEHLRISYNVYLAVYSIAHALQDIYTCLPGRGLFTNGSC 449

Loop IV Human 450 ADIKKVEAWQVLKHLRHLNFTNNMGEQVTFDECGDLVGNYSIINW 494 Bovine 451 ADIKKVEAWQVLKHLRHLNFTSNMGEQVTFDECGDLAGNYSIINW 495 Rat 450 ADIKKVEAWQVLKHLRHLNFTNNMGEQVTFDECGDLVGNYSIINW 494

Dog 450 ADIKKVEAWQVLKHLRHLNFTNNMGEQVTFDECGDLMGNYSIINW 494 Mouse 450 ADIKKVEAWQVLKHLRHLNFTNNMGEQVTFDECGDLVGNYSIINW 494 Human 495 HLSPEDGSIVFKEVGYYNVYAKKGERLFINEEKILWSGFSREVPF 539 Bovine 496 HLSPEDGSIVFKEVGYYNVYAKKGERLFINDEKILWSGFSREVPF 540 Rat 495 HLSPEDGSIVFKEVGYYNVYAKKGERLFINEEKILWSGFSREVPF 539 Dog 495 HLSPEDGSIVFKEVGYYNVYAKKGERLFINEEKILWSGFSREMPF 539 Mouse 495 HLSPEDGSIVFKEVGYYNVYAKKGERLFINEGKILWSGFSREVPF 539 Human 540 SNCSRDCLAGTRKGIIEGEPTCCFECVECPDGEYSDETDASACNK 584 Bovine 541 SNCSRDCLAGTRKGIIEGEPTCCFECVECPDGEYSDETDASACDK 585 Rat 540 SNCSRDCQAGTRKGIIEGEPTCCFECVECPDGEYSGETDASACDK 584 Dog 540 SNCSRDCLAGTRKGIIEGEPTCCFECVECPDGEYSDETDASACDK 584 Mouse 540 SNCSRDCQAGTRKGIIEGEPTCCFECVECPDGEYSGETDASACDK 584 Human 585 CPDDFWSNENHTSCIAKEIEFLSWTEPFGIALTLFAVLGIFLTAF 629 Bovine 586 CPDDFWSNENHTSCIAKEIEFLSWTEPFGIALTLFAVLGIFLTAF 630 Rat 585 CPDDFWSNENHTSCIAKEIEFLAWTEPFGIALTLFAVLGIFLTAF 629 Dog 585 CPDDFWSNENHTSCIAKEIEFLSWTEPFGIALTLFAVLGIFLTAF 629 Mouse 585 CPDDFWSNENYTSCIAKEIEFLAWTEPFGIALTLFAVLGIFLTAF 629

TM1

Human 630 VLGVFIKFRNTPIVKATNRELSYLLLFSLLCCFSSSLFFIGEPQD 674 Bovine 631 VLGVFIKFRNTPIVKATNRELSYLLLFSLLCCFSSSLFFIGEPQD 675 Rat 630 VLGVFIKFRNTPIVKATNRELSYLLLFSLLCCFSSSLFFIGEPQD 674 Dog 630 VLGVFIKFRNTPIVKATNRELSYLLLFSLLCCFSSSLFFIGEPQD 674 Mouse 630 VLGVFIKFRNTPIVKATNRELSYLLLFSLLCCFSSSLFFIGEPQD 674

TM2

Human 675 WTCRLRQPAFGISFVLCISCILVKTNRVLLVFEAKIPTSFHRKWW 719 Bovine 676 WTCRLRQPAFGISFVLCISCILVKTNRVLLVFEAKIPTSFHRKWW 720 Rat 675 WTCRLRQPAFGISFVLCISCILVKTNRVLLVFEAKIPTSFHRKWW 719 Dog 675 WTCRLRQPAFGISFVLCISCILVKTNRVLLVFEAKIPTSFHRKWW 719 Mouse 675 WTCRLRQPAFGISFVLCISCILVKTNRVLLVFEAKIPTSFHRKWW 719

TM3

Human 720 GLNLQFLLVFLCTFMQIVICVIWLYTAPPSSYRNQELEDEIIFIT 764 Bovine 721 GLNLQFLLVFLCTFMQIVICAIWLNTAPPSSYRNHELEDEIIFIT 765 Rat 720 GLNLQFLLVFLCTFMQILICIIWLYTAPPSSYRNHELEDEIIFIT 764 Dog 720 GLNLQFLLVFLCTFMQIVICVIWLYTAPPSSYRNHELEDEIIFIT 764 Mouse 720 GLNLQFLLVFLCTFMQIVICIIWLYTAPPSSYRNHELEDEIIFIT 764

TM4

Human 765 CHEGSLMALGFLIGYTCLLAAICFFFAFKSRKLPENFNEAKFITF 809 Bovine 766 CHEGSLMALGFLIGYTCLLAAICFFFAFKSRKLPENFNEAKFITF 810 Rat 765 CHEGSLMALGSLIGYTCLLAAICFFFAFKSRKLPENFNEAKFITF 809 Dog 765 CHEGSLMALGFLIGYTCLLAAICFFFAFKSRKLPENFNEAKFITF 809 Mouse 765 CHEGSLMALGSLIGYTCLLAAICFFFAFKSRKLPENFNEAKFITF 809

TM5

Human 810 SMLIFFIVWISFIPAYASTYGKFVSAVEVIAILAASFGLLACIFF 854 Bovine 811 SMLIFFIVWISFIPAYASTYGKFVSAVEVIAILAASFGLLACIFF 855 Rat 810 SMLIFFIVWISFIPAYASTYGKFVSAVEVIAILAASFGLLACIFF 854 Dog 810 SMLIFFIVWISFIPAYASTYGKFVSAVEVIAILAASFGLLACIFF 854 Mouse 810 SMLIFFIVWISFIPAYASTYGKFVSAVEVIAILAASFGLLACIFF 854

TM6 TM7 Human 855 NKIYIILFKPSRNTIEEVRCSTAAHAFKVAARATLRRSNVSRKRS 899 Bovine 856 NKVYIILFKPSRNTIEEVRCSTAAHAFKVAARATLRRSNVSRQRS 900 Rat 855 NKVYIILFKPSRNTIEEVRSSTAAHAFKVAARATLRRPNISRKRS 899 Dog 855 NKVYIILFKPSRNTIEEVRCSTAAHAFKVAARATLRRSNVSRKRS 899 Mouse 855 NKVYIILFKPSRNTIEEVRSSTAAHAFKVAARATLRRPNISRKRS 899

Human 900 SSLGGSTGSTPSSSISSKSNSEDPFPQ—-PERQKQQQPLALTQQE 942 Bovine 901 SSLGGSTGSTPSSSISSKSNSEDPFPQQQPKRQKQPQPLALSPHN 945 Rat 900 SSLGGSTGSIPSSSISSKSNSEDRFPQ--PERQKQQQPLSLTQQE 942 Dog 900 GSLGGSTGSTPSSSISSKSNSEDPFPQ--PERQKQQQPLALTQRE 942 Mouse 900 SSLGGSTGSNPSSSISSKSNSEDRFPQ--PERQKQQQPLALTQQE 942

Human 943 -QQQQP---LTLPQQQRSQ-QQPRCKQKVIFGSGTVTFSLSFDEP 982 Bovine 946 AQQPQPRPPSTPQPQPQSQ-QPPRCKQKVIFGSGTVTFSLSFDEP 989 Rat 943 –QQQQP---LTLHPQQQQQPQQPRCKQKVIFGSGTVTFSLSFDEP 983 Dog 943 QQPPQP---LTLPPQPQ-----PRCKQKVIFGSGTVTFSLSFDEP 979 Mouse 943 -QQQQP---LTLQPQQQQQPQQPRCKQKVIFGSGTVTFSLSFDEP 983 Human 983 QKNAMAHRNSTHQNSLEAQKSSDTLTRHQPLLPLQCGETDLDLTV 1027 Bovine 990 QKTAVAHRNSTHQTSLEAQKNNDALTKHQALLPLQCGETDSELTS 1034 Rat 984 QKNAMAHRNSMRQNSLEAQRSNDTLGRHQALLPLQCADADSEMTI 1028 Dog 980 QKSAAAPRNSTLQHSLEAQRSPEPPARPQALLPPQGGDTDAELPA 1024 Mouse 984 QKNAMAHRNSMRQNSLEAQKSNDTLNRHQALLPLQCAEADSEMTI 1028 Human 1028 QETGLQGPVGGDQRPEVEDPEELSPALVVSSSQSFVISGGGSTVT 1072 Bovine 1035 QETGLQGPVGEDHQLEMEDPEEMSPALVVSNSRSFVISGGGSTVT 1079 Rat 1029 QETGLQGPMVGDHQPEMESSDEMSPALVMSTSRSFVISGGGSSVT 1073 Dog 1025 QEPGLQGPGGADRRPEMRDPEELSPALVVSSSQSFVISGGGSTVT 1069 Mouse 1029 QETGLQGPMVGDHQPEIESPDEMSPALVVSTSRSFVISGGGSSVT 1073 Human 1073 ENVVNS 1078 Bovine 1080 ENMLRS 1085 Rat 1074 ENVLHS 1079 Dog 1070 ENILHS 1075 Mouse 1074 ENILHS 1079 Figure 1.1: A comparison of the amino acid sequences of mammalian CaRs. The sequences of human, bovine, rat, dog and mouse have been aligned with conserved residues highlighted in black ( X ). The site of signal peptide cleavage has been highlighted in blue ( X ). The 19 conserved cysteines in the extracellular domain have been highlighted in green ( X ). The three predicted sites of phosphorylation in the intracellular tail have been highlighted in yellow ( X ). The loops within the Venus-flytrap are indicated with a red line (―).The regions of the transmembrane domain that span the membrane are indicated with a blue line (―).

(Fredriksson et al. 2003). The Glutamate family, named after the first GPCR identified

of this class, the mGluR, is alternatively known as Family C or 3 and is characterised by

members that have very large extracellular domains (Bockaert and Pin 1999). While the

CaR was the first member of Family C identified that differed from the mGluR, the

number of family members has grown to the point that Family C has been subdivided

into the following six subgroups; mGluRs, CaRs, γ-aminobutyric acidB receptors

(GABAB), pheromone receptors, taste receptors and orphan receptors (Pin et al. 2003).

1.4 Properties of the Calcium-Sensing Receptor

As the receptor’s name suggests, the principal physiological ligand of the CaR is

calcium (Brown et al. 1994). It has been hypothesised that 3 to 5 Ca2+ ions can bind

cooperatively to the CaR, based on the Hill coefficient (Quinn et al. 2004). Detection of

the CaR by Western blotting revealed that the receptor produced three different protein

bands between 100 and 200 kDa that represent different monomeric forms of the CaR

(Bai et al. 1996). The lowest band, at ~120 kDa, is a non-glycosylated form of the CaR

that is expressed at a much lower level than the other two forms and is not always

detected in Western blots (Bai et al. 1996). A doublet of bands equivalent to molecular

masses of ~130-140 and ~150-160 kDa correspond to the immature form of CaR, which

is glycosylated with carbohydrates containing high mannose content, and a mature form

of the receptor, which is glycosylated with complex carbohydrates, respectively (Bai et

al. 1996). Only the mature form of the receptor is expressed at the cell surface (Bai et al.

1998a). Frequently, Western blotting would also detect bands greater than 200 kDa that

were eventually shown to represent dimeric and oligomeric forms of the receptor (Bai et

al. 1998a).

1.4.1 Calcium-Sensing Receptor Dimerisation

Bai et al. demonstrated that the CaR was normally expressed at the cell surface as a

homodimer, although there have been instances where the CaR has been detected in

heterodimeric complexes with another receptor, such as the mGluR (Bai et al. 1998a;

Gama et al. 2001). The endoplasmic reticulum (ER) has been identified as the site of

CaR dimer formation and dimerisation is essential but not sufficient, for the release of

the receptor from the ER (Pidasheva et al. 2006). Although CaR dimerisation occurs

prior to cell surface expression, studies of detergent solubilised CaR indicated that the

receptor undergoes conformational changes after binding to cations that favoured the

6

oxidation of free sulfhydryl groups and promoted CaR dimer formation, suggesting

ligand binding may have a stabilising effect on the receptor dimer (Ward et al. 1998).

1.5 Calcium-Sensing Receptor Structure

As a GPCR the CaR is comprised of the three main structural features of this receptor

family, an extracellular domain, a seven-transmembrane domain and an intracellular tail

(Brown et al. 1993). Studies examining both the biochemical and functional properties

of the CaR have provided insight into how the receptor’s three distinct structural regions

influence its expression, dimerisation and function (Bai 2004; Hu and Spiegel 2007).

Characteristics of the three domains will be discussed below, primarily in relation to the

human CaR.

1.5.1 The Extracellular Domain

As with all the members of GPCR Family C, the CaR contains a very large extracellular

domain, which covers 612 amino acids and includes two clusters of acidic residues at

amino acids 216-251 and 557-611 (Garrett et al. 1995). The importance of the

extracellular domain to the function of the CaR is highlighted by the fact that the

majority of naturally occurring mutations identified in subjects with calcium

homeostatic disorders are located in this domain (Bai 2004). While the extracellular

domain of the CaR can be subdivided into a bilobed Venus-flytrap and a cysteine-rich

domain, it also contains a number of other structural elements which will be discussed

below (Bai 2004).

1.5.1.1 Bilobed Venus-Flytrap

The amino acid sequence of the extracellular domain of the human CaR was aligned

with that of the mGluR and the bacterial periplasmic-binding protein, from which the

extracellular domains of the Family C GPCRs are proposed to be derived, in order to

better understand the properties of this region (Ray et al. 1999). The resultant model

consisting of amino acids 36-513 of the CaR produced a bilobed Venus-flytrap structure

with the N-terminal Lobe I connected to the C-terminal Lobe II by three strands (Ray et

al. 1999). Although the Venus-flytrap structure can exist in two states-opened and

closed, it has been proposed that ligand binding stabilises the closed state and triggers

the transmission of signal from the extracellular to the transmembrane domain

(Parmentier et al. 2002). Both the CaR and mGluR contain four segments within Lobe I

that do not align with the sequence of the bacterial periplasmic-binding protein and are

7

modelled as unstructured loops designated I-IV (Ray et al. 1999). Following the

determination of the three-dimensional crystal structure of the mGluR1 in both the

unliganded and ligand-bound forms, it was noted that Loops I, III and IV connected

regions of secondary structure within the extracellular domain (Kunishima et al. 2000).

Loop I consists of residues 39-67 and based on the proposed structural model connects a

β-sheet to an α-helix (Reyes-Cruz et al. 2001). CaR constructs with deletion of residues

48-59 and 50-59 were expressed at equivalent levels to the wild-type CaR, but had

reduced biological activity, while a CaR construct lacking residues 42-47 was unable to

respond to extracellular calcium and was only expressed as the incompletely processed

130 kDa form that did not reach the cell surface (Reyes-Cruz et al. 2001). Residues 117-

137 form Loop II and deletion of this region severely diminished biological activity of

the receptor with only a very minor fraction of the mutated receptor reaching the cell

surface (Reyes-Cruz et al. 2001). The longest of the loops is Loop III, which covers

residues 356-416 and a deletion construct removing residues 365-385 had no impact on

the receptor’s biological activity and expression (Reyes-Cruz et al. 2001). Loop IV is

the shortest loop spanning only 12 residues between 437 and 449 (Reyes-Cruz et al.

2001). The three CaR constructs with deletions of residues 438-445, 440-444 and 447-

453 all expressed at levels equivalent to wild-type, but all had a lower biological

activity (Reyes-Cruz et al. 2001).

1.5.1.2 Ca2+-Binding Pocket

It has been proposed that like the mGluRs the extracellular domain of the CaR contains

the sites of ligand-binding (Brauner-Osborne et al. 1999; Reyes-Cruz et al. 2001).

Identification of the residues of the CaR involved in Ca2+-binding has been hindered by

the lack of both a crystal structure for the receptor and a method to directly measure

Ca2+-binding to the receptor (Hu and Spiegel 2003). Several groups have generated

models based on the x-ray structures of a related Family C GPCR, the mGluR1, to help

identify the Ca2+-binding pocket (Huang et al. 2007b; Silve et al. 2005). Silve et al.

proposed that residues Ser147, Ser170, Asp190, Gln193, Tyr218, Phe270, Ser296 and

Glu297 were critical to the binding of Ca2+ in the CaR (Silve et al. 2005). In

experiments where these amino acids, with the exception of Glu297, had been mutated

to alanines it had been shown that CaR activity was impaired (Silve et al. 2005; Zhang

et al. 2002)). Glu297 was not mutated to an alanine but instead the mutations E297K

and E297D were made to mimic the naturally occurring mutations detected in patients

with FHH and ADH, respectively (Pollak et al. 1993; Silve et al. 2005). Experiments

8

examining the functionality of these mutant CaRs revealed that the E297K mutation

impaired the function of the CaR, while the E297D mutation enhanced the receptor’s

activity (Silve et al. 2005). In the model proposed by Huang et al. there are three

predicted sites of Ca2+-binding within the extracellular domain (Huang et al. 2007b).

The first site is in lobe 2 and contains the residues Glu224, Glu228, Glu229, Glu231

and Glu232. At the second site, located in lobe 1, the residues Glu378, Glu379, Thr396,

Asp398 and Glu399 have been indicated as key residues involved in Ca2+-binding

(Huang et al. 2007b). Ser147, Ser170, Asp190, Tyr218 and Glu297 are predicted to be

the amino acids that are important to Ca2+-binding at the third site that is positioned in a

crevice between the two lobes. It should be noted that the five key residues for Ca2+-

binding identified at the third site in the model from Huang et al. were also identified as

being critical for Ca2+-binding in the model presented by Silve et al. (Huang et al.

2007b; Silve et al. 2005). Experiments using CaR constructs in which glutamates had

been mutated to isoleucines revealed that mutation of the residues Glu228, Glu229,

Glu224, Glu378, Glu379 and Glu297 impaired CaR function while a mutation of

Glu398 and Glu399 enhanced CaR responsiveness (Huang et al. 2007b). To further

validate the model put forward by Huang et al, fragments of the CaR containing the first

(Gly222-Ile235) and second (Gly383-Ile408) putative Ca2+-binding sites were inserted

into the CD2 protein to be used in metal binding studies (Huang et al. 2007b). These

studies indicated that with the addition of the CaR fragments the CD2 fusion proteins

were capable of binding metal ions, but alanine mutation of residues of the CaR

fragments predicted to be critical for Ca2+-binding led to weaker metal ion binding

(Huang et al. 2007b).

1.5.1.3 Signal Peptide Cleavage Site

A site of signal peptide cleavage was identified at Tyr20 (Figure 1.1) through amino

acid sequencing of the N-terminus of a CaR extracellular domain that had been

expressed and purified from HEK293 cells, a cell line that does not endogenously

express the receptor (Goldsmith et al. 1999).

1.5.1.4 Cysteines

While there is a cluster of cysteines between residues 542 and 598, there are a total of

19 highly conserved cysteines spread throughout the extracellular domain of the CaR

following the signal peptide cleavage site as indicated in Figure 1.1 (Fan et al. 1998).

The expression in HEK293 cells of 19 mutant CaR constructs, in which individual

9

cysteines of the extracellular domain had been mutated to serines, revealed that 14

cysteines (Cys60, Cys101, Cys236, Cys358, Cys395, Cys542, Cys546, Cys561,

Cys562, Cys565, Cys568, Cys582, Cys585 and Cys598), which are conserved in

mGluR, were critical to the cell surface expression and biological activity of the

receptor (Fan et al. 1998). Further studies using CaR mutants with cysteine to serine

substitutions revealed that a single residue change was not sufficient to disrupt the

covalent disulfide bonds which mediate the formation of receptor dimers (Pace et al.

1999; Ray et al. 1999). However, two groups demonstrated that the mutation of two

cysteines, as in the case of the C101S/C239S and C129S/C131S CaR constructs,

eliminated dimerisation (Pace et al. 1999; Ray et al. 1999). A later study confirmed that

the combination of Cys129 and C131S mutations leads to the CaR losing the capacity to

form disulfide bonds, but that this was not sufficient to disrupt dimerisation, suggesting

that the CaR is also able to dimerise through other intermolecular interactions (Zhang et

al. 2001). With the exception of the GABAB receptors, all members of the GPCR

Family C contain nine cysteines that are located near the C-terminal end of the

extracellular domain in a region that has been referred to as the nine-cysteine domain of

family 3 GPCRs (Yu et al. 2004). In the CaR this region has been shown to be critical

for CaR-mediated signalling (Hu et al. 2000).

1.5.1.5 Peptide Linker

Immediately following this cysteine-rich domain there are 14 amino acids, residues

599-612, that have been described as a peptide linker connecting the extracellular

domain to the seven transmembrane domain (Ray et al. 2007). While all members of the

GPCR Family C with a nine-cysteine domain contain a peptide linker that is 14 amino

acids long, there is variability between the sequence of the peptide linker of each

member (Ray et al. 2007). Insertion or deletion of amino acids within the peptide linker

of the CaR had a negative impact on the cell surface expression of the receptor and

abrogated its signalling capacity in response to extracellular calcium (Ray et al. 2007).

The CaR’s cell surface expression and signalling was impaired by the substitution of an

alanine at Leu606 but not by the replacement of the CaR’s peptide linker with the 14

amino acids corresponding to the mGluR’s peptide linker (Ray et al. 2007).

15.1.6 N-Linked Glycosylation Sites

Upon cloning of the human CaR, 11 putative N-linked glycosylation sites, Asn-Xaa-

Ser/Thr, were identified in its extracellular domain, the majority of which are highly

10

conserved amongst species (Garrett et al. 1995; Ray et al. 1998). Indirect evidence from

experiments using CaR constructs with natural occurring mutations showed that

glycosylation of the receptor was necessary for the CaR to be fully biologically active

(Bai et al. 1996). In later studies it was revealed that inhibition of glycosylation of the

CaR by tunicamycin treatment blocked normal cell surface expression of the receptor,

which would impair its biological activity, as seen in the experiments using mutated

CaR constructs (Fan et al. 1997). Glycosylation of eight conserved N-linked

glycosylation sites, Asn90, Asn130, Asn261, Asn287, Asn446, Asn468, Asn488 and

Asn541, was demonstrated to be important for cell surface expression in experiments

using CaR constructs in which the asparagines of predicted N-linked glycosylation sites

had been substituted with glutamines (Ray et al. 1998). Further experiments with

mutant CaR constructs containing multiple asparagine to glutamine substitutions

revealed that glycosylation at a minimum of three sites was necessary for cell surface

expression of the receptor (Ray et al. 1998).

1.5.2 The Transmembrane Domain

1.5.2.1 Membrane Spanning Region

Garrett et al. proposed that the transmembrane domain of the human CaR spans residues

613-862 that include seven hydrophobic regions (labelled TM1-TM7 in Figure 1.1),

which form helices linked by alternating intracellular and extracellular loops, a feature

present in all GPCRs (Garrett et al. 1995). There is currently no clear consensus as to

exactly which amino acids comprise the helices and loops but only an estimate as to

where the helices and loops of the transmembrane region begin and end. Although there

is low sequence homology between the transmembrane domains of Family C GPCRs

and those of the Rhodopsin GPCR Family, there is evidence that there is similarity

between the three dimensional structure of the two families’ transmembrane domains

including the conserved disulfide bond linking the top of TM3 to the second

extracellular loop and the seven highly conserved residues between Family C and the

Rhodopsin Family members that are likely to act in a similar manner in both types of

receptors (Pin et al. 2003). As the transmembrane domains of the two GPCR families

are structurally similar it is likely that the mechanisms of G-protein coupling via the

transmembrane, as outlined by Wess in regards to rhodopsin, are valid for the CaR (Pin

et al. 2003; Wess 1997). It has been hypothesised that ligand binding to the extracellular

domain of a GPCR, leads to conformational changes in TM3 and TM4 that induce the

activation of different types of G protein (Wess 1997). In support of conformational

11

changes to the transmembrane helices leading to CaR activation was the identification

of a mutation in TM7 that leads to a constitutively active receptor (Zhao et al. 1999).

The A843E mutation is currently the only CaR mutation identified that results in

constitutive activation of the receptor and is proposed to alter the conformational state

of the the transmembrane to promote G protein coupling (Zhao et al. 1999). Mutations

such as the A843E that cause constitutive activation have been identified in other

GPCRs, including the L457R mutation identified in TM3 of the luteinizing hormone

receptor (Latronico et al. 1998). In addition to its role in signal transduction, the

transmembrane domain is believed to be involved in receptor dimerisation through

noncovalent interactions (Zhang et al. 2001). The examination of two naturally

occurring truncation mutations of the CaR, P747fs and A877Stop, revealed that dimer

formation was abolished in the P747fs mutant, which lacks TM5, while the A877Stop,

which contains TM5, had the capacity to form a dimer (Pearce et al. 1996; Zhang et al.

2001). A consensus dimerisation motif for noncovalent hydrophobic interactions that

was originally identified in the β2-adrenergic receptor has also been identified in TM5

of the CaR (Hebert et al. 1996).

1.5.2.2 Intracellular Loops

As the intracellular loops of other GPCRs have been implicated in the coupling of

receptors to G proteins, the intracellular loops of the CaR were examined in order to

deduce their importance in G protein-mediated signalling (Chang et al. 2000). A tandem

alanine scan throughout the second and third intracellular loops of the bovine CaR

identifed residues critical to the receptor’s ability to signal (Chang et al. 2000). The

third intracellular loop, which is highly homologous to the third intracellular loop of the

mGluRs, contained more residues involved in CaR signalling than the second

intracellular loop, which has poor homology with its mGluR amino acid counterparts

(Chang et al. 2000). Only the N-terminal portion of the second intracellular loop,

particularly amino acids Leu704 and Phe707, was found to be important to the

signalling capabilities of the CaR (Chang et al. 2000). Mutagenesis of any one of the

three residues of the third intracellular loop - Leu798, Phe802 and Glu804, completely

abrogated CaR signalling, while alanine mutation of any one of the following residues

in the third intracellular loop - Lys794, Arg796, Lys797, Pro799, Asn801, Asn803,

Lys806 and Phe807, resulted in an impairment of CaR signalling (Chang et al. 2000).

12

1.5.2.3 Extracellular Loops

Within the first and second extracellular loops of the CaR are two cysteines, Cys677

and Cys765, which have been identified as residues critical in maintaining the

conformation of the CaR (Ray et al. 2004). Mutation of either cysteine resulted in

incorrect processing of the CaR, which has been hypothesised to be due to the

disruption of disulfide bonds (Ray et al. 2004). Experiments using a chimeric receptor

with the extracellular domain of rhodopsin fused to the transmembrane domain and

intracellular tail of the CaR showed that although this chimeric receptor lacked the

extracellular domain of the CaR it was still expressed at the cell surface and was

capable of responding to extracellular calcium (Hauache et al. 2000). Even though the

chimeric receptor was only able to respond to calcium in the presence of an allosteric

modulator, this data suggested that the transmembrane domain contained additional

sites for calcium binding (Hu et al. 2002). As the extracellular loops are the only parts

of the transmembrane domain exposed to the extracellular environment and contain a

number of acidic residues that may be involved in calcium binding, Hu et al. mutated

the eight acidic residues found in extracellular loops 1-3 to alanines to examine their

possible role in calcium binding (Hu et al. 2002). Substitution of either of the two acidic

residues in extracellular loop 1 - Glu671 or Asp674, did not significantly alter the

biological activity of the CaR (Hu et al. 2002). The individual mutation of three of the

five acidic amino acids in extracellular loop 2 - Asp758, Glu759 and Glu767, to

alanines increased the sensitivity of the CaR to extracellular calcium, while mutations at

Glu755 and Glu 758 had no significant impact on CaR signalling (Hu et al. 2002).

Alanine substitution of the remaining acidic residue in the third extracellular loop,

Glu837, resulted in a mutant CaR that responded to extracellular calcium, albeit with a

lower maximal response compared to the wild-type CaR, but unlike the other seven

mutant CaRs was not potentiated by NPS R-568, a phenylalkylamine (Hu et al. 2002).

Later studies revealed that the negative charge of the glutamate was required for the

interaction between the CaR and phenylalkylamines, leading to the hypothesis that a salt

bridge formed between the glutamate and the positively charged central amine of these

compounds (Hu et al. 2005). These observations led to a series of experiments

attempting to identify the binding site of allosteric modulators.

1.5.2.4 Binding of Allosteric Modulators

Several types of allosteric modulator are known to influence the CaR’s activity, but of

particular interest are compounds referred to as calcimimetics and calcilytics because

13

they act as allosteric agonists and antagonists, respectively (Nemeth et al. 2001; Nemeth

et al. 1998). Homology modelling of the transmembrane domain of the CaR based on

the crystal structure of rhodopsin predicted that the residues Phe668, Arg680, Phe684

and Glu837 were important for the binding of phenylalkylamines to the CaR (Miedlich

et al. 2004). It was shown experimentally that all four of these residues were involved in

the binding of a calcilytic, NPS 2143, but only Phe668, Phe684 and Glu837 were

essential for binding of the calcimimetic NPS R-568 (Miedlich et al. 2004). A separate

group, also applying the rhodopsin structure to model the CaR transmembrane domain,

demonstrated experimentally that while the binding sites of calcimimetics and

calcilytics overlap they are not identical (Petrel et al. 2004). It has also been reported

that structurally different calcimimetics and calcilytics interact with specific sets of

residues in the second and third extracellular loops that share some commonality, but

are distinct (Hu et al. 2006; Petrel et al. 2004).

1.5.3 The Intracellular Tail

Unlike the extracellular and the transmembrane domains, very few naturally occurring

mutations have been identified in the 216 amino acids that comprise the intracellular tail

of the CaR (Ray et al. 1997). The first mutation identified in the intracellular tail was an

Alu sequence insertion at codon 877 (Janicic et al. 1995a). In 2000 Lienhardt et al

identified a large in-frame deletion, S895-V1075, while Carling et al identified the first

point mutation, F881L, located in the intracellular tail of the CaR (Carling et al. 2000;

Lienhardt et al. 2000). In addition to these mutations, the cytoplasmic tail of the CaR

contains three polymorphisms, A986S, G990R and Q1011E (Heath et al. 1996). As can

be seen in Figure 1.1 the intracellular tail of the CaR is the least conserved domain

between mammalian species and the divergence in sequence is even greater in

comparison to non-mammalian CaRs (Loretz et al. 2004). However, there remain

portions of the intracellular tail that are highly homologous between species including a

membrane proximal region spanning residues 863-925 and a region comprised of amino

acids 960-984. The former has been shown to be essential to the cell surface expression

and activity of the receptor, while the latter has been shown to be involved in binding to

accessory proteins (Awata et al. 2001; Hjalm et al. 2001; Ray et al. 1997).

1.5.3.1 Membrane Proximal Region

Investigation of the role of the intracellular tail in expression and activity of the CaR

began with the examination of the aforementioned Alu insertion mutant, which resulted

14

in a truncated receptor with decreased cell surface expression and an inability to

respond to extracellular calcium (Bai et al. 1997). This was followed by a series of

experiments that used a set of CaR mutants that included truncation mutants, 1-865, 1-

874, 1-888 and 1-903, as well as a pair of alanine scan substitutions between Ser875 and

Val883 to further examine the impact of the carboxyl terminal tail on the receptor’s

functionality (Ray et al. 1997). Although all truncation mutants were expressed at the

cell surface, the 1-865 and 1-874 truncation mutants were expressed at a lower level

than the wild-type CaR (Ray et al. 1997). Despite cell surface expression of both the 1-

865 and 1-874 truncation mutants, neither mutant responded to stimulation by

extracellular calcium, while both 1-888 and 1-903 truncation mutants showed biological

activity comparable to wild-type (Ray et al. 1997). Both alanine scan mutants, Ala875-

879 and Ala881-883, were expressed at lower levels on the cell surface when compared

to the wild-type CaR (Ray et al. 1997). The Ala875-879 was unresponsive to

extracellular calcium, while the biological activity of the Ala881-883 mutant was much

lower than the wild-type receptor even when both constructs were expressed at the cell

surface to the same degree as wild-type CaR (Ray et al. 1997). A separate group

examined a set of CaR truncation mutants that included 1-868, 1-886, 1-908 and 1-1024

using an alternate method of measuring CaR activity and found that although all

mutants responded to extracellular calcium, only the 1-868 truncation mutant had a

significantly decreased response compared to wild-type CaR (Gama and Breitwieser

1998). The 1-868 truncation mutant also had an increased rate of desensitisation

compared to the wild-type CaR, while the four other truncation mutants were

desensitised at a rate comparable to wild-type CaR (Gama and Breitwieser 1998). A

third group, using bovine CaR truncation mutants 1-866, 1-895 and 1-929, found that

only the 1-866 truncation mutant was unable to respond to extracellular calcium, as

measured in an assay similar to the one used by Ray et al. (Chang et al. 2001). An

alanine scan between Arg867 and Val895 using the 1-895 truncation mutant indicated

that alanine substitutions throughout the regions 879-883 and 892-895 resulted in a

significant decrease in CaR activity (Chang et al. 2001). Individual alanine substitution

at His880 and Phe882 in full-length bovine CaR also resulted in a reduction in the

biological activity of the receptor (Chang et al. 2001). A large proportion of the H880A

and F882A bovine CaRs expressed in HEK293 cells was retained in the ER, although

they both had glycosylation patterns similar to the wild-type receptor (Chang et al.

2001). It should be noted that both the H880A and F882A mutations occur in a region

of the intracellular tail predicted to form an α-helix. Chang et al. have raised the

15

possibility that an ER retention motif (RXR) beginning at Arg897 might regulate

normal trafficking of the CaR from the ER to the cell surface (Chang et al. 2007). The

work from these three studies highlights the importance of this membrane proximal

region of the CaR’s carboxyl-tail to the overall function of the receptor.

1.5.3.2 Phosphorylation Sites

Prior to the discovery of the CaR, experiments conducted in dissociated bovine

parathyroid cells revealed that protein kinase C (PKC) modulated PTH secretion and the

mobilisation of intracellular calcium (Racke and Nemeth 1993a; Racke and Nemeth

1993b). The regulation of these two processes would later be attributed to the CaR

(Brown et al. 1993). The intracellular tail of the human CaR is predicted to contain three

PKC phosphorylation sites at Thr888, Ser895 and Ser915 (Bai et al. 1998b). Treatment

of HEK293 cells expressing either wild-type CaR or CaR mutated at S895A or S915A,

with the PKC activator, phorbol myristate acetate (PMA), led to an attenuation of the

response to extracellular calcium by the wild-type or mutant CaRs (Bai et al. 1998b).

However, the response to extracellular calcium in HEK293 cells expressing the mutant

CaR construct, T888V, was unaffected by PKC activity, suggesting that PKC

phosphorylation of Thr888 inhibits the biological activity of the CaR (Bai et al. 1998b).

The substitution of Thr888 with hydrophobic residues such as valine, alanine and

tryptophan produced CaRs with an increased responsiveness to its agonists.

Alternatively, CaRs containing mutations at T888D, T888E and T888G were less

responsive than the wild-type CaR to agonist stimulation (Jiang et al. 2002). However,

both sets of mutant CaR constructs exhibited significantly reduced sensitivity to PKC

activity in comparison to the wild-type CaR (Jiang et al. 2002). Treatment of HEK293

cells expressing CaR with the PKC specific inhibitor, GF109203X, negated the

inhibitory effect that activated PKC had on the CaR (Bosel et al. 2003). Experiments

using an antibody specific for the CaR phosphorylated at Thr888 revealed that an

increase in extracellular calcium or acute treatment of the CaR with a calcimimetic

increased phosphorylation of the CaR at Thr888, an effect ablated by treatment with a

calcilytic (Davies et al. 2007). This suggests that the CaR is able to activate PKC, which

in turn phosphorylates the CaR, leading to an inhibition of CaR activity, forming a

negative feedback loop (Davies et al. 2007). It was also observed that after

phosphorylation the Thr888 residue could be dephosphorylated, a process that was

inhibited in the presence of the phosphatase inhibitors calyculin or endothall

thiohydride, suggesting that protein phosphatase 2 is responsible for the

16

dephosphorylation of the CaR (Davies et al. 2007). The antibody specific for the CaR

phosphorylated at Thr888 was able to detect both the mature and immature forms of the

receptor by Western blotting, indicating that both forms are phosphorylated (Davies et

al. 2007). In addition to the PKC phosphorylation sites there are two predicted protein

kinase A (PKA) phosphorylation sites, Ser899 and Ser900, within the intracellular tail

of the CaR (Bosel et al. 2003). Experiments using a PKA specific inhibitor, H-89,

suggested that PKA has only a minor role in the regulation of the CaR (Bosel et al.

2003).

1.6 Calcium-Sensing Receptor Signalling

Just as the major structural elements of the CaR were discussed as either extracellular or

intracellular in the previous section, so too can the receptor’s function be divided by the

plasma membrane into an extracellular component, “sensor”, and an intracellular

component, “transmitter”. The “sensory” aspect of the CaR relates to its ability to detect

changes in the extracellular environment through binding to its agonists, while the

“transmitter” characteristics of the receptor relate to its ability to modulate intracellular

signalling events. These two key facets of the CaR will be outlined below.

1.6.1 Calcium-Sensing Receptor Stimuli

Although extracellular calcium is considered the primary physiological agonist of the

CaR, there are a host of different stimuli to which the receptor is responsive (Hofer and

Brown 2003). The various physiological and pharmacological agonists of the CaR can

be divided into those that can directly induce CaR activation and the allosteric

modulators that sensitise the CaR to its agonists (Brown and MacLeod 2001).

1.6.1.1 Cations

While Ca2+ and Mg2+ may be the only endogenous divalent cations that activate the CaR

there is a growing list of divalent and trivalent cations that are capable of acting as CaR

agonists (Riccardi 2002). In 1990, Nemeth conducted experiments to determine the

CaR’s sensitivity to known cation agonists and found their rank order of potency to be

as follows: La3+ > Gd3+ > Be2+ > Ca2+ = Ba2+ > Sr2+ > Mg2+ (Nemeth and Carafoli

1990). Evidence suggests that not all CaR agonist cations bind to the same region of the

receptor (Hammerland et al. 1999).

17

1.6.1.2 Amino acids

Amino acids were originally shown to act as allosteric modulators of CaR activity in

HEK293 cells stably expressing the CaR (Conigrave et al. 2000). The CaR was

stereoselective for L-amino acids and exhibited greater affinity for large aromatic L-

amino acids (Conigrave et al. 2000). The rank order of potency displayed by the L-

amino acids for the CaR is as follows; L-Phe=L-Trp > L-His > L-Ala > L-Glu > L-Arg

= L-Leu (Conigrave et al. 2000). Although the site of amino acid binding in the CaR is

in the Venus-flytrap domain, it is believed to be distinct from the Ca2+ binding site

(Mun et al. 2005).

1.6.1.3 Pharmacological Agents

Two types of compounds have been designed to act on the CaR, the calcimimetics,

which enhance the sensitivity of the CaR to extracellular calcium, and the calcilytics,

which act as CaR antagonists (Trivedi et al. 2008). The first calcimimetics generated

were the phenylalkylamines, NPS R-467 and NPS R-569, which were shown to be

potent, stereoselective allosteric modulators of the CaR (Nemeth et al. 1998). Currently,

only Cinacalcet-HCl, which is pharmacokinetically more stable than either NPS R-467

and NPS R-569, is commercially available for therapeutic use (Evenepoel 2008). The

calcilytic, NPS 2143, was the first substance, ionic or molecular, identified that was able

to act as a CaR antagonist (Nemeth et al. 2001). Subsequently, structurally different

calcilytics have been designed including Calhex 231 and compound 3 (Brauner-

Osborne et al. 2007). Investigations to determine the binding sites of selected

calcimimetics and calcilytics have revealed that they bind to distinct, but overlapping

regions of the transmembrane domain (Miedlich et al. 2004; Petrel et al. 2004).

1.6.1.4 Polyamines

A number of endogenous polyamines have been experimentally shown to activate the

CaR (Quinn et al. 1997). The concentrations of polyamines used in these experiments to

elicit a CaR-mediated response are higher than their physiological concentrations, but it

should be noted that 0.5 mM of extracellular calcium was routinely used (Riccardi

2002). However, even a modest rise in the concentration of extracellular calcium

dramatically increased the responsiveness of the CaR to polyamines, suggesting that

polyamines might contribute to CaR signalling physiologically (Riccardi 2002). Quinn

et al. measured the efficacies of a group of polyamines and found their order of potency

18

to be as follows: spermine > spermidine >> putrescine, indicating that polyamines with

a higher positive charge were more potent activators of the CaR (Quinn et al. 1997).

1.6.1.5 Polypeptides

Prior to the identification of the CaR, studies examining the effects of polyarginine,

polylysine and protoamine on bovine parathyroid cells revealed that these polypeptides

mimicked the cellular responses observed with extracellular calcium stimulation

(Brown et al. 1991b). Experiments using Chinese hamster ovary cells transiently

expressing the receptor, subsequently confirmed that polypeptides act through the CaR

(Ruat et al. 1996). Amyloid-β peptide, which is excessively produced in the brain of

patients with Alzheimer’s disease, has also been shown to stimulate the CaR and is

proposed to act on the receptor in a fashion similar to spermine, as both molecules have

a similar spacing of positive charges (Brown and MacLeod 2001; Ye et al. 1997a).

1.6.1.6 Aminoglycoside Antibiotics

Like the polypeptides, the polyvalent aminoglycoside antibiotics were originally found

to mimic the effects of extracellular stimulation on cultured bovine parathyroid cells

(Brown et al. 1991a). The aminoglycoside antibiotics were confirmed to act via the CaR

in experiments using HEK293 cells stably expressing the receptor (McLarnon et al.

2002). The order of potency of the aminoglycoside antibiotics tested is as follows:

neomycin > tobramycin > gentamicin > kanamycin, suggesting that their efficacies

positively correlate with the number of attached amino groups (McLarnon et al. 2002).

1.6.1.6 Ionic Strength

Alterations in ionic strength have been shown to influence the sensitivity of the CaR for

its agonists (Brown and MacLeod 2001). It was demonstrated that the CaR expressed in

cultured cells treated with an increase in ionic strength became less sensitive to its

agonists (Quinn et al. 1998). The influence of ionic strength on the CaR’s affinity for

spermine was found to be greater than that of extracellular calcium, suggesting that the

binding efficacies of agonists with a greater number of positive charges would be more

affected by changes in ionic strength (Quinn et al. 1998).

1.6.1.7 pH

Quinn et al. demonstrated that increasing the pH above the physiological range, pH 7.0-

7.8, also increased the agonist sensitivity of the CaR and that a decrease in pH had the

19

reverse effect (Quinn et al. 2004). Part of the variation in CaR agonist affinity by pH

modulation is a result of changes in the ionisation of the charges on the CaR agonists

(Quinn et al. 2004). However, in experiments using HEK293 cells transiently

transfected with any one of the following activating CaR mutants - E191K, F128L,

C129F, Del 543, E127K, and E127A, pH modulation altered CaR agonist sensitivity

(Quinn et al. 2004). These results and the fact that the CaR affinity for extracellular

calcium and magnesium was also influenced by changes in pH suggest that the

alterations in CaR agonist sensitivity by pH is partially the result of molecular changes

to the receptor (Quinn et al. 2004).

1.6.2 Intracellular Signalling Pathways Regulated by the Calcium-Sensing Receptor

The CaR is capable of modulating an extensive and complex array of intracellular

signalling pathways as outlined in Figure 1.2 (Ward 2004). An overview of the better

characterised CaR-mediated pathways will be given below.

1.6.2.1 Phospholipase Signalling

In 1993, studies examining the recently cloned bovine CaR expressed in Xenopus laevis

oocytes identified the phospholipase C (PLC) pathway as being upregulated upon

stimulation of the CaR with extracellular calcium (Brown et al. 1993). CaR activation of

the PLC pathway has been shown to be pertussis toxin sensitive in CaR expressing

Xenopus laevis oocytes and AtT-20 cells but in bovine parathyroid and CaR expressing

HEK293 cells the opposite has been confirmed (Brown et al. 1993; Emanuel et al. 1996;

Kifor et al. 1997b). This suggests that in some cells exhibiting pertussis toxin

insensitive CaR-mediated PLC activity the receptor is coupled to Gq/11 (Kifor et al.

1997b). As indicated in Figure 1.2 an activated G protein subunit interacts with PLC,

leading to the cleavage of phosphatidyl inositol 4,5-bisphosphonate (PIP2) into IP3 and

DAG (Malarkey et al. 1995). IP3 acts as an agonist for the IP3 receptor that upon

activation mobilises calcium from intracellular stores into the cytoplasm, while DAG

can activate PKC (Malarkey et al. 1995; Supattapone et al. 1988).

In addition to PLC, the CaR is also able to activate two other phospholipases,

phospholipase D (PLD) and phospholipase A2 (PLA2) (Kifor et al. 1997a). There are

conflicting reports on how extracellular calcium regulates PLD activity via the CaR.

Kifor et al. showed that downregulation of PKC by pre-treatment with PMA in

parathyroid cells or HEK293 cells stably expressing the CaR abolished PLD activity

20

caused by 2 mM extracellular calcium (Kifor et al. 1997a). In contrast, Huang et al.

demonstrated that PKC down regulated by the same treatment in MDCK cells stably

overexpressing the CaR, did not reduce PLD activity in response to 5 mM extracellular

calcium (Huang et al. 2004). The difference in observed outcomes might be due to the

differences in cell types or levels of extracellular calcium used by the two groups.

Huang et al. went on to show that Rho was involved in CaR-mediated PLD activation

by using the Rho family inhibitor C3 exoenzyme. This led to the abolition of CaR-

mediated PLD activity (Huang et al. 2004). PKC activity is only partially responsible

for CaR-mediated activation of PLA2 (Handlogten et al. 2001). The CaR primarily

regulates PLA2 activity by increasing intracellular calcium levels via Gq activation of

the PLC pathway (Handlogten et al. 2001). The calcium influx causes the PLA2 to

translocate to membranes where it can hydrolyse phospholipids (Purkiss and Boarder

1992). The increase in intracellular calcium also activates calmodulin, which in turn

activates calmodulin-dependent protein kinase (CaMK) (Muthalif et al. 1996). The

activation of these two proteins has been shown to be required for CaR-mediated

activation of PLA2 (Handlogten et al. 2001). However, there is also evidence that CaR-

induced activation of the extracellular signal regulated kinase (ERK) can lead to the

phosphorylation and activation of PLA2 (Kifor et al. 2001). Both PLC and PLD are able

to break down phospholipids to generate phosphatidic acid, which can then be

subsequently hydrolysed by PLA2 to produce free arachidonic acid (Purkiss and

Boarder 1992; Yang et al. 1967).

1.6.2.2 Mitogen Activated Protein Kinase Signalling

The CaR has been implicated in the activation of the following subtypes of mitogen

activated protein kinase (MAPK) signalling cascades: ERK, c-Jun NH2 terminal kinase

(JNK) and p38 MAPK (Ogata et al. 2006).

1.6.2.2.1 Extracellular Signal Regulated Kinase

In 1998 experiments investigating stimulation of rat fibroblasts with either Ca2+ or Gd3+

showed that there was increased activity of the tyrosine kinase, Src, in response to either

cation (McNeil et al. 1998). Rat fibroblasts coexpressing a dominant negative mutant

CaR, R796W, with wild-type CaR exhibited lower Src kinase activity in response to

stimulation with either Ca2+ or Gd3+ than fibroblasts expressing only wild-type CaR,

indicating that this effect was mediated by the CaR (McNeil et al. 1998). As Src kinase

can induce the signalling cascade leading to ERK activation, the effects of Ca2+ or Gd3+

22

stimulation on fibroblasts expressing wild-type CaR, either alone or with the R796W

mutant were examined (McNeil et al. 1998). Not only did these experiments reveal that

ERK activation was mediated through the CaR, but by using a tyrosine kinase inhibitor

selective for Src, herbimycin, McNeil et al. were also able to demonstrate that the CaR

activation of ERK was mediated through the Src kinase (McNeil et al. 1998). A separate

group confirmed that ERK activation by the CaR via the Src kinase was possible in

experiments where HEK293 cells transiently expressing the CaR were stimulated by

extracellular calcium or the calcimimetic, NPS R-467, with or without treatment

involving either of the tyrosine kinase inhibitors, herbimycin and genistein (Kifor et al.

2001). Agonist induced CaR activation of ERK was inhibited in rat ovarian surface

epithelial cells by the transfection of dominant negative mutants of Ras, Raf and MEK,

indicating that the CaR activated ERK via a signalling cascade through the CaR-Src-

Ras-Raf-MEK-ERK pathway as presented in Figure 1.2 (Hobson et al. 2000). However,

Kifor et al. also demonstrated through the use of a PLC specific inhibitor, U-73122, and

a PKC selective inhibitor, GF109203X, that CaR signalling through the PLC pathway

leading to PKC activation could also activate ERK (Kifor et al. 2001). As the effects of

the PLC pathway inhibitors and pertussis toxin treatment on HEK293 cells expressing

the CaR had a synergistic inhibitory effect on ERK activation in response to

extracellular calcium, it can be concluded that the CaR can initiate the PLC pathway

and activate tyrosine kinases to increase ERK activity presumably via coupling to the

Gq/11 and Gi subunits, respectively (Kifor et al. 2001). Phosphatidyl inositol 3 kinase

(PI3K) has been implicated as a component of CaR-mediated ERK activation in CaR

expressing HEK293 cells and ovarian surface epithelial cells in a study that treated both

cell types with the PI3K inhibitors wortmannin and LY294009, resulting in the

inhibition of CaR induced ERK activity (Hobson et al. 2003).

Recently, there have been a number of studies linking the CaR to triple-membrane-

spanning signalling through the EGF receptor (EGFR) (MacLeod et al. 2004; Yano et

al. 2004b). Research into this mode of signalling via the EGFR began when it was

observed that the EGFR undergoes phosphorylation following GPCR activation (Daub

et al. 1996). A chimeric receptor with the extracellular portion of the EGFR and the

platelet derived growth factor receptor’s transmembrane and cytoplasmic domains was

used to show that an EGFR specific extracellular ligand was necessary for EGFR

transactivation by GPCRs (Prenzel et al. 1999). Heparin-binding epidermal growth

factor (HB-EGF) is present on the cell surface as a precursor protein proHB-EGF and

23

can act as a ligand for the EGFR (Dethlefsen et al. 1998). Experiments using inhibitors

to block either HB-EGF specifically or proteolytical processing of proHB-EGF revealed

that HB-EGF was essential for GPCR transactivation of the EGFR (Prenzel et al. 1999).

CaR-mediated ERK activation via triple-membrane-spanning signalling was first

examined in PC-3 cells and subsequently studied in HEK293 cells stably expressing the

CaR, using a combination of inhibitors and neutralising antibodies (MacLeod et al.

2004; Yano et al. 2004b). Treatment of PC-3 and CaR expressing HEK293 cells with

either AG1478, to inhibit the EGFR or GM6001, a broad-spectrum inhibitor of matrix

metalloproteases (MMPs) to inhibit HB-EGF production, reduced the level of CaR

induced stimulation of ERK (MacLeod et al. 2004; Yano et al. 2004b). Antibodies to

HB-EGF and the extracellular domain of EGF receptor also diminished the CaR-

mediated increases in ERK phosphorylation in both cell types (MacLeod et al. 2004;

Yano et al. 2004b). The results of these experiments indicate that stimulation of the CaR

can utilise the key components of triple-membrane-spanning signalling to phosphorylate

ERK (MacLeod et al. 2004; Yano et al. 2004b).

1.6.2.2.2 c-Jun NH2 Terminal Kinase

Studies investigating the role of the CaR in NIH/3T3 cells, a fibroblast cell line, found

that stimulation of the receptor leads to the phosphorylation of JNK, a protein

alternatively known as the stress-activated protein kinase (Hoff et al. 1999). Initiation of

the JNK signalling cascade by the CaR caused morphological changes in the NIH/3T3

cells that were inhibited by the introduction of a dominant-negative kinase that is

upstream of JNK in the pathway (Hoff et al. 1999). Ogata et al. would later show that

CaR agonist stimulation of fibroblasts isolated from rat jaw cysts enhanced

cyclooxygenase-2 expression (Ogata et al. 2006). Western blotting with

phosphospecific antibodies and inhibition experiments with SP600125, a JNK specific

inhibitor, revealed that the CaR-induced expression of cyclooxygenase-2 in fibroblasts

was via JNK signalling (Ogata et al. 2006).

1.6.2.2.3 p38 Mitogen Activated Protein Kinase

Phosphospecific antibodies were used in Western blots to detect increased

phosphorylation of the p38 MAPK in the CaR expressing mouse osteoblastic MC3T3-

E1 cell line in response to treatment with a variety of CaR agonists (Yamaguchi et al.

2000). By measuring the amount of [3H]-thymidine incorporated during DNA synthesis

Yamaguchi et al. was able to demonstrate that CaR induction of the p38 MAPK

24

signalling cascade generated mitogenic responses within the MC3T3-E1 cell line

(Yamaguchi et al. 2000). Using a combination of Western analysis and inhibition

studies, CaR-mediated p38 MAPK signalling has since been detected in rat H-500

Leydig cancer cells where p38 MAPK phosphorylation following CaR stimulation

results in the release of PTH-related protein (PTHrP) and in HK-2G cells, a proximal

tubule human kidney epithelial cell line, in which vitamin D receptor expression levels

are regulated by CaR-mediated p38 signalling (Maiti et al. 2008; Tfelt-Hansen et al.

2003).

1.6.2.3 Inhibition of Cyclic AMP

Prior to the identification of the CaR, it was observed that in bovine parathyroid cells a

range of divalent cations, including Ca2+, Mg2+, Ba2+ and Sr2+, inhibited the dopamine

stimulated accumulation of cyclic adenosine monophosphate (cAMP) in a pertussis

toxin sensitive manner (Chen et al. 1989). Treatment of the cortical thick ascending

limb of rat kidney with antidiuretic hormone leads to the production of cAMP (Ferreira

and Bailly 1998). Stimulation of the endogenously expressed CaR within the cortical

thick ascending limb with either extracellular calcium or neomycin inhibited the

antidiuretic hormone-induced accumulation of cAMP (Ferreira and Bailly 1998).

Another study examined cAMP formation in response to forskolin in HEK293 cells

stably expressing the CaR and noted that stimulation with the cations Ca2+, Mg2+ and

Gd3+ or the calcimimetic NPS R-467 significantly decreased cAMP content in this cell

type (Chang et al. 1998).

1.6.2.4 Rho Signalling

As mentioned above Huang et al. used an inhibitor to demonstrate the involvement of

Rho in CaR-mediated PLD activation (Huang et al. 2004). It was also shown that

extracellular calcium stimulation of MDCK cells stably overexpressing the CaR caused

Rho to translocate to the membrane (Huang et al. 2004). Further evidence of the CaR

acting via the Rho pathway has been reported by other groups examining two separate

CaR-mediated signalling events, the activation of serum response element (SRE)-

mediated gene transcription (Pi et al. 2002) and the production of intracellular Ca2+

oscillations by amino acid stimulation (Rey et al. 2005). The Rho family inhibitor C3

exoenzyme was used in experiments to demonstrate that Rho inhibition leads to the

abolition of CaR-stimulated SRE activity in HEK293 cells stably expressing the CaR

(Pi et al. 2002). When a mutant Rho, that was unaffected by C3 exoenzyme, was also

25

expressed in these cells, CaR-mediated SRE activity was partially restored (Pi et al.

2002). It has also been shown that activation of Rho by the CaR involved the

recruitment of the Rho-guanine nucleotide exchange factor (Rho-GEF) Lbc and filamin

(Pi et al. 2002). Transient calcium oscillations in CaR expressing HEK293 cells caused

by amino acid stimulation were also abrogated by C3 exoenzyme treatment (Rey et al.

2005). Interestingly, the sinusoidal calcium oscillations caused by extracellular calcium

stimulation were unaffected by the addition of C3 exoenzyme (Rey et al. 2005). A

dominant negative filamin peptide was used to block the production of transient calcium

oscillations caused by amino acid stimulation of the CaR. This confirmed the earlier

findings that filamin enhances activation of Rho by the CaR (Rey et al. 2005).

1.7 The Biological Roles of the Calcium-Sensing Receptor

Maintaining systemic calcium homeostasis is regarded as the major physiological role

of the CaR (Brown and MacLeod 2001). Essentially, systemic calcium homeostasis is

the balance within the body between the ingestion and absorption of calcium,

circulating calcium and excreted calcium, which involves the gastrointestinal tract, bone

and kidney (Brown 2000). The CaR expressed in these tissues, as well as in the

parathyroid and thyroid glands, is responsible for regulating the levels of calcium

absorbed or excreted by the body primarily by detecting circulating levels of calcium

within the extracellular fluid (Brown and MacLeod 2001). The CaR responds to a

decrease in the concentration of extracellular calcium by increasing the secretion of

PTH from the parathyroid gland and decreasing the secretion of calcitonin from the

thyroid (Brown 2000). The higher concentration of circulating PTH stimulates bone

resorption, leading to an increased calcium efflux from the skeleton and increased 1,25-

dihydroxyvitamin D3 production from the kidney, which increases calcium absorption

in the gastrointestinal tract (Quarles 2003). As calcitonin inhibits bone resorption and

decreases calcium excretion from the kidneys the CaR-mediated decrease in calcitonin

secretion in response to lower circulating calcium increases the overall level of calcium

retained by the body (Brown 2000).

Following the discovery of the CaR and its role in calcium homeostasis several

inherited disorders of calcium homeostasis were linked to functional abnormalities of

the receptor (Hauache 2001). Primarily, the calcium homeostatic disorders are

associated with mutations of the CaR, which are catalogued on an online database at

http:/data.mch.mcgill.ca/casrdb/, but there are also cases where antibodies to the CaR

26

are believed to interfere with the function of receptor (Thakker 2004). Over 100 CaR

mutations have been identified that are associated with caclium homeostatic disorders

(Pidasheva et al. 2004). First characterised in 1972, familial hypocalciuric

hypercalcaemia (FHH) is an autosomal dominant disorder associated with mild to

moderate hypercalcaemia, inappropriately normal PTH levels and low rates of urinary

calcium excretion that is the result of a loss-of-function CaR mutation on a single allele

(Hendy et al. 2000). There is emerging evidence that the biochemical severity of FHH is

linked to the dominant negative effect CaR mutations have on wild-type CaR activity

(Ward et al. 2006). Inactivating mutations in both copies of the CaR gene result in a

more serious condition known as neonatal severe hyperparathyroidism (NSHPT), which

is characterised by life-threatening severe hypercalcaemia, failure to thrive and

undermineralisation of bone (Tfelt-Hansen and Brown 2005). Without treatment by

parathyroidectomy, NSHPT can lead to multiple fractures, ribcage deformity and

neurodevelopmental disorders (Hendy et al. 2000). Gain-of-function CaR mutations

result in autosomal dominant hypocalcaemia, a generally asymptomatic condition with

patients experiencing mild hypocalcaemia and possibly seizures during childhood

(Hendy et al. 2000). Bartter syndrome type V, with symptoms including hypokalaemic

metabolic alkalosis, hyper-reninaemia, hyperaldosteronism and hypocalcaemia, is also

due to activating CaR mutations (Thakker 2004). Some patients with either autoimmune

hypocalciuric hypercalcaemia or autoimmune acquired hypoparathyroidism have been

shown to have circulating antibodies that recognise the extracellular domain of the CaR,

which are believed to be responsible for their respective conditions (Thakker 2004).

Although the CaR plays a vital role in the maintenance of calcium homeostasis, the

receptor has been detected in a host of tissues unrelated to calcium homeostasis,

including brain, breast and skin (Bikle et al. 1996; Chattopadhyay et al. 1997; Cheng et

al. 1998). Within the different CaR expressing cell types the receptor has been observed

to regulate a multitude of cellular processes including secretion, proliferation,

differentiation, apoptosis and gene expression (Buchan et al. 2001; Komuves et al.

2002; Peiris et al. 2007; Rutten et al. 1999; Tu et al. 2008). Below, the cellular

processes mediated by the CaR will be discussed in relation to several of the tissue

types in which there has been substantial investigation of the receptor’s function.

27

1.7.1 Calcium-Sensing Receptor in the Parathyroid

As mentioned earlier in this chapter, the CaR was originally cloned from parathyroid

cells, where the receptor mediates the inhibition of PTH secretion in response to agonist

stimulation (Brown et al. 1993). A large fraction of the CaR expressed in parathyroid

cells is localised to caveolae, which are regions of the cell membrane containing

signalling molecules and scaffolding proteins (Kifor et al. 1998). As the first cells to be

identified as expressing the CaR, parathyroid cells have been used extensively to

examine the cell signalling properties of the receptor. As outlined above, the CaR

expressed in parathyroid cells was shown to regulate PLC, PLA2, PLD and MAPK

signalling (Kifor et al. 1998; Kifor et al. 2003). However, the link between the induction

of intracellular signals by the CaR and the exocytotic apparatus that secretes PTH from

the cells is still largely unknown (Brown and MacLeod 2001). There is recent data

showing CaR signalling decreases the level of PTH mRNA produced in parathyroid

cells, suggesting an additional means by which the receptor limits the level of

circulating PTH (Carrillo-Lopez et al. 2008). The CaR is also hypothesised to regulate

the proliferation of parathyroid cells based on the evidence of greater parathyroid

hyperplasia in the parathyroid glands associated with decreased CaR function as seen in

patients with NSHPT, or reduced expression as seen in the parathyroid adenomas of

patients with primary hyperparathyroidism (Chen and Goodman 2004; Farnebo et al.

1998). Marked parathyroid cellular hyperplasia has also been observed in CaR knockout

mice (Ho et al. 1995).

1.7.2 Calcium-Sensing Receptor in the Kidney

Following the cloning of the CaR from parathyroid glands, the CaR expression was

detected in the kidney, another organ with a critical role in calcium homeostasis

(Riccardi et al. 1995). A combination of immunohistochemistry and RNA isolation

techniques was used to demonstrate that the CaR is expressed throughout the majority

of the nephron, including the proximal tubule, thick ascending limb, distal tubule and

collecting ducts (Butters et al. 1997; Riccardi et al. 1998). Interestingly, the expression

pattern of the CaR differs between the segments of the nephron (Ba and Friedman

2004). In proximal tubules the CaR is localised to the base of apical brush-border

membranes, in thick ascending limbs it is expressed on basolateral membranes, while in

the collecting ducts it is found at apical plasma membranes (Butters et al. 1997;

Riccardi et al. 1998). As each segment of the nephron has a specialised function it was

postulated that the role of the CaR in each segment would be specific (Brown and

28

Hebert 1997). Inorganic phosphate absorption by proximal tubules is inhibited by PTH,

but can be restored by stimulation of the CaR (Ba and Friedman 2004). In the thick

ascending limb, CaR was found to reduce the activity of the 70-pS apical membrane

potassium channel, which would lead to a reduction in potassium recycling (Wang et al.

1996). Aslanova et al, demonstrated that the intracellular pH within the thick ascending

limb is regulated by the CaR in a chloride-dependent manner (Aslanova et al. 2006).

PTH-dependent absorption of calcium by the cortical ascending limb was inhibited by

stimulation of the CaR, possibly by inhibiting the accumulation of cAMP (Ferreira et al.

1998; Motoyama and Friedman 2002). CaR agonist treatment of collecting duct cells

stably expressing aquaporin 2, inhibited the effects of forskolin-induced trafficking of

aquaporin 2, by reducing cAMP levels, activating the PKC signalling pathway and

increasing the level of actin fibre assembly (Procino et al. 2004).

1.7.3 Calcium-Sensing Receptor in the Gastrointestinal Tract

The gastrointestinal tract consists of a system of organs designed to cope with the

nutrient, electrolyte and fluid absorption requirements of the body, as well as the

secretion of excess electrolytes and fluids (Kirchhoff and Geibel 2006). The CaR has

been identified in a number of the organs that constitute the gastrointestinal tract,

including the oesophagus, stomach, small intestines and colon (Chattopadhyay et al.

1998a; Cheng et al. 1997). Recent experiments using the human oesophageal epithelial

cell line, HET-1A, have provided some insight into the role of the CaR in the

oesophagus, revealing that CaR stimulation led to increased phosphorylation of ERK,

intracellular calcium mobilisation and the secretion of IL-8 (Justinich et al. 2008). In the

stomach, the CaR is expressed in several specialised cells, including mucous epithelial

cells, G cells and parietal cells (Buchan et al. 2001; Busque et al. 2005; Rutten et al.

1999). In human gastric mucous epithelial cells, stimulation of the CaR, which is

primarily expressed at the basolateral membrane, results in increases in both

intracellular calcium levels and the rate of proliferation (Rutten et al. 1999). The CaR

expressed in G cells modulates the secretion of gastrin via the PLC signalling pathway

(Buchan et al. 2001). In response to either multivalent cations or amino acids, the CaR

expressed in parietal cells regulates the activity of a key component of gastric acid

secretion, the H+-K+-ATPase (Busque et al. 2005; Dufner et al. 2005). Research into the

role of the CaR in the intestine has focussed on the colon despite the receptor being

expressed in epithelial cells throughout the intestine (Hebert et al. 2004). Cheng et al.

showed that the colonic CaR in both surface and crypt cells responds to multivalent

29

cations by inducing PLC signalling (Cheng et al. 2002). Activation of the CaR inhibited

the forskolin-stimulated fluid secretion observed in isolated perfused colonic crypt cells,

presumably by inhibiting the cAMP pathway (Cheng et al. 2004). In colonic

myofibroblasts, stimulation of the CaR upregulated the expression and secretion of bone

morphogenetic protein-2 in a PI3K-dependent manner, but decreased the expression of

the bone morphogenetic protein-2 antagonist, Noggin (Peiris et al. 2007). Experiments

examining cultured intestinal cell lines found that the CaR regulated markers of cell

proliferation and differentiation (Chakrabarty et al. 2003; Kallay et al. 1997). The role

of the CaR in the regulation of cell proliferation and differentiation, as well as results of

several studies examining the preventive effects of a high calcium diet against colon

cancer has led to the proposal that the use of therapeutic CaR agonists may reduce the

risk of colon cancer (Kirchhoff and Geibel 2006; Rodland 2004).

1.7.4 Calcium-Sensing Receptor in Bone

Evidence of CaR expression in bone was first obtained in 1997 when a combination of

techniques was used to detect the CaR in the osteoblastic cell line MC3T3-E1

(Yamaguchi et al. 1998). Chang et al would later show that that the CaR is expressed in

osteoblasts of mouse, rat and bovine origin (Chang et al. 1999). It was demonstrated

that stimulation of the CaR expressed in osteoblasts led to an increase in proliferation

(Dvorak et al. 2004). The signalling mechanisms involved in CaR-induced proliferation

observed in osteoblasts is unclear, as one study has reported that JNK signalling was

necessary, while another reported the requirement for ERK, Akt and glycogensynthase

kinase 3β phosphorylation (Chattopadhyay et al. 2004; Dvorak et al. 2004). Following

the detection of the CaR in osteoclasts it was found that stimulation of the receptor

resulted in an inhibition of the bone resorbing activity of the osteoclasts (Kameda et al.

1998). CaR activity has been shown to promote both differentiation and apoptosis

within osteoclasts via the PLC pathway (Mentaverri et al. 2006). Initially the generation

of a CaR knockout mouse was believed to demonstrate a clear functional role of the

receptor in bone as these mice exhibited bone abnormalities and rickets (Garner et al.

2001; Ho et al. 1995). However, when the CaR knockout mice were bred with mice

lacking parathyroid glands a normal bone phenotype was observed, which suggested

that the bone abnormalities detected in the CaR knockout mice resulted from the loss of

CaR expression in the parathyroid glands (Tu et al. 2003). Evidence of alternative

splicing of the CaR supported this notion as the region of the CaR disrupted to generate

the CaR knockout mice could be spliced out without affecting the functionality of the

30

receptor (Oda et al. 2000; Rodriguez et al. 2005). In vivo evidence of the functional

relevance of the CaR in bone has only recently been presented with the generation of

conditional knockout mice that specifically do not express the CaR in bone, which

revealed a profound lack of postnatal growth and skeletal development (Chang et al.

2008). Most of the mice not expressing the CaR in bone did not survive past the first

three weeks after birth and exhibited undermineralisation of the skull, vertebrae and

long bones, as well as fractures of the ribs and long bones (Chang et al. 2008). The

postnatal growth deficiency, undermineralisation and fractures observed in mice deleted

in bone CaR is consistent with the symptoms observed in a patient with NSHPT

involving the effective so-called knockout of the CaR (Ward et al. 2004)

1.7.5 Calcium-Sensing Receptor in the Nervous System

Immunocytochemisty and in situ hybridization studies have revealed a wide distribution

of the CaR throughout the central nervous system with an extremely varied expression

pattern (Ferry et al. 2000; Rogers et al. 1997; Ruat et al. 1995). Not only does the

expression pattern of the CaR within the nervous system overlap with those of two other

Family C GPCRs, the mGluR and GABAB receptors, but it has also been shown in

coimmunoprecipitation experiments that the CaR heterodimerises with these receptors

(Chang et al. 2007; Gama et al. 2001). The highest level of CaR expression is within the

region of the brain known as the subfornical organ which, due to an absence of a blood-

brain barrier, is exposed to systemic fluid (Yano et al. 2004a). There is experimental

evidence indicating that a population of the subfornical organ neurons exhibit a

subthreshold, hyperpolarisation-activated inward current that is potentiated by CaR

stimulation (Washburn et al. 2000a). Furthermore, it has been proposed that the CaR-

mediated current regulates the bursting of action potentials and that subsequent

depolarising afterpotentials of neurones of the subfornical organ can also be modulated

by the CaR (Washburn et al. 2000b). There is also abundant expression of the CaR in

the hippocampus, but there has been little evidence of the role, if any, the receptor plays

in this part of the brain (Yano et al. 2004a). Due to an increase in CaR expression within

the hippocampus, corresponding to the time when long-term potentiation first occurs, it

has been hypothesised that the CaR may be involved in cognitive functions such as

memory and learning (Chattopadhyay et al. 1997). Recently, CaR activity was found to

promote axon growth and branching in developing neurons (Vizard et al. 2008).

Research examining the gonadotropin-releasing hormone neuron cell line, GT1-7,

demonstrated that CaR activation within these cells caused the secretion of cytokines

31

that promoted chemotaxis in astrocytes (Bandyopadhyay et al. 2007). CaR expressed in

the neurons of the hippocampus have been shown to regulate the opening of both

calcium permeable, nonselective cation channels and calcium activated potassium

channels (Vassilev et al. 1997; Ye et al. 1997b). Further studies have revealed that the

CaR-mediated activation of calcium permeable, non-selective cation channels can be

induced by the CaR agonist, amyloid-β peptide, which is excessively produced in

patients with Alzheimer’s disease (Ye et al. 1997a). The expression of the CaR within

the nervous system is not limited to neuronal cells, as the receptor has been detected in

glial cells as well (Yano et al. 2004a). CaR identified in oligodendrocytes and microglia

have also been found to regulate calcium activated potassium channels (Chattopadhyay

et al. 1998b; Chattopadhyay et al. 1999). Chattopadhyay et al. also demonstrated that

agonist stimulation of CaR expressed in astrocytes promoted PTHrP secretion

(Chattopadhyay et al. 2000).

1.7.6 Calcium-Sensing Receptor in Breast

The CaR has been identified in both normal and malignant breast tissue by Northern

analysis and immunohistochemistry (Cheng et al. 1998). In mice, it has been observed

that there is a constant low level of CaR expression in mammary glands throughout

development from prepubertal to adult glands, but the receptor is downregulated during

pregnancy and subsequently upregulated to its highest level of expression during

lactation (VanHouten et al. 2004). It has been demonstrated that PTHrP secretion is

regulated by the CaR in cultured cells derived from the mammary glands (Sanders et al.

2000; VanHouten et al. 2004). However, in normal breast cell lines, CaR stimulation

inhibits PTHrP secretion, while in breast cancer cell lines PTHrP secretion is increased

by CaR stimulation (Sanders et al. 2000; VanHouten et al. 2004). The opposite CaR-

mediated effects on PTHrP secretion have recently been revealed to be a result of the

CaR coupling to Gαi in normal breast cells and Gαs in malignant breast cells

(Mamillapalli et al. 2008). Aside from regulating PTHrP production in normal breast

cells, the CaR has been shown to be important for the transport of calcium into milk

during lactation (Ardeshirpour et al. 2006). The proposed role of the CaR in breast

cancer relates to the metastasis of the breast cancer to bone where the increase in CaR-

mediated PTHrP secretion causes greater bone resorption, which raises the level of

extracellular calcium and further stimulates the CaR leading to further PTHrP secretion,

resulting in a vicious cycle leading to worsening osteolysis (Sanders et al. 2000).

32

1.7.7 Calcium-Sensing Receptor in Epidermal Cells

In 1996, the CaR was identified in cultured human keratinocytes (Bikle et al. 1996).

Results from experiments using mice that were either unable to express full-length CaR

or overexpressed the CaR, suggested that the receptor has a role in the proliferation and

differentiation of keratinocytes (Komuves et al. 2002; Oda et al. 2000; Turksen and

Troy 2003). Recently, knockdown technology was used to decrease the expression

levels of the CaR in human keratinocytes, revealing that aside from its role in

differentiation the CaR is important in cell survival as a decrease in CaR expression in

keratinocytes correlated with an in increase in apoptosis (Tu et al. 2008).

1.8 Interacting Protein Partners of the Calcium-Sensing Receptor

In order to have a better understanding of the mechanisms that govern the CaR’s

functionality, recent work in the CaR field has aimed at identifying proteins that interact

with the receptor (Huang and Miller 2007). As with all GPCRs, the CaR signals through

its interaction with heterotrimeric G proteins, but recently several other proteins have

been identified that interact with the CaR and influence its signalling characteristics

(Huang and Miller 2007). Several identified interacting protein partners of the CaR will

be discussed below.

1.8.1 Filamin

In 2001, two groups performed yeast two-hybrid library screens using the CaR

intracellular tail as bait in order to discover proteins that interact with the CaR and both

studies revealed filamin A as an interaction target for the receptor (Awata et al. 2001;

Hjalm et al. 2001). Following the isolation of a filamin A cDNA fragment from a

human kidney cDNA library, Awata et al. used the yeast two-hybrid system to delineate

a region of filamin A comprised of amino acids 1566-1875 that interacted with the CaR

(Awata et al. 2001). Following the screening of a bovine parathyroid cDNA library

Hjalm et al. performed yeast two-hybrid mapping studies to localise the site of filamin

A to which the CaR binds to residues 1534-1719 (Hjalm et al. 2001). Taken together,

these two studies define filamin A’s minimal CaR binding site as amino acids 1566-

1719. The interaction between the CaR and filamin A was confirmed in mammalian

cells via confocal microscopy and a series of coimmunprecipitation studies (Awata et al.

2001; Hjalm et al. 2001). Zhang and Breitwieser performed a series of

coimmunoprecipitation experiments using truncation and deletion mutants of the CaR

that revealed that the site of interaction for filamin A was between residues 962-981, a

33

domain predicted to form two β-strands in the CaR tail (Zhang and Breitwieser 2005).

Filamin A is a cytoskeletal scaffold protein that has been implicated in the organisation

of signalling molecules (Li et al. 2005). Due to the possible role of filamin A in

intracellular signalling, the effect of disrupting the interaction between the CaR and

filamin A on the ERK pathway was examined using inhibitory peptides (Awata et al.

2001; Hjalm et al. 2001). Awata et al. blocked the binding between the CaR and filamin

A using a myc-tagged peptide corresponding to the filamin A binding site in the CaR

between residues 907-1022 and found that CaR-mediated ERK activity was reduced in

a dose-dependant manner (Awata et al. 2001). Conversely, Hjalm et al. generated a

filamin A fusion construct that contained the CaR interaction domain, amino acids

1534-1719, that was able to significantly disrupt CaR coupling to the ERK pathway

(Hjalm et al. 2001). Peptides corresponding to the CaR binding site in filamin A that

disrupted the interaction between the two proteins were also used to show that this

interaction was important for CaR-mediated Rho signalling (Pi et al. 2002; Rey et al.

2005). Silencing of the filamin A gene in HEK293 cells stably expressing the CaR with

siRNA caused a significant decrease in CaR-mediated JNK activation, indicating the

necessity of the interaction between the CaR and filamin A for the induction of the JNK

signalling cascade by the CaR (Huang et al. 2006a). Experiments that examined the

expression levels of the CaR in M2 cells, a cell line that does not express filamin A,

revealed that CaR expression was almost doubled when filamin A was transfected into

the M2 cells (Zhang and Breitwieser 2005). Inhibition of CaR expression with CaR

antisense cDNA 48 hours after the transfection of CaR with or without filamin A

resulted in a lower level of CaR expression in cells not expressing filamin A, suggesting

that filamin A protects the CaR against degradation (Zhang and Breitwieser 2005).

1.8.2 Potassium Channels

In addition to filamin A, the inwardly rectifying potassium channel Kir4.2 was

identified as a binding partner to the CaR intracellular tail in the yeast two-hybrid

kidney library screen described above (Huang et al. 2007a). The Kir4 family of proteins,

consisting of Kir4.1 and Kir4.2, are channels expressed on the basolateral membrane of

the distal nephron within the kidney and are believed to be involved in the regulation of

membrane potential and recycling potassium for Na,K-ATPases (Lourdel et al. 2002).

The CaR coimmunoprecipitated with both Kir4.1 and Kir4.2 from kidney cortex and

liver, respectively, and confirmed the initial findings of the yeast two-hybrid system

(Huang et al. 2007a). Kir4.2 and the CaR were observed to colocalise at the cell

34

membrane of HEK293 cells stably expressing both proteins, while endogenous CaR and

Kir4.1 colocalised at the basolateral membrane of the distal convoluted tubule in rat

kidney sections (Huang et al. 2007a).

1.8.3 Dorfin

In a third independent yeast two-hybrid library screen that used the CaR intracellular

tail as bait to probe a human kidney cDNA library, an E3 ubiquitin ligase, dorfin, was

identified as an interacting protein partner of the CaR (Huang et al. 2006b). By

interacting with both E2-ubiquitin and a target protein, dorfin is able to transfer the

ubiquitin to the target protein (Niwa et al. 2001; Wojcikiewicz 2004). The yeast two-

hybrid system was used to localise the sites of CaR and dorfin interaction to residues

880-900 in the CaR and amino acids 660-838 of dorfin (Huang et al. 2006b). It should

be noted that residues 660-838 form the carboxyl terminus of dorfin and that removal of

either amino acids between 660-720 or 780-838 disrupted the interaction between dorfin

and the CaR (Huang et al. 2006b). In HEK293 cells transiently expressing the CaR, an

increase in the ubiquitination of the receptor was observed when dorfin was

overexpressed in these cells (Huang et al. 2006b). Further evidence of dorfin regulating

the ubiquitination of the CaR was the reduction in ubiquitination of the receptor

detected in HEK293 cells cotransfected with the CaR and a dominant negative dorfin

construct incapable of catalysing the ubiquitination of substrates (Huang et al. 2006b).

Individual mutation of any of the 16 lysines present either in the intracellular loops or

tail to arginines had no significant impact on the level of CaR ubiquitination (Huang et

al. 2006b). However, when all 16 lysine residues were converted to arginines CaR

ubiquitination was abolished, indicating that the receptor is polyubiquitinated (Huang et

al. 2006b). Increasing the level of dorfin expressed in HEK293 cells transiently

expressing the CaR led to an increased rate of CaR degradation, while the reciprocal

result was observed when a dominant negative dorfin was expressed at increasing levels

(Huang et al. 2006b). Dorfin-mediated degradation of the CaR was shown to occur via

the proteosome when treatment with the proteasomal inhibitor MG132 abolished the

increase in CaR degradation associated with dorfin overexpression (Huang et al.

2006b). Coimmunoprecipitation experiments indicated that the CaR and dorfin are part

of a protein complex that includes the valosin-containing protein, suggesting that the

dorfin-mediated degradation of the CaR proceeds through the ER-associated

degradation (ERAD) pathway (Huang et al. 2006b).

35

1.8.4 Associated Molecule with SH3 Domain of STAM (AMSH)

A more recent screen of a human kidney cDNA yeast two-hybrid library used a CaR-tail

that contained a deletion of the region S895-V1075, which mimicked a naturally

occurring mutation, and identified associated molecule with SH3 domain of STAM

(AMSH) as an interacting partner of the CaR (Herrera-Vigenor et al. 2006). AMSH is

an ubiquitin isopeptodase that is a key regulatory component of endosomal sorting of

the EGFR (McCullough et al. 2004). The AMSH cDNA clone identified in the yeast

two-hybrid library screen corresponded to an AMSH splice variant that was able to bind

to both wild-type CaR and the CaR deletion mutant in a mammalian system following

the cloning of the cDNA fragment into a suitable expression vector (Herrera-Vigenor et

al. 2006). Full-length AMSH was also found to interact with the CaR in HEK293 cells

transfected with both proteins and increasing the level of AMSH expression diminished

the amount of CaR expressed (Herrera-Vigenor et al. 2006). Reduced CaR expression

due to its interaction with AMSH was observed in HEK293 cells transiently expressing

both the CaR and AMSH following stimulation with extracellular calcium (Reyes-

Ibarra et al. 2007).

1.8.3 Receptor-Activity-Modifying Proteins

The receptor-activity-modifying protein (RAMP) family are single-transmembrane

spanning protein that have been shown to affect receptor trafficking, glycosylation,

ligand specificity and second messenger production in several cell types (Morfis et al.

2003). While investigating a new method to examine cell surface expression of the CaR,

Bouschet et al. observed that the receptor was not expressed at the cell surface of COS7

cells (Bouschet et al. 2005). They proposed that the lack of CaR at the cell surface was

due to COS7 cells not expressing any members of the RAMP family and this was tested

by cotransfection of the CaR and the three different RAMPs into COS7 cells (Bouschet

et al. 2005). When the CaR was coexpressed in COS7 cells with either RAMP1 or

RAMP3 the receptor was delivered to the cell surface, but when coexpressed with

RAMP2 there was still no CaR detected at the cell surface (Bouschet et al. 2005).

Colocalisation and coimmunoprecipitation experiments provided evidence that both

RAMP1 and RAMP3 shared the same subcellular location as the CaR and that both

interact with the receptor. As RAMP3 had a greater influence on the cell surface

expression of the CaR than RAMP1, only the effects of RAMP3 on receptor trafficking

and glycosylation, were examined but the results observed are believed to also be valid

for RAMP1 (Bouschet et al. 2005). A high level of CaR was present in the ER and a

36

negligible amount in the Golgi apparatus in the absence of RAMPs, but when

coexpressed with RAMP3 a high level of the receptor was colocalised with RAMP3 in

the Golgi apparatus suggesting that trafficking of the CaR from the ER to Golgi is

reliant on RAMPs (Bouschet et al. 2005). CaR transiently expressed in both COS7 cells

transfected with RAMP3 and in HEK293 cells, which express endogenous RAMP1,

was glycosylated to a greater extent than CaR expressed in COS7 cells without any

RAMPs (Bouschet et al. 2005).

1.8.4 β-Arrestins

β-arrestins are ubiquitously expressed proteins that are involved in the desensitisation

and internalisation of most GPCRs leading to the eventual endocytosis of the receptors

in clathrin-coated pits (DeWire et al. 2007). Measuring luciferase activity in HEK293

cells cotransfected with rat CaR and a SRE-luciferase reporter construct in the presence

and absence of β-arrestins 1 and 2, revealed that both β-arrestin isoforms reduce the

level of CaR-induced luciferase activity (Pi et al. 2005). Interaction between β-arrestin 1

and the CaR was demonstrated in mammalian cells by coimmunoprecipitation

experiments, while colocalisation of the CaR and β-arrestin 2 was shown to occur

following stimulation of the receptor in U2OS cells and was enhanced by the

overexpression of G protein receptor kinase (GRK) 4 (Pi et al. 2005). In yeast two-

hybrid studies, both β-arrestin isoforms were found to interact with a region of the rat

CaR intracellular tail between amino acids 877 and 1079, but neither isoform interacted

with the residues 636-805 of the CaR, which contain the intracellular loops of the

receptor (Pi et al. 2005). In COS7 cells transiently transfected with constructs for β-

arrestin 1 and a fragment of the rat CaR corresponding to residues 877-1079, it was

observed that in cells that were additionally transfected with GRK2 the amount of β-

arrestin 1 coimmunoprecipitated with the CaR fragment was greater than in the absence

of ectopically expressed GRK2 (Pi et al. 2005). Lorenz et al. showed that

overexpression of both β-arrestin 1 and 2 had a negative influence on CaR-mediated

inositol phosphate production in GripTite293 cells, a HEK293 cell line genetically

engineered to have greater adherence. This effect could be abolished by either a PKC

inhibitor or by mutations of all the intracellular PKC phosphorylation sites of the CaR

(Lorenz et al. 2007).

37

1.9 Statement of Aims

As outlined in this chapter the CaR plays a crucial role linking a diverse range of stimuli

to different signalling pathways that in turn lead to a variety of tissue specific cellular

processes. A major determining factor of the biological responses initiated by the CaR

upon stimulation is likely to be the binding of accessory proteins to the intracellular tail

of the receptor. While several of these CaR-tail targets and their functional significance

have already been elucidated from a number of yeast two-hybrid screens using the CaR-

tail as bait, this number is almost certainly not exhaustive and it is anticipated that there

remain many more targets awaiting identification. Moreover, we had the opportunity to

screen a haemopoietic cell line library, not previously used in a yeast two-hybrid screen

with the CaR-tail, which had the potential to reveal many new and exciting targets. This

then forms the underlying hypothesis of this thesis:

That many novel CaR-tail protein targets that potentially influence CaR signalling

and other processes remain to be identified and that at least some of these targets will

be revealed by screening this unique library.

In order to address this hypothesis, other investigators in our laboratory used the LexA

yeast two-hybrid system to screen a mouse pluripotent haemopoietic cell line library

using the CaR-tail as bait. This screen revealed a large number of “potentially

interacting” clones when plated on selective medium. Approximately 130 of these

clones were further confirmed as “potentially interacting” using a Lac Z reporter assay.

The aims of this thesis were then:

AIM 1) To examine 60 of these “potentially interacting” clones to determine that they

are “true positives”. This was performed by plasmid rescue from unique clones,

cotransformation of the purified library plasmid with the CaR-tail plasmid into yeast

and verification of interaction using a Lac Z reporter system. Library plasmids

confirmed as positive underwent sequence analysis and database assessment. A

secondary aim was to determine the unique binding site in the CaR-tail for each of the

confirmed interacting proteins by CaR-tail deletion mapping using the yeast two-hybrid

system.

AIM 2) To examine in more detail two of the partner binding proteins identified above,

(1) Filamin A, a cytoskeletal protein shown previously to interact with the CaR and

influence CaR-mediated cell signalling

38

and

(2) Testin, a LIM domain containing, focal adhesion protein also known to have effects

on the cytoskeleton.

For filamin A, two interacting clones, found to be different to those published

previously, were examined for their ability to directly bind to the CaR-tail using

pulldown techniques and the implications for multiple filamin A binding sites for the

CaR on receptor function discussed.

For testin the aims were to:

(a) Determine the importance of the integrity of the second zinc finger of LIM domain 1

on testin – CaR binding using alanine scan site directed mutagenesis studies.

(b) Examine the ability of testin to interact with the CaR both directly, using pulldown

studies, and in a mammalian system by coimmunoprecipitation studies.

(c) Examine the colocalisation of the CaR and testin in mammalian cells using confocal

microscopy.

(d) Examine the effect of testin on CaR-mediated signalling pathways, specifically the

ERK and Rho signalling pathways using a Western blot-based technique and SRE-

luciferase reporter system, respectively.

(e) Examine the effect of testin on cell morphology and the cytoskeleton, specifically

focal adhesions and actin stress fibre assembly, using testin shRNA knockdown studies

in conjunction with fluorescence microscopy.

39

CChhaapptteerr 22 Materials and Methods

2.1 Materials

2.1.1 Reagents

Item Supplier

Acrylamide (30% Acrylamide/Bis Solution) Bio-Rad Laboratories

Agarose Promega

Ampicillin Sigma-Aldrich

Ammonium persulphate Sigma-Aldrich

Antipain dihydrochloride Sigma-Aldrich

Aprotinin Sigma-Aldrich

Bacto Agar Becton Dickinson

Bacto Peptone Becton Dickinson

Bacto Tryptone Becton Dickinson

Bacto Yeast Extract Becton Dickinson

BCA Protein Assay kit Pierce

Benzamidine (hydrochloride:hydrate) Sigma-Aldrich

β-mercaptoethanol Sigma-Aldrich

BigDye Terminator version 3.1 PerkinElmer

5-Bromo-4-chloro-3indolyl Sigma-Aldrich

B-Dgalactopyranoside (X-gal)

Bovine Serum Albumin New England Biolabs

Bromophenol blue Sigma-Aldrich

Calcium Chloride BDH Chemicals

Coomassie Brilliant Blue R-250 Sigma-Aldrich

Deoxynucleotide triphosphates Promega

Dimethyl sulphoxide BDH Chemicals

Dithiothreitol Sigma-Aldrich

DNA ladder (1 Kb Gibco BRL) Invitrogen

DMEM Thermo Electron Company

DMEM – Calcium Free Invitrogen

Enhanced Chemiluminescence Reagent PerkinElmer

(Western Lightning Plus)

40

Ethylenediaminetetra-acetic Acid AnalaR

Ethanol (Absolute and 95%) Biolab

Ethidium Bromide Calbiochem

Expand High Fidelity PCR System Roche Diagnostics

Fetal calf serum (FCS) Invitrogen

G418 (disulfate salt, cell culture tested) Sigma-Aldrich

Glacial Acetic Acid BDH Laboratory Supplies

Glass beads, acid washed Sigma-Aldrich

D-(+)-Glucose Sigma-Aldrich

DO Supplement -Leu/-Trp/-Ura Clontech

Glutathione Sigma-Aldrich

Glutathione Sepharose 4B Amersham Biosciences

Glycerol Ajax Finechem

Glycine ICN Biomedicals

Goat serum Sigma-Aldrich

HEPES Sigma-Aldrich-USA

Hydrochloric acid (32% (v/v)) Ajax Finechem

Hygromycin B Sigma-Aldrich

Isopropanol Rowe Scientific

Hybond-C super nitrocellulose membrane Amersham

Hyperfilm™ ECL Amersham

Isopropanol β-thiogalactopyranoside Promega

Kanamycin sulphate Sigma-Aldrich

Leupeptin Sigma-Aldrich

Lipofectamine 2000 Reagent Invitrogen

Lithium acetate (dihydrate) Sigma-Aldrich

Luciferase Assay Substrate Promega

Lysozyme Sigma-Aldrich

Magnesium Chloride Sigma-Aldrich

Magnesium Sulphate Sigma-Aldrich

Methanol Ajax Finechem

Opti-MEM Invitrogen

Penicillin-Streptomycin (cell culture tested) Sigma-Aldrich

Pepstatin A Sigma-Aldrich

Phenylmethylsulfonyl Fluoride Roche Diagnostics

41

Polyethylene Glycol 3350 Sigma-Aldrich

Ponceau S George T. Gurr Ltd.

Precision Plus Protein Dual Color Standards Bio-Rad

Prolong Gold Reagent Invitrogen

Protein G sepharose Amersham Biosciences

Protein low molecular weight markers Amersham Biosciences

Potassium Chloride Merck

Potassium Phosphate BDH Chemicals

Qiagen PCR cloning kit Qiagen

Qiagen Maxi Prep kit Qiagen

Qiagen QIAEXII Gel Extraction kit Qiagen

QIAquick PCR purification kit Qiagen

QuikChange Site-Directed Mutagenesis kit Stratagene

Salmon sperm DNA Sigma

Skim milk powder Diploma (Bonland Dairies Pty Ltd)

Sodium Acetate Crown Scientific, WA

Sodium Bicarbonate Ajax Chemicals

Sodium Chloride BDH Chemicals

Sodium Fluoride BDH Chemicals

di-Sodium Hydrogen Orthophosphate BDH Chemicals

Sodium dodecyl sulphate MP Biomedicals, Inc.

NaH2PO4.H2O Merck Pty. Ltd, VIC

Sodium Hydroxide APS Finechem

Sodium Fluoride Ajax Chemicals

Sodium Molybdate BDH AnalaR

Sodium vanadate Sigma-Aldrich

Sodium Dodecyl Sulphate (SDS) MP Biomedicals

N,N,N1,N1-tetramethylethylene (TEMED) Sigma-Aldrich

Tetracycline hydrochloride Sigma-Aldrich

Triton X-100 Roche Diagnostics

Trizma® base Sigma-Aldrich

Trypsin SAFC Biosciences

Tryptophan Sigma-Aldrich

TWEEN 20 Sigma-Aldrich

Urea Sigma

42

Xylene cyanole FF Sigma

Yeast nitrogen base w/o amino acids Becton Dickinson

2.1.2 Plasmids

Item Supplier

pBTM116 Dr Schickwann Tsai, Fred Hutchinson

Cancer Research Centre

pBTM116/CaR-tail Dr Bryan Ward, Sir Charles Gairdner

Hospital

pcDNA3.0/EGFP Karin Kroeger, WAIMR

pcDNA3.1/CaR. FLAG Aaron Magno, Sir

Charles Gairdner Hospital

pET28a/CaR-tail Nuella Cattalini, Sir Charles Gairdner

Hospital

pET-NusA Dr Evan Ingley, WAIMR

pGEX4T.1 Amersham

pSRE-luciferase Prof Jeffrey Pessin, SUNY

pSUPERIOR.retro.neo+gfp Prof Peter Leedman, WAIMR

pVP16 Dr Schickwann Tsai, Fred Hutchinson

Cancer Research Centre

2.1.3 Enzymes

Item Supplier

Alkaline Phosphatase Promega

BamHI Promega

BglII Promega

EcoRI Promega

HindIII Promega

KpnI Promega

NcoI Promega

NotI New England Biolabs

PstI Promega

SalI Promega

T4 DNA ligase Promega

Taq DNA Polymerase Promega

XcmI New England Biolabs

43

XhoI Promega

2.1.4 Cell lines

Item Supplier

HEK293 Assoc Prof Karin Eidne, WAIMR

HEK293-CaR (G418 resistant) Prof Arthur Conigrave, University of

Sydney

HEK293-CaR (Hygromycin resistant ) Dr Donald Ward, Manchester

University

PA317 Prof Peter Leedman, WAIMR

2.1.5 Antibodies

Item Supplier

Goat anti-mouse-Alexa Fluor 546 antibody Invitrogen

Goat anti-mouse-HRP antibody Sigma-Aldrich

Goat anti-rabbit-HRP antibody Promega

Mouse anti-α-Tubulin antibody Sigma-Aldrich

Mouse anti-FLAG M2 monoclonal antibody Sigma-Aldrich

Rabbit anti-ERK antibody Promega

Rabbit anti-GFP antibody Santa Cruz Biotechnology

Rabbit anti-P-ERK antibody Promega

Rabbit anti-P-Y118 paxillin antibody Invitrogen

Rabbit anti-mouse Alexa Fluor-568 antibody Invitrogen

Rabbit anti-mouse HRP antibody Sigma-Aldrich

2.1.6 Equipment

Item Supplier

Centrifuge, Beckman AvantiTM J-301 Beckman Coulter

DNA Thermal Cycler Perkin Elmer

Microcentrifuge IEC Micromax Model

#100,120,220 and 240 IEC

Microscope (Model IMT-2) Olympus

Olympus IX81 inverted microscope Olympus

PTC-100 Programmable Thermal Cycler MJ Research, Inc.

POLARstar OPTIMA plate reader BMG Labtechnologies

44

Varian Spectrophotometer Series 634 Varian Technologies

Scion Imaging Software Scion Corp

Sonifier Cell Disruptor B15 Branson

X-Ray Processor, AGFA CP1000 Scanner AGFA

2.1.7 Commercial Suppliers

Company Address

AGFA Morstel, Belgium

Ajax Chemicals NSW, Australia

Amersham Buckinghamshire, UK

BDH Chemicals Vic, Australia

Becton Dickinson CA, USA

Beckman Coulter USA

Biolab Vic, Australia

Bio-Rad Laboratories CA, USA

BMG Labtechnologies Offenburg, Germany

Branson CT, USA

Calbiochem USA

Clontech USA

Crown Scientific NSW, Australia

Diploma Vic, Australia

George T. Gurr Ltd. London, England

ICN USA

IEC USA

Invitrogen CA, USA

Merck Vic, Austalia

MJ Research Inc. (Bio-Rad) MA, USA

MP Biomedicals, Inc OH, USA

New England Biolabs, Inc MA, USA

Olympus Japan

Perkin Elmer MA, USA

Pierce IL, USA

Promega Wisconsin, USA

Qiagen Hilden, Germany

Roche Diagnostics IN, USA

45

SAFC Biosciences KS, USA

Santa Cruz Biotechnology CA, USA

Scion Corp USA

Sigma-Aldrich MO, USA

Stratagene CA, USA

Thermo Electron Company Vic, Australia

Varian Technologies CA, USA

Whatman® International Ltd Maidstone, England

2.2 Methods

2.2.1 General Methods

2.2.1.1 Tissue Culture Methodology

2.2.1.1.1 Maintenance of Cell Lines

Frozen 1 ml stocks of cells stored in liquid nitrogen were rapidly thawed at 37oC and

added aseptically to prewarmed DMEM (Doublecco’s Modified Eagle Medium)

supplemented with 10% fetal calf serum (FCS), 100 units/mL penicillin and 10 μg/mL

streptomycin in a 75 cm2 cell culture flask.

Mammalian cell lines were generally maintained in DMEM supplemented with FCS

and antibiotics in 75 cm2 flasks and incubated at 37°C with 5% CO2. Cells were

passaged at subconfluent levels of approximately 90%. Medium was aspirated from the

flasks containing the cells, which were then washed twice with sterile PBS. After

washing, 2 mL of 1 x trypsin/EDTA solution was added to the cells and incubated for 5

min at 37°C. The trypsin/EDTA was then deactivated with 10 mL DMEM supplemented

with FCS and antibiotics and transferred to a sterile 15 mL falcon tube for

centrifugation at 1, 000 rpm for 1 minute at room temperature. Following this, the

medium was aspirated and the cells were resuspended in 10 mL of fresh medium and 1

mL of the cell suspension transferred to a new flask containing fresh DMEM

supplemented with FCS and antibiotics.

To generate frozen aliquots of cells, following trypsinisation, as described above, the

cell pellets were resuspended in a volume of freezing medium (DMEM with 25% FCS

and 10% DMSO) and aliquoted into cryotubes. The cryotubes were placed in a

polystyrene rack and placed at -70°C overnight before being placed in liquid nitrogen

for long-term storage.

46

2.2.1.1.2 Transfection

Cells were grown to approximately 50-60% confluency in 25 cm2 flasks. Routinely, 5

μg of plasmid DNA was diluted into 500 μL of Opti-MEM and combined with 500 μL

of Opti-MEM containing 15 μL of Lipofectamine2000. This mixture was incubated for

20 min at room temperature. Media was then aspirated from the cells and replaced with

4 ml of DMEM containing 10% FCS but no antibiotics. The 1 ml reactions containing

the DNA and Lipofectamine2000 were then added to the cells and incubated for 4 hr at

37oC with 5% CO2. The medium was again aspirated from each flask and replaced with

5 ml of DMEM supplemented with FCS and antibiotics. Generally, cells were

incubated for 48 hr at 37oC with 5% CO2 prior to cell lysis or other studies.

2.2..1.1.3 Lysis of Cultured Mammalian Cells

Following transfection cells were grown for 48 hr in DMEM with 10% FCS, 100

units/mL penicillin and 100 μg/mL streptomycin before culture medium was removed

from flasks and cell monolayers washed twice with 1 ml of ice-cold PBS, while on ice.

Cells were then lysed in 500 μl of cell lysis buffer (150 mM NaCl, 20 mM Tris pH 6.8,

10 mM EDTA, 1 mM EGTA, 1% Triton X-100,) with 100 mM iodoacetamide and the

protease inhibitors 1 mM PMSF, 0.01 mg/ml aprotinin, antipain, and leupeptin, and 0.1

mg/ml pepstatin A. Lysates were transferred to eppendorf tubes and passed through a 25

gauge needle 10 times on ice, prior to centrifugation at 14 000 rpm for 30 min at 4 °C.

Cleared lysates were transferred to fresh eppendorf tubes for the determination of

protein concentration using the Pierce BCA Protein Assay kit prior to

coimmunoprecipitation.

2.2.1.2 Transformation of Competent Cells

An appropriate aliquot, between 2 and 10 μL of plasmid DNA was added to either 100

or 200 μL of competent bacterial cells, either XL1-Blue or BL21 codon (+) and kept on

ice for 10 min before being heat shocked for 90 sec at 42oC. The competent bacterial

cells were then placed on ice for 2 min before having 800 μL of warmed 2xYT media

added. The reaction was then incubated for 1 hr at 37oC with shaking. Following the

incubation, the cells were spun down at 7,500 rpm for 30 sec in a microcentrifuge at

room temperature and 900 μL of the supernatant was removed and the pellet was

47

resuspended in the remaining 100 μL before being plated out onto LB or 2xYT agar

plates with the appropriate antibiotic.

2.2.1.3 Plasmid DNA Preparation

Small scale production of plasmid DNA was performed using the Wizard® Plus SV

Miniprep DNA Purification System Kit (Promega). A single isolated bacterial colony

was used to inoculate 5 mL of LB with appropriate antibiotic selection and grown

overnight at 37oC with shaking. The next day the culture was centrifuged and DNA

extracted from the pellet according to the manufacturer’s instructions. Purified DNA

was generally eluted into 80 μL of sterile ddH2O except when the DNA was to be used

in the cotransformation of yeast when it was eluted into 50 μL of sterile ddH2O.

Large scale production of plasmid DNA was performed using the Qiagen Maxiprep Kit

according to the manufacturer’s instructions with some minor alterations. A single

isolated bacterial colony was used to inoculate a starter culture of 5 mL of LB with

appropriate antibiotic selection that was grown for approximately 4 hr at 37oC with

shaking. At the end of this incubation the starter culture was transferred to a 3 L flask

containing 500 mL of LB with appropriate antibiotic selection and grown overnight at

37oC with shaking. The following day the cells were pelleted by centrifugation at 4,000

rpm in a Sorvall RC-3 Centrifuge for 20 min at 4°C. The supernatant was removed and

the pellet of cells processed for plasmid DNA extraction in accordance with the kit’s

instructions. The sample was then centrifuged at 4,000 rpm in a Sorvall RC-3

Centrifuge for 30 min at 4°C and the supernatant added to a QIAGEN-tip 500 column,

which had been equilibrated with Buffer QBT.

After the sample had passed through, the column was washed twice with Buffer QC.

Plasmid DNA was eluted from the column with 15 mL of Buffer QF and then

precipitated with 10.5 mL of isopropanol. The DNA was centrifuged at approximately

11,000 rpm for 30 min at 4 °C in an Avanti J-30I Beckman centrifuge using a JA30.50

rotor. The supernatant was removed and the pellet was resuspended in 5 mL of 70%

ethanol and aliquotted into eppendorfs to be centrifuged at 13,200 rpm in a

microcentrifuge at 4oC. The supernatant was removed and the DNA pellet was allowed

to air-dry at room temperature. Once all of the ethanol had evaporated, the DNA in each

eppendorf was redissolved in ddH2O and pooled together into a final volume of 600 μL.

The DNA was quantitated and its quality verified by agarose gel electrophoresis.

48

2.2.1.4 Quantitation of DNA

DNA was quantitated by spectrophotometric analysis using a Varian spectrophotometer.

DNA was diluted with sterile ddH2O and placed in a quartz cuvette where the

absorbance was measured at the ultraviolet wavelength of 260 nm and where 1 OD260

was specified as 50 μg/mL for double stranded DNA and 33 μg/mL for single stranded

oligonucleotides. A reading at the wavelength 280 nm was also taken to measure the

purity of the DNA preparation. An OD260/OD280 value of approximately 1.8 indicated a

pure DNA preparation.

2.2.1.5 Agarose Gel Electrophoresis

DNA was routinely run on a 1% (w/v) agarose gel with agarose dissolved in 1 x TAE

buffer and containing 0.4 μg/mL ethidium bromide for DNA visualisation. A 1 Kb

DNA ladder was run alongside the DNA samples for size determination. Gels were

electrophoresed in a DNA electrophoresis mini-sub DNA tank (Bio-Rad) in 1 x TAE

buffer at 100 volts (V). DNA bands were visualised on a UV transilluminator and

photographed using an IBI Quick Shooter Polaroid camera.

2.2.1.6 Purification of DNA

2.2.1.6.1 Purification of DNA from Agarose Gels

Following electrophoresis on an agarose gel, DNA bands were extracted and purified

using the QIAGEN QIAEX II Gel Extraction Kit according to the manufacturer’s

specifications. Briefly, up to 250 mg of agarose containing the appropriate DNA band

was excised from the gel using a sterile scalpel and placed into eppendorf tubes for

processing. The agarose was first solubilized in 500 μl of QX1 buffer. The DNA was

then absorbed to 20 μl QIAEX II beads and washed once in QXI buffer, then twice with

wash solution containing ethanol before the pellet was dried. DNA was eluted with 20

μl of ddH2O.

2.2.1.6.2 Purification of DNA Using the QIAquick PCR Purification Kit

DNA was purified from restriction enzyme digestions, dephosphorylation reactions or

PCR reactions using the QIAGEN PCR Product Purification kit according to the

manufacturer’s specifications. Briefly, 5 volumes of Buffer PB were added to 1 volume

of the reaction and mixed by inversion. This mixture was then applied to the supplied

spin column and centrifuged at 13,200 rpm for 1 min. DNA bound to the column

49

membrane was then washed in 0.75 ml of Buffer PE and centrifuged at 13,200 rpm for

1 min. Finally, DNA was eluted in 30 μl of ddH20.

2.2.1.7 Ethanol precipitation of DNA

The volume of DNA suspension was made up to 20 μL with ddH2O and 2.5 volumes of

ethanol and 0.1 volumes of 3 mM sodium acetate pH 5.2 were then added and the

sample was incubated for 15 min at room temperature. The sample was then spun for 30

min at 32,000 rpm in a microcentrifuge at room temperature. Following the spin, the

supernatant was removed and the pellet was washed with 70 μL of 75% ethanol. The

sample was then centrifuged at 32,000 rpm in a microcentrifuge for 10 min at room

temperature and the supernatant was again removed. The pellet was then placed into a

desiccator to dry for 30 min.

2.2.1.8 Restriction Enzyme Digestion

Routinely, restriction enzyme digests were performed either as 20 μL or 40 μL reactions

containing 1 x reaction buffer, 1.5 to 5 μg DNA and 10 to 20 units of enzyme.

Reactions were incubated for between 3 to 18 hr at 37oC. Digests requiring two

different restriction enzymes were performed either simultaneously using a mutually

compatible reaction buffer or sequentially with either an ethanol precipitation or agarose

gel extraction step to purify the initial restriction enzyme digestion products as

described in 2.2.1.7 and 2.2.1.6.1 respectively.

2.2.1.9 Dephosphorylation of 5’-Ends

To prevent re-circularisation during ligation, vector DNA digested with a single

restriction enzyme was treated with 20 units of alkaline phosphatase for 1 hr at 37oC to

dephosphorylate 5’ ends. The alkaline phosphatase was inactivated by incubation at

75oC for 10 min. The DNA was then purified from the dephosphorylation reaction

using the QIAGEN PCR Product Purification Kit as described in 2.2.1.6.2.

2.2.1.10 Ligations

DNA ligations were performed using T4 DNA ligase (Promega) in 10 µL reactions

containing 1 x reaction buffer, 3 units of T4 ligase and a vector:insert molar ratio of 1:3.

Control ligations, where insert DNA was excluded, were run alongside all test ligations.

Reactions were incubated at 15ºC overnight and then transformed into XL1-Blue

competent cells as described in 2.2.1.2.

50

Ligation of PCR products into the pDrive cloning vector (Qiagen PCR cloning kit) was

performed according to the manufacturer’s instructions. Four µL (300-400 ng) of DNA

was added to 1 µL (50 ng) pDrive and 5 µL of the 2 x master mix. Reactions were

incubated at 15ºC overnight and then transformed into XL1-Blue competent cells as

described in 2.2.1.2.

2.2.1.11 Reverse Transcriptase-PCR (RT-PCR)

Reverse transcription was performed using the Sensiscript RT-PCR kit (QIAGEN)

according to the manufacturer’s instructions to generate cDNA from mRNA extracted

from selected cell lines that had been diluted to approximately 25 ng/μl in RNAse free

ddH2O. The 20 μL RT-PCR mix contained 50 ng of mRNA (first denatured at 65oC for

5 min), 1x Buffer RT, 500 nM dNTP, 250 ng of random hexamers, 10 U of RNasin and

1 μL of Sensiscript Reverse Transcriptase. The reverse transcription reactions were

incubated at 37oC for 90 min in a Perkin Elmer DNA Thermal Cycler. The cDNA

generated was amplified by PCR as outlined in 2.2.1.7 with 10 μL of cDNA.

2.2.1.12 PCRs Using a Proofreading Enzyme.

For the amplification of DNA fragments that were used for cloning purposes the

Expand High Fidelity PCR system (Roche) was used as described in the manufacturer’s

instructions. The 50 μL PCR reactions contained in 1 x reaction buffer in ddH2O, 7.5 U

of High Fidelity enzyme, 1.5 mM MgCl, 300 μM dNTP, 400 nM of each of the two

appropriate complementary primers (Appendix 1) and either 10 μL of cDNA or 2 ng of

plasmid DNA. Reactions were overlayed with oil and cycled in the Perkin Elmer DNA

Thermal Cycler for 40 cycles as follows:

Phase Temperature Duration Number of Cycles

A: T ep94oC 1 min 40

T ep68oC 1 min

T ep72oC 2 min

B: Te p72oC 10 min 1

C: Te p4oC indefinite 1

51

2.2.1.13 Site-Directed Mutagenesis

Site-directed mutagenesis was performed using the QuickChange site-directed

mutagenesis kit, which is a polymerase chain reaction (PCR) based system. Primers

used in site-directed mutagenesis are outlined in Appendix 1. The 50 μL PCR reaction

contained 5 μL of 10x reaction buffer, 50 ng of template plasmid DNA, 125 ng of each

of the two complementary mutagenesis primers, 1 μL of dNTP mix and 1 μL of Pfu

Turbo DNA polymerase. As the PCR reaction was conducted in a Perkin Elmer DNA

Thermal Cycler it was necessary to overlay the reaction mixtures with mineral oil to

prevent evaporation. The cycling parameters used were as follows:

Phase Temperature Duration Number of Cycles

A: T ep95oC 30 sec Number 1

B: T ep95oC 30 sec Num 16 or 18

T ep55oC 1 min

Te p68oC 2 min per kb of plasmid length

C: Te p4oC indefinite Number1

It should be noted that in Phase B of the PCR reaction the number of cycles is

dependent on whether a single amino acid is being changed (16 cycles) or multiple

amino acids are being mutated (18 cycles). Following the PCR, the reaction mixture

was incubated at 37oC with 1 μL of Dpn I restriction enzyme for 1 hr to remove

methylated parent DNA and then used to transform XL1-Blue competent cells as

outlined in 2.2.1.2

2.2.1.14 DNA Sequencing

DNA sequencing was performed using the Big Dye Terminator Version 3.1 mix. The 10

μL sequencing reactions contained 2 μL of Big Dye Terminator Version 3.1 mix,

approximately 200 ng of plasmid DNA and 25 ng of the appropriate sequencing primer

(see Appendix 1) made up to a final volume of 10 μL with ddH2O. The PCR reactions

were conducted in a MJ Research, Inc., PTC-100 Programmable Thermal Cycler using

the following cycling conditions:

Temperature Duration Number of Cycles

T ep95oC 30 sec Num 25

T ep49oC 15 sec

Te p59oC 4 min

52

Following the PCR the reaction mixture was purified by ethanol precipitation as

outlined in 2.2.1.X. Samples were sent to the Department of Clinical Immunology,

Royal Perth Hospital to be processed using an ABI Prism 3730 48 capillary sequencer.

Sequences generated were compared to published sequences using ClustalW2 at EBI

Tools (http://www.ebi.ac.uk/Tools/clustalw2/index.html) (Chenna et al. 2003) to align

sequences and the Chromas program (version 1.45) to view the chromatograms

generated.

2.2.1.15 Quantification of Protein Concentration Using a BCA Assay Kit

Protein samples were diluted in the appropriate buffer to a final volume of 50 μL. A

standard curve covering a range of protein standards from 500 μg/mL to 15.6 μg/mL

was generated by performing serial two-fold dilutions of bovine serum albumin (BSA)

in the same buffer in a final volume of 50 μL. By adding 50 parts of Reagent A to 1 part

of Reagent B, the BCA reagent was constituted and then 1 mL of it was added to the

prepared protein samples and standards. This solution was then mixed by gentle

inversion and incubated for 30 min at 37oC. After incubation, samples were allowed to

come to room temperature and the protein was quantitated using a Varian

spectrophotometer measuring the absorbance at a wavelength of 562 nm. The protein

sample concentrations were determined using a standard curve relating to BSA

concentration at OD562 reading.

2.2.1.16 Quantification of Protein Concentration Using the Bradford Assay

BSA was serially diluted in the appropriate buffer to create a standard curve including

points between 3 mg/mL and 0.5 mg/mL. To 1 mL of Bradford Reagent, which had

been freshly filtered and was at room temperature, 5 μL of sample or protein standard

was added. After the solution was mixed by gentle inversion it was incubated for 5 min

at room temperature before its absorbance was measured on a Varian spectrophotometer

at a wavelength of 595 nm. The BSA standard curve was used to assess the protein

concentration of samples.

2.2.1.17 Preparation of Gels and Electrophoresis

Gels were prepared and run on a Mini-PROTEAN® II Dual Slab Cell (Bio-Rad). The

separating gel was prepared with 1 x separating buffer (1.5 M Tris pH 8.8, 0.4% (w/v)

SDS), 7.5%, 10% or 15% acrylamide, 0.03% (w/v) APS, 0.1% (v/v) TEMED made up

in ddH2O to a final volume of 7.5 mL. Following the addition of APS and TEMED the

53

gel was immediately poured into the gel cast. The stacking gel was prepared with 1 x

stacking buffer (0.5 M Tris pH 6.8, 0.4% (w/v) SDS), 4% acrylamide, 0.1% APS (w/v)

and 0.1% (v/v) TEMED made up in ddH2O to a final volume of 3.5 mL. Following the

addition of the APS and TEMED the stacking gel was poured immediately into the gel

cast, overlaying the separating gel, and a comb inserted. Both the separating and

stacking gels set within 1 hr at room temperature. Proteins were electrophoresed in

freshly prepared 1 x SDS-polyacrylamide gel electrophoresis (SDS-PAGE) running

buffer (25 mM Tris, 0.1% (w/v) SDS, 192 mM glycine) at 180 V.

2.2.1.18 Western Blotting

After proteins had been separated on a suitable SDS-PAGE percentage gel, they were

transferred to a Hybond-C super nitrocellulose membrane (Amersham) using a Mini

Trans-Blot Electrophoretic Transfer Cell (Bio-Rad) according to manufacturer’s

instructions. The membrane was placed into the appropriate blocking solution (see

Appendix 2 for solutions and incubation times used for specific antibodies) and

incubated with rotation for 1 hr at room temperature. The blocking solution was then

removed and the membrane incubated with the primary antibody in the appropriate

buffer. Following the incubation with the primary antibody the membrane was washed 3

times in the appropriate buffer and then incubated with a secondary antibody conjugated

to horseradish peroxidise in the appropriate buffer for 1 hr. After incubation with the

secondary antibody the membrane was again washed 3 times and exposed to enhanced

chemiluminescience reagent for 1 min. The membrane was then exposed to Hyperfilm

Film for a range of times from 10 sec to 5 min. Membranes that were to be probed again

using different antibodies were placed in 50 mL of membrane stripping buffer (62.5

mM Tris-HCl ph 6.8, 100 mM β-mercaptoethanol, 2% SDS) for 30 min at 50oC and

then rinsed in ddH2O prior to undergoing the same treatment as above with the different

antibodies.

2.2.1.19 Densitometry

The Western films were scanned using a Cannon Scanjet scanner and saved as grey

scale tif files. Density of bands was measured using Scion Image software with

correction for background.

54

2.2.1.20 Statistical Analysis

Statistical significance was determined by an ANOVA analysis (p<0.05) using SPSS

version 15.0 software.

2.2.2 Identification of Positive Clones from a Yeast Two-Hybrid Library Screen

2.2.2.1 DNA Extraction from Yeast

In the initial screen of the yeast two-hybrid screen of the EMLC.1 mouse pluripotent

haemopoietic cell line library 130 yeast colonies were identified as potentially

containing library proteins that interact with the CaR-tail and were stored as glycerol

stocks. These glycerol stocks were used to inoculate 4 mL of uracil and leucine

deficient media and grown overnight at 30oC with vigorous shaking at 200 rpm. The

next day 1.5 mL of the overnight culture was spun down in a microcentrifuge at 10,000

rpm for 30 sec at room temperature. The supernatant was removed and the pellet

resuspended in 200 μL of Yeast Lysis Buffer (10 mM Tris pH 8.0, 100 mM NaCl, 1

mM EDTA, 1 % SDS). To the resuspended pellet 200 μL of phenol/chloroform-isoamyl

alcohol (1:1 v/v) and approximately 300 mg of acid washed glass beads were added.

This mixture was then vortexed at high speed for 2 min and then spun down in a

microcentrifuge at 14,000 rpm for 10 min at room temperature. The aqueous phase was

transferred to a fresh tube. 8 μL of 10 M ammonium acetate and 300 μL of chilled

ethanol were added prior to being placed at -70oC for a minimum of 1 hr. The solution

was then spun down at 14,000 rpm in a microcentrifuge for 30 min at 4oC. The

supernatant, containing the extracted plasmid, was then removed and stored at -70oC for

future use. The DNA pellet was washed with 75% ethanol, centrifuged and dried in a

dessicator prior to resuspension in 50 μL ddH2O

2.2.2.2 Profiling of Plasmids by Restriction Enzyme Digestion

Plasmid DNA extracted from yeast colonies was used in PCR reactions to amplify the

library inserts using the M13F and VP16-2 primers (see appendix 1 for details). The

PCR mix contained 5 μL of plasmid DNA, 1 x Promega Taq Polymerase Buffer, 2 mM

magnesium chloride, 300 μM dNTP, 125 ng of both the M13F primer and VP16-2

primers and 0.5 μL Taq polymerase. The PCR conditions used were as follows, 1 cycle

of 2 min at 94oC, 35 cycles of denaturation at 94oC for 15 sec, annealing at 60oC for 20

sec and extension at 72oC for 2 min. Amplified DNA was then digested by the

restriction enzyme Hae III at 37oC for a minimum of 3 hr. Both digested and undigested

PCR products were separated on 1% agarose gels. Both the size of the library insert and

55

its HaeIII digestion pattern were examined to determine which clones were unique and

worthy of further investigated.

2.2.2.3 Plasmid Recovery of Library Clones

An aliquot of 7.5 μL of plasmid DNA from each unique library clone was added to 200

μL of HB101 competent cells on ice and incubated for the 30 min and then heat

shocked for 90 sec at 42oC. The HB101 cells were then placed back on ice for 2 min

before having 800 μL of SOC media added and incubated for 1 hr at 37oC with shaking.

Following incubation the cells were washed twice by resuspension in 1 mL of M9

media after microcentrifugation at 7,500 rpm for 30 sec at room temperature. After the

final wash 850 μL of the supernatant was removed and the pellet was resuspended in the

remaining M9 medium before being plated out onto leucine deficient M9 plates that

contain 100 μg/mL of ampicillin. Plates were incubated overnight at 37oC and colonies

picked the next day for DNA extraction using the Promega Wizard Plus SV Miniprep

DNA Purification System according to the manufacturer’s specifications. The purified

plasmid was digested by the restriction enzyme, Not I, to release inserts. Digestion

products were separated on a 1% agarose gel to check the size of the inserts.

2.2.2.4 Cotransformation of Bait and Library Plasmids with Yeast L40

Verification of the interaction between selected library proteins and the CaR-tail in β-

galactosidase colony lift assays required the transformation of yeast with a combination

of constructs. Briefly, L40 yeast were transformed with one of the following

combinations of constructs:

Library clone/pVP16 and CaR-tail/pBTM116 - test

Library clone/pVP16 and ARLE-1/pBTM116 - negative control

Hsp90(520-724)/pVP16 and Cyp40(185-370)/pBTM116 - positive control

Hsp90(520-724)/pVP16, ARLE-1/pBTM116 - negative control

Library clone/pVP16 alone - negative control

The L40 yeast transformed with only the library clone/pVP16 construct acted as a

control to show that the construct could not intrinsically activate the LacZ gene. The

Hsp90 and Cyp40 cotransformation provided a positive control for the system as these

two proteins have previously been shown to interact using the β-galactosidase colony

lift assay (Carrello et al. 1999). ARLE-1 is commonly used as a negative control to test

for non-specific binding and eliminate false positives (Carrello et al. 1999). Prior to

contransformation, L40 yeast were grown on YPAD plates for roughly 3 days at 30oC.

56

After the L40 yeast had grown to an appropriate size colonies were inoculated into 5

mL of YPAD media. The yeast was grown overnight at 30oC with shaking and were

diluted into 50 mL of YPAD media containing 2% glucose to give an optical density

(OD) between 0.3 and 0.4 at an absorbance of 600 nm. The yeast was incubated at 30oC

with shaking until the OD at absorbance 600 nm reached 0.6. The 50 mL of culture was

then spun down at 2500 rpm for 5 min at room temperature in a Sorvall RC-3

Centrifuge, after which the supernatant was removed and the pellet resuspended in 40

mL of TE buffer (10mM Tris-HCl pH 7.5, 1mM EDTA). The resuspended cells were

centrifuged as before and the supernatant was again removed. The pellet, containing the

L40 cells, was then resuspended in 2 mL of TE buffer containing 100 mM lithium

acetate and left at room temperature for 10 min. Approximately 1 μg of each plasmid

DNA and 10 μL of 10 mg/mL salmon sperm DNA (freshly boiled for 10 min to

denature) was added to 100 μL of L40 competent cells. After the addition of 700 μL of

TE buffer containing 40% polyethylene glycol 3350 and 100 mM of lithium acetate the

L40 competent cells were vigorously vortexed for 10 sec and incubated for 30 min at

30oC with shaking. Following the 30 min incubation 88 μL of dimethyl sulphoxide

(DMSO) was added to the cells which were mixed by inversion. The L40 competent

cells were then heat shocked for 7 min at 42oC and put on ice to quickly bring them to

room temperature. Once at room temperature the cells were spun down in a

microcentrifuge at 7500 rpm for 30 sec at room temperature and then the supernatant

was removed. The pellet was then resuspended 1 mL of TE buffer and microcentrifuged

as before and the supernatant once again removed. This was repeated but only 900 μL

of the supernatant was removed with the pellet being resuspended in the remaining 100

μL of TE buffer and spread on appropriate amino acid deficient plates. All transformed

yeast, with the exception of those transformed only with library clone/pVP16, were

grown on uracil, tryptophan and leucine deficient media to select for yeast that

contained both plasmids prior to the β-galactosidase colony lift assay. As yeast

transformed with library clone/pVP16 alone did not contain the pBTM116 vector they

did not have the capacity to produce tryptophan and were grown on medium lacking

only uracil and leucine. The plates were incubated at 30oC for a period of time ranging

between 3 to 6 days before colonies were of a sufficient size to be used in a β-

galactosidase assay.

57

2.2.2.5 β-galactosidase Colony Lift Assays

Once colonies had grown to a sufficient size a piece of Whatman #5 filter paper, which

has been cut to size, is placed onto the plates containing the colonies. Pressure is evenly

applied to the filter paper to ensure that it becomes evenly wetted and that the colonies

stuck to it. The filter is then removed and placed into liquid nitrogen. The filter paper is

initially placed onto the surface of the liquid nitrogen with the colonies facing up for

several sec and then submerged for 10 sec. The filter paper was removed and allowed to

thaw at room temperature before being placed with the colonies facing up onto

Whatman #1 filter paper that has been soaked with 2 mL of Z buffer (60 mM Na2HPO4,

40 mM NaH2PO4.2H20, 10 mM KCl, 1 mM MgSO4.7H20 38.6 mM β-mercaptoethanol,

25 μM X-Gal). The colonies were monitored for colour change representing β-

galactosidase activity over a period of 4 hr.

2.2.3 Protein Interaction Studies

2.2.3.1 Baculoviral Expression and Purification of His-Tagged CaR-Tail

The BacPAK Baculovirus Expression System (Clontech) was used to generate a His-

tagged CaR-tail as per the manufacturer’s instructions. Briefly, Bacfectin was used to

cotransfect His-tagged CaR-tail/BacPAK9 transfer plasmids and linearised BacPAK6

viral DNA into Sf9 insect cells. After 72 hr the primary virus was collected and

subsequently used to infect 300 mL cultures of exponentially growing Sf9 cells. After 3

days cells were divided into 50mL aliquots and collected by centrifugation in an Avanti

J-30I Beckman centrifuge using a JA30.50 rotor at approximately 7,000 rpm for 5 min.

Once the supernatant had been removed the pellet was stored at -80oC. This work was

conducted by Kendall Walker, Laboratory for Molecular Genetics (Neuromuscular

Diseases), Western Australian Institute for Medical Research.

Purification of the His-tagged CaR-Tail began with the resuspension of an aliquotted

pellet of Sf9 insect cells in 2 mL of Buffer 1 (50 mM NaPO4, 300 mM NaCl and 10 mM

imidazole, pH 8.0). The protease inhibitors phenylmethylsulphonylflouride (PMSF) and

benzamidine were immediately added to the resuspended cells at concentrations of 1

mM and 5 mM respectively. Subsequent to the addition of the protease inhibitors the

cells were incubated with 1 mL of 5 mg/mL of lysozyme in Buffer 1 containing 1% of

Triton X-100 on ice for 5 min. Following the incubation the resuspension was subjected

to sonication by pulsing 5 times at 50% duty cycle with a Branson Sonifier Cell

Disruptor B15 on ice and then centrifuged at 32,000 rpm for 1 hr at 4oC in a Sorval RC-

58

90 ultracentrifuge using a Kontron 65.13 rotor. After the centrifugation PMSF and

benzamidine were added to a final concentration of 1 mM and 5 mM, respectively, to

the supernatant. The supernatant was subsequently divided into 2 aliquots. The aliquots

were then incubated for 1 hr at 4oC with rotation in eppendorfs containing

approximately 200 μL of nickel-nitriloacetic acid (Ni-NTA) agarose beads that had

been washed in Buffer 1 3 times. Between the consecutive washings, the Ni-NTA beads

were centrifuged at 1,200 rpm for 1 min at 4oC in a microcentrifuge. The beads were

then washed 8 times in Buffer 2 (50 mM NaPO4, 300 mM NaCl and 20 mM imidazole,

pH 8.0) with 0.2% of Triton X-100. This was followed by a further 8 washes in Buffer 2

without Triton X-100, before being resuspended in 200 μL of renaturation buffer. The

Ni-NTA beads were then divided into 50 μL aliquots to be used in the pulldown assays

outlined in 2.2.3.5.

2.2.3.2 Bacterial Expression and Purification of His-Tagged CaR-Tail

The CaR-tail/pET28a plasmid construct was expressed in BL21 Codon (+) cells. An

isolated colony containing the plasmid was used to inoculate a 100 mL of 2xYT media

containing 100 μg/mL of kanamycin and incubated overnight at 37oC with shaking at

220 rpm. Following the incubation the overnight culture was centrifuged for 10 minutes

in a Sorvall RC-3 Centrifuge at 4,000 rpm. The cell pellet was resuspended in 2 mL of

2xYT and added to 1 L of 2xYT containing 100 ug/mL of kanamycin. The 1 L culture

was incubated for 90 min prior to the addition of isopropanol β-thiogalactopyranoside

(IPTG) to a final concentration of 0.4 mM and further incubated for 1 hr at 37oC. After

the incubation the cells were pelleted at 3,500 rpm for 15 min. The cells were then

resuspended in 25 mL of denaturatiuon buffer (8 M urea, 137 mM NaCl, 2.7 mM KCl,

4.3 mM Na2PO4, 1.4 mM KH2PO4, 20 mM imidazole, 0.2% Triton X-100 and 2.5 mM

β–mercaptoethanol). Cells were lysed on ice by sonication, 5 pulses at 50% duty cycle,

using a Branson Sonifier Cell Disruptor B15. The lysed cells were then ultracentrifuged

for 1 hr at 32,000 rpm in a Sorval RC-90 ultracentrifuge using a Kontron 65.13 rotor.

The supernatant was incubated with pre-washed Ni-NTA agarose beads for 3 hr at 4oC

with rotation. The beads were then washed 5 times with denaturation buffer, leaving a

pellet to which renaturation buffer (137 mM NaCl, 2.7 mM KCl, 4.3 mM Na2PO4, 1.4

mM KH2PO4, 20 mM imidazole, 0.2% Triton X-100 and 2.5 mM β–mercaptoethanol)

was added dropwise while the beads remained on ice. The resuspended Ni-NTA beads

were then centrifuged at 3,000 rpm in the Sorvall RC-3 Centrifuge and washed 5 times

in renaturation buffer before being resuspended in 200 μL of renaturation buffer. The

59

Ni-NTA beads were then divided into 50 μL aliquots to be used in the pulldown assays

outlined in 2.2.3.5.

2.2.3.3 Bacterial Expression and Purification of Glutathione S-Transferase (GST)-

Fusion Proteins

The pGEX4T-1 expression plasmid was used to generate both full length proteins and

protein fragments that contained an N-terminal GST-tag. These GST-fusion protein

plasmid constructs were expressed in BL21 Codon (+) cells. An isolated colony

containing a GST fusion protein plasmid was used to inoculate a 100 mL of 2xYT

media containing 100 μg/mL of ampicillin and incubated overnight at 37oC with

shaking at 220 rpm. Following the incubation the overnight culture was centrifuged for

10 minutes in a Sorvall RC-3 Centrifuge at 4,000 rpm. The cell pellet was resuspended

in 2 mL of 2xYT and added to 1 L of 2xYT containing 100 ug/mL of ampicillin. The 1

L culture was incubated for 90 min prior to the addition of IPTG to a final concentration

of 0.4 mM and a further incubation for 1 hr at 37oC. After the incubation the cells were

pelleted at 3,500 rpm for 15 min. The supernatant was removed and the pellet

resuspended in 15 mL of mouse tonicity PBS (MTPBS) to which was added 100 μL of

100 mM PMSF. The cell suspension was stored at -70oC until required.

To begin the purification of the GST-fusion proteins the pellets were thawed and 130

μL of 1 M DTT, 105 μL of 0.5 M EDTA (pH 8.0), 260 μL of 100 mM PMSF and a

complete protease inhibitor tablet (Roche) was added. The bacterial pellet was lysed

with 1 mL of 20 mg/mL of lysozyme on ice for 5 min followed by sonication on ice (3

pulses 50% duty cycle, using a Branson Sonifier Cell Disruptor B15). Insoluble debris

was sedimented by centrifugation at 32,000 rpm for 1 hr at 4oC in a Sorval RC-90

ultracentrifuge using a Kontron 65.13 rotor. The supernatant was then collected and

placed onto a 400 μL 1:1 suspension of glutathione sepharose beads that had been

washed 4 times with MTPBS. The cell lysate was incubated with the beads for 2 hr at

4oC with rotation. Following the incubation the beads were washed 5 times with

MTPBS containing 1 % Triton X-100, 5 mM DTT, 2 mM EDTA and complete protease

inhibitor and subsequently washed 5 more times with MTPBS containing 5 mM DTT, 2

mM EDTA and complete protease inhibitor but no 1% Triton X-100. The beads were

transferred to eppendorfs and protein was eluted from the beads with 500 μL of 10 mM

glutathione in 50 mM Tris pH 8.0 for 15 min at 4oC. Eluates were both purified and

concentrated in Buffer A (10 mM Tris-HCl pH 7.3, 100 mM KCl) using a Centricon

60

microconcentration column. Concentrations of recovered protein were then measured by

using the Bradford Protein Assay.

2.2.3.4 Alternate Purification Method for GST-Testin

An isolated colony containing the plasmid encoding the GST-testin fusion protein was

used to inoculate 50 mL of 2xYT media containing 100 μg/mL of ampicillin and

incubated overnight at 37oC with shaking at 220 rpm. Following overnight incubation, 5

mL of the culture was used to inoculate a fresh 500 mL of 2xYT containing 100 μg/mL

of ampicillin, which was then incubated at 37oC until the OD at absorbance 600 nm

reached 0.6. IPTG, to a final concentration of 0.4 mM, was added and the culture

incubated for 3 hr at 37oC with shaking at 220 rpm prior to being centrifuged for 10

minutes in a Sorvall RC-3 Centrifuge at 4,000 rpm. The supernatant was removed and

the cell pellet was frozen at -70oC. After thawing on ice, the pellet was resuspended in

10 ml of ice-cold STE buffer (10 mM Tris-HCl, pH 8.0, 1 mM EDTA, 150 mM NaCl)

containing 0.1 mg/ml lysozyme and 10 mM DTT and placed on ice for 15 min. The

resuspension was sonicated 3 times at 25 % duty cycle with a Branson Sonifier Cell

Disruptor B15 on ice and then centrifuged at 32,000 rpm for 1 hr at 4°C to pellet debris.

The supernatant was then topped up to 20 ml with STE buffer containing of 2 % v/v

Triton X-100. This solution was incubated for 30 min at room temperature with

agitation. A volume of 1 mL of glutathione sepharose 4B beads pre-washed 3 times in

ice-cold PBS was incubated with the supernatant for 1 hr with rotation at room

temperature. The beads were pelleted and then washed three times in PBS. The GST

fusion protein was eluted in 500 μl of 20 mM glutathione in Tris-HCl, pH 8.0 for 15

min at room temperature and then concentrated using a Centricon microconcentration

column.

2.2.3.5 Pulldown Assay with His-Tagged CaR-Tail

Aliquots of Ni-NTA agarose beads (50 μL) with or without bound baculovirally or

bacterially derived His-tagged CaR-tail were resuspended in 500 μL of Buffer A and 25

μg of a GST-fusion protein or GST alone was added. The mixtures were incubated

overnight at 4oC with rotation. The following day, samples were washed 5 times with 1

mL of Buffer A containing 0.2% Triton 100-X and complete protease inhibitor and then

5 times with 1 mL of Buffer A containing only complete protease inhibitor. After the

final wash, bound proteins were eluted from the NI-NTA agarose beads with 50 μL of

Laemmli sample buffer and boiled for 5 min. The NI-NTA agarose beads were spun

61

down in a microcentrifuge and 40 μL of eluted protein was loaded onto a 15% SDS-

PAGE gel to be separated by electrophoresis.

2.2.3.6 Staining and Preservation of Polyacrylamide Gels

The staining of SDS-PAGE gels was performed by incubation of the gel in Coomassie

Brilliant Blue R-250 stain (0.5% (w/v) Coomassie Brilliant Blue R, 10% (v/v) acetic

acid glacial, 30% (v/v) isopropyl alcohol) for 20 minutes at room temperature with

gentle agitation. Gels were then rinsed with Coomassie destain solution (20% (v/v)

methanol, 5% (v/v) acetic acid glacial) to remove excess Coomassie stain before being

incubated with Coomassie destain solution at room temperature with gentle agitation

until protein bands were visible and background staining was minimal.

2.2.3.7 Coimmunoprecipitation

In order to first remove non-specific binding proteins, protein G sepharose beads (40 μl

of a 1:1 slurry) were prepared by washing 3 times in 1 ml of cell lysis buffer and 2 mg

of protein from the extracted cell lysate, was added and the volume made up to 1 ml

with cell lysis buffer containing protease inhibitors. The lysate-bead suspension was

incubated for 1 hr at 4oC with rotation before centrifugation at 14,000 rpm for 2 min at

4oC in a microcentrifuge and the supernatant transferred to a fresh eppendorf tube. The

cleared cell lysate were incubated overnight with rotation at 4oC with 5 μg of either

anti-Flag antibody or anti-GFP antibody for immunoprecipitation of CaR-FLAG or

EGFP-testin respectively. Lysate containing antibody was then incubated with 40 μl of

a 1:1 slurry of pre-washed (as above) protein G sepharose beads for 1 hr at 4 °C. The

bead mixtures was then centrifuged for 1 min at 8,000 rpm at 4οC in a microcentrifuge

and the supernatant discarded. The protein G sepharose beads were washed 6 times

with cell lysis buffer containing protease inhibitors. After the final wash, the

supernatant was discarded and the bound antibody and proteins were eluted into 50 μl

Laemmli sample buffer for 5 min at room temperature. Following centrifugation at

13,200 rpm for 1 min at room temperature the supernatant was loaded onto an SDS-

PAGE gel for electrophoretic separation of protein before transferring to a nitrocellulose

membrane and immunodetection of specific protein.

62

2.2.4 Confocal Microscopy

2.2.4.1 Detection of CaR-FLAG and EGFP-Testin by Confocal Microscopy

Cells transiently expressing CaR-FLAG and EGFP-testin were plated out onto poly-L-

lsine coated coverslips in a 6 well plate 24 hr after transfection. Following a further 24

hr incubation the cells were washed 3 times for 3 min in PBS at room temperature and

then fixed using 4% (v/v) formaldehyde in PBS for 20 min at room temperature. This

was followed by another round of washing in PBS after which the cells were

permeabilized in PBS containing 0.2% (v/v) Triton X-100 at room temperature for 20

min. After permeabilization the cells were again washed in PBS and then blocking

buffer containing 10% (v/v) goat serum and 1% (w/v) BSA in PBS was placed onto the

cells for 1 hr at room temperature. The blocking buffer was removed and the cells were

incubated overnight at room temperature with FLAG antibody diluted 1/250 in PBS

containing 10% (v/v) goat serum and 1% (w/v) BSA. The next day the cells were

washed in PBS and Alexa546-conjugated goat anti-mouse antibody diluted 1/400 in

PBS containing 10% (v/v) goat serum and 1% (w/v) BSA was put onto the cells for 1 hr

at room temperature. After incubation with the secondary antibody the cells were

washed 5 times in PBS for 3 min at room temperature. The coverslips were then

mounted onto slides with antifade mounting media (50 mM Tris-PO4, 50 mM

NaH2PO4.2H2O, 20% (w/v) polyvinyl alcohol, 30% (w/v) glycerol) and sealed with nail

polish. CaR-FLAG was detected at wavelength 568 nm, while EGFP-testin was

detected at 488 nm using a Bio-Rad 1024 UV confocal microscope.

2.2.5 Detection of Signalling Pathway Activity

2.2.5.1 ERK Assay

HEK293 cells stably expressing the CaR were grown in 25 cm2 flasks and upon

reaching approximately 60% confluency were transfected with either EGFP-

testin/pcDNA3 or the vector control, EGFP/pcDNA3. On the day after transfection the

cells were trypsinised and resuspended in 10 mL of DMEM without CaCl2 (Invitrogen)

that had been supplemented with 10% BSA, 100 units/mL penicillin and 100 μg/mL

streptomycin and 0.5 mM CaCl2. The resuspended cells were then plated into 24 well

plates that had been coated with poly-L-lysine and incubated overnight at 37oC with 5%

CO2. 48 hr after transfection the medium was aspirated and replaced with physiological

saline solution. The cells were incubated in physiological saline solution supplemented

with BSA and 0.5mM CaCl2 for 1 hr at 37oC with 5% CO2 before stimulation.

Duplicate wells of cells were stimulated for 5 min with a range of Ca2+ concentrations

63

with or without phenylalanine in physiological saline solution supplemented with BSA

at 37oC with 5% CO2. After the 5 min the cells were placed on ice and washed once in

ice-cold PBS before addition of 100 μL of MAPK lysis buffer (20 mM Tris-HCl ph 7.4,

150 mM NaCl, 25 mM NaF, 2.5 mM EDTA, 35 mM β-glycerophosphate, 1% (v/v)

Triton X-100, 10% (v/v) glycerol) to each well. The 24 well plates were then stored at -

80oC for at least 16 hr. Once the lysates were thawed the duplicates were spun down

and pooled. Protein concentrations measured using the Pierce BCA Protein Assay kit as

described previously in section 2.2.1.15. Following quantification of protein

concentrations, 25 μg of protein was separated on a 12.5% SDS-PAGE gel and

transferred to a Hybond-C super nitrocellulose membrane (Amersham) prior to

detection of ERK and phosphorylated ERK by western analysis as described in section

2.2.1.18.

2.2.5.2 SRE-Luciferase Assay

EGFP-testin/pcDNA3 or the vector control, EGFP/pcDNA3, was cotransfected with the

SRE-luciferase reporter construct into HEK293 cells stably expressing the CaR grown

in 25 cm2 flasks when they reached approximately 60% confluency. On the day after

transfection the cells were trypsinised and resuspended in 10 mL of DMEM

supplemented with FCS and antibiotics prior to being plated into a 24 well plate that

had been coated with poly-L-lysine. The cells were then incubated overnight at 37oC

with 5% CO2. The following day triplicate wells of cells were dosed with a range of

calcium concentrations for 8 hr at 37oC with 5% CO2 in serum-free DMEM

supplemented with antibiotics. At the end of the incubation the cells were placed on ice

and washed once in ice-cold PBS prior to the addition of 180 μL of luciferase lysis

buffer (30 mM Tris-HCl pH 7.8, 2mM EDTA, 10% (v/v) glycerol, 0.1% Triton X-100)

containing 2 mM DTT to the wells. The 24 well plates were then stored at -80oC for at

least 16 hr after which the lysed cells were thawed and protein concentrations of the

lysates were determined using the Bradford Protein Assay, as described in 2.2.1.16. For

each sample 50 μL of lysate was aliquotted into a well of an OptiPlate™ white 96-well

plate. The plate was then placed into a POLARstar OPTIMA plate reader (BMG

Labtechnologies), which dispensed 50 μL of Luciferase Assay Substrate (Pierce) to

each sample and measured the resulting luminescence produced. Luminescence was

normalised to protein concentrations.

64

2.2.6 Generation of the Testin Knockdown HEK293-CaR Stable Cell Line

2.2.6.1 Cloning of Knockdown Target Sequences

Oligonucleotides (Appendix 1) previously shown to knockdown testin in mammalian

cells (Griffith et al. 2005) were purchased from Sigma-Proligo. The sense and antisense

oligonucleotide were first annealed to each other by adding 3 μg of each oligonucleotide

to annealing buffer (50 mM HEPES pH7.4, 100 mM NaCl) in a total volume of 50 μL

that was incubated at 90oC for 4 min, 70oC for 10 min and 37oC for 20 min before being

cooled at 4oC for 15 min. The annealed oligonucleotides were ligated into the

pSUPERIOR.retro.neo+gfp vector which had been linearised by restriction enzyme

digestion using Hind III and Bgl II in the following reaction: 1 μL linearised

pSUPERIOR.retro.neo+gfp, 2 μL annealed oligonucleotides, 0.2 units of Invitrogen T4

DNA ligase and 2 μL of 5 x reaction buffer, made up to a final volume of 10 μL with

sterile ddH2O. The reaction was incubated overnight at room temperature and then

transformed into 200 μL of XL1 competent cells. DNA extracted from transformed

bacterial colonies was tested for the presence of insert by restriction enzyme digestion

with EcoRI and HindIII, which should release an approximately 287 bp fragment

containing the oligonucleotide insert. The insert of the pSUPERIOR construct was

verified by sequencing with the recommended primers (Appendix 1).

2.2.6.2 Generating the Stable Packaging Cell Line

The PA317 viral packaging cell line produces amphotropic virions upon transfection

with the pSUPERIOR.retro.neo+gfp construct containing insert which are suitable for

infecting human cell lines. PA317 cells were maintained in DMEM supplemented with

10% FCS, 100 units/mL penicillin and 10 μg/mL streptomycin. Lipofectamine2000 was

used to transfect 10 μg of the pSUPERIOR.retro.neo+gfp construct containing insert

that had been linearised by restriction enzyme digestion with ScaI into PA317 cells at

approximately 80% confluency in a 75 cm2 flask. A control flask of PA317 cells were

treated in an identical manner but without the addition of vector. The media from both

flasks was replaced 24 hr after transfection with media containing 400 μg/mL G418.

The G418 containing medium was replaced every two days until all the non-transfected

PA317 cells had died.

2.2.6.3 Retroviral Infection of HEK293-CaR Stable Cell Lines

The PA317 viral packaging cells were taken off G418 selection two days prior to

infecting HEK293-CaR stables. The HEK293-CaR stable cells used for the knockdown

65

studies were hygromycin resistant and generously supplied by Dr Donald Ward, from

Manchester University. PA317 cells at approximately 70% confluency were washed

with PBS and incubated with 7 mL of DMEM supplemented with 10% FCS for 24 hr at

37°C with 5% CO2. Following incubation, the medium referred to as ‘viral soup’, was

removed and filtered through a 0.22 μm Millex filter unit into a sterile 15 mL tube. The

filtered viral soup was placed onto HEK293-CaR stable cells at approximately 70%

confluency and the cells incubated for 24 hr at 37°C with 5% CO2. A control flask of

HEK293-CaR stable cells (without the addition of viral soup) grown in 7 mL of DMEM

supplemented with 10% FCS was incubated alongside the test flask. A second viral

soup was generated by again incubating PA317 viral packaging cells with 7 mL of

DMEM supplemented with 10% FCS for 24 hr at 37°C with 5% CO2. The following

day, the second viral soup was removed and processed as before. The first viral soup

was then aspirated from the HEK293-CaR stable cells and the cells were washed with 5

mL of PBS. As before the filtered viral soup was placed onto the cells and incubated for

24 hr at 37°C with 5% CO2. The media on the control HEK293-CaR stable cells was

replaced with 7 mL of DMEM supplemented with 10% FCS. After the second 24 hr

incubation with the second dose of viral soup, the HEK293 cells were passaged into 2

75 cm2 flasks and maintained in DMEM supplemented with 10% FCS, 100 units/mL

penicillin, 10 μg/mL streptomycin, 400 μg/mL G418 and 200 μg/mL hygromycin.

Initially, medium containing the G418 and hygromycin was replaced daily due to the

high confluency of cells but as the number of surviving cells decreased medium was

replaced every two days. As acute stimulation of the CaR with aminoglycoside

antibiotics, such as G418, has been linked to an increase in cell death, after the first

week, the concentration of G418 was reduced to 100 μg/mL, which is routinely used for

selection of G418-resistant HEK293-CaR stable cells. Approximately 2 weeks after the

viral infection all the non-infected HEK293 cells had died. At this point the cells in the

test flask were sorted by flow cytometry, selecting for EGFP-containing cells

2.2.6.4 Enrichment of EGFP-positive Cells Verification of Testin Knockdown

The expression of EGFP by pSUPERIOR.retro.neo+gfp allowed the infected cells to be

identified by cell sorting. Cells were grown to 90% confluency in a 75 cm2 flask,

trypsinised and resuspended in 5 mL of sorting buffer (25 mM HEPES pH 7.0, 2 mM

EDTA, 1% FCS in 1 x PBS). The cells were then sorted using a Fluorescence Activated

Cell Sorter Vantage Cytometer and cells expressing high levels of EGFP were collected

into a small volume of FCS. Collected cells were then placed into a 25 cm2 flask with 5

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mL DMEM supplemented with 10% FCS, 100 units/mL penicillin, 100 μg/mL

streptomycin, 100 μg/mL G418 and 200 μg/mL hygromycin and incubated for 2 weeks

at 37oC and 5% CO2. Cells were then sorted a second time, as described above. After

the second round of sorting, which produced over 90% of EGFP positive cells,

knockdown of testin expression was examined by Western analysis as described in

2.2.1.18.

2.2.7 Studies of Morphological and Cytoskeletal Changes.

Cells at 30% to 40% confluency in either 25 cm2 flasks (for morphology studies) or on

fibronectin-coated coverslips in 6 well plates (for cytoskeletal studies) were incubated

in DMEM without supplementation for 3 hr with or without the addition of magnesium.

Magnesium was used in favour of calcium as the CaR agonist because the media used

during treatment would have to be change from DMEM to an alternative HEPES buffer

if calcium was used as calcium chloride precipitates in DMEM at the concentrations

used for stimulation. For the morphological studies, multiple fields of cells were

photographed using a Sony digital camera attached to an Olympus phase contrast

microscope. Actin stress fibres and focal adhesions were detected by

immunofluorescence staining of cells on fibronectin-coated coverslips as outlined

below. HEK293-CaR stable cells were dual stained with both phalloidin-Alexa Fluor-

568, to detect actin stress fibres, and anti-phospho-paxillin-(Y118) antibody followed

by an Alexa Fluor-488 goat anti-mouse secondary antibody, to detect focal adhesions.

As testin knockdown HEK293-CaR stable cells expressed EGFP, they were stained

individually with either phalloidin-Alexa Fluor-568 only or with anti-phospho-paxillin-

(Y118) antibody followed by an Alexa Fluor-568 rabbit anti-mouse secondary antibody.

Following the 3 hr treatment, cells were washed with PBS and then fixed using 4%

(v/v) formaldehyde in PBS for 5 min at 37oC. Cells were then permeabilized in PBS

containing 0.25% (v/v) Triton X-100 at room temperature for 20 min and washed in 100

mM glycine for 10 min before being washed 5 times with TBS for 5 min. Blocking

buffer containing 10% (v/v) goat serum and 1% (w/v) BSA in TBS was placed onto the

cells for 20 min at room temperature with or without the addition of phalloidin-Alexa

Fluor-568. The blocking buffer was removed and the cells incubated for 1 hr at room

temperature with 2.5 μg/mL anti-phospho-paxillin-(Y118) antibody with 10% (v/v) goat

serum and 1% (w/v) BSA in TBS. This was followed by 3 washes with 1% (v/v) BSA

in TBS for 10 min at room temperature. The cells were then stained with either an

Alexa Fluor-488 or Alexa Fluor-568 rabbit anti-mouse secondary antibody with 10%

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(v/v) goat serum and 1% (w/v) BSA in TBS for 1 hr at room temperature. This was

followed by 3 washes with 1% (w/v) BSA in TBS for 10 min at room temperature. The

coverslips were then mounted onto slides using Prolong Gold reagent and stored at 4oC.

Images of cells were taken using the Olympus IX81 inverted microscope.

68

CChhaapptteerr 33 Identification of Proteins that Interact with the Intracellular Tail of the

Calcium-Sensing Receptor in a Yeast Two-Hybrid Library Screen

3.1 Introduction

It was originally believed that GPCRs were only capable of interacting with

heterotrimeric G-proteins. However, the limited number of G-protein subunits, 16 Gα, 5

Gβ and 12 Gγ, suggested that this theory was inadequate to account for the ability of

GPCRs to precisely regulate physiological outcomes via specific signalling pathways

(Bockaert and Pin 1999). More recently, it has been shown that GPCRs are capable of

interacting with a vast array of proteins and it is these interactions that govern various

aspects of receptor biology, including subcellular compartmentalisation, trafficking and

degradation, as well as the regulation of signalling events (Bockaert et al. 2004). While

the intracellular loops of GPCRs have been shown to be important in receptor binding

to G proteins, it is the intracellular tail of GPCRs that has proven to be the most

important region for interaction with binding partners (Bockaert et al. 2003).

As summarised in Chapter 1, a number of proteins have been found to interact with the

CaR using a variety of techniques. To identify proteins that interact with the CaR

intracellular tail, several groups have utilised the yeast two-hybrid system to screen both

parathyroid and kidney cDNA libraries using the C-terminal tail of the CaR (Awata et

al. 2001; Herrera-Vigenor et al. 2006; Hjalm et al. 2001; Huang et al. 2006). The yeast

two-hybrid system has been widely used to examine protein-protein interactions in vivo.

In general, the protein of interest is fused to the DNA binding domain of a transcription

factor, forming the “bait”, while proteins generated from the cDNA clones of the library

are fused to the activation domain of the transcription factor, creating the “prey”. If the

protein of interest and a protein generated from a cDNA clone interact, then the

transcription factor is reconstituted as a functional unit and able to induce transcription

of a reporter gene as outlined in Figure 3.1 (Fields and Song 1989). The design of this

system does however mean that only interactions between two proteins can be observed.

Other drawbacks of the yeast two-hybrid system are the possibility of many false

positives or negatives and as a yeast-based system it is not ideal for the detection of

protein interactions that require post-translational modifications (Bockaert et al. 2003).

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To date, the yeast two-hybrid cDNA libraries screened with the CaR intracellular tail as

“bait” have been generated from tissues that are important to whole body calcium

homeostasis. In order to identify proteins that may interact with the CaR and affect its

function in tissues and processes that are unrelated to calcium homeostasis our

laboratory performed a LexA-based yeast two-hybrid screen of an EMLC.1 mouse

pluripotent haemopoietic cell line library using the intracellular tail of the human CaR,

amino acids 865-1078, as bait. The EMLC.1 cell line library was generated by Dr Evan

Ingley from the Western Australian Institute for Medical Research and has been

successfully used by a number of groups to identify novel interacting protein partners of

a variety of “bait” proteins (Hunter et al. 2005; Ingley et al. 2000; Lim et al. 2002).

Established by Tsai et al. in 1994, the EMLC.1 cell line is a unique model system for

studying the survival, proliferation and differentiation of haemopoietic stem cells (Tsai

et al. 1994). Evidence of CaR expression in haemopoietic stem cells was obtained in

immunocytochemistry studies using a CaR specific antiserum (House et al. 1997). It

was observed in CaR-deficient mice that, despite being able to successfully migrate

from the liver to bone, haemopoietic stem cells failed to become lodged in the endosteal

niche of bone marrow (Adams et al. 2006). In vitro experiments found that the ability of

haemopoietic stem cells derived from CaR-deficient mice to adhere to either fibronectin

or collagen I was significantly lower compared to those from wild-type mice (Adams et

al. 2006). It has been hypothesised that agents that modulate CaR function could be

employed to alter erythropoiesis in a controlled manner (Drueke 2006).

3.2 Results

In the initial screen of the EMLC.1 cell line library performed by Dr Bryan Ward,

approximately 15,000 interacting clones were generated on selective media (-THULL).

A total of 129 of the larger colonies that tested positive for β-galactosidase activity in a

colony lift assay were stored as glycerol stocks. As part of this thesis 60 of the 129

colonies were examined to distinguish between colonies containing true interacting

proteins of the CaR and false positives.

3.2.1 Verification of Clones

The EMLC.1 cell line library had been cloned into the VP16 “prey” vector. Primers

specific for the VP16 vector were used to generate a PCR product from plasmid DNA

extracted from the yeast colonies that corresponded to the inserted cDNA library clone.

The PCR products were subsequently digested with Hae III and by examining both the

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size of the PCR products and Hae III digestion profiles run on 1% agarose gels (Figure

3.2) unique clones could be identified from the library screen. Plasmids believed to be

unique, based on the profile of the PCR product and Hae III digestion profiles, were

selected for plasmid rescue and tested in the yeast two-hybrid system by examining β-

galactosidase activity. It would later become apparent that selection of unique clones

according to their PCR product and Hae III digestion profiles did not eliminate multiple

copies of clones being selected for plasmid rescue. This was primarily due to many

colonies containing two different clones that may have resulted in PCR products of the

non-interacting clone being produced in favour of the interacting clone. Library clones

were considered as genuine interacting partners of the CaR if yeast colonies

cotransformed with the library clone and CaR-tail exhibited β-galactosidase activity

equivalent to or greater than the positive control with no significant β-galactosidase

activity observed in the negative controls. A colour change in colonies of the positive

control was routinely observed after approximately 2.5-3 hr. Yeast colonies containing

the CaR and an interacting clone that exhibited a colour change at a time point similar to

the positive control were denoted by a “+”. When test colonies displayed β-

galactosidase activity greater than the positive control they were assigned with either

++, for colonies undergoing a colour change after 1-2.5 hr, or +++, for colonies that

turned blue in the first hour. The combination of DNA profiling and verification of the

interactions in β-galactosidase colony lift assays reduced the 60 isolated clones to 14

unique clones that were identified by sequence analysis. The 14 clones represented

seven different proteins that are presented in Table 3.1 alongside their β-galactosidase

activities relative to the positive control. The seven proteins identified in the yeast two-

hybrid screen as being protein binding partners of the CaR were filamin A, filamin B,

testin, 14-3-3 θ, OS-9, Ubc9 and MPc2.

3.2.2 Mapping of Verified Interacting Proteins of the CaR

Overlapping clones discovered in the library screen aided in defining the region of the

identified protein that is required for binding to the CaR. To more precisely define the

regions of the CaR intracellular tail that are important for the interaction of the receptor

with its interacting partners, deletion mapping studies were conducted using the yeast

two-hybrid system. The relative strength of an interaction between the CaR and its

binding partners is expressed as outlined above.

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3.2.2.1 Filamin A

Two clones of filamin A identified in the yeast two-hybrid library screen that do not

overlap are illustrated in Figure 3.3A. The two clones exhibited greater β-galactosidase

activity (++) than the positive control when tested with full-length CaR-tail by the yeast

two-hybrid system. One filamin A clone predominantly corresponded to the filamin

repeat 11, while the second clone was an unusually large clone that was over 2000 bp in

length and encompassed filamin repeats 16-22. Both clones interacted with the same

minimal region of the CaR intracellular tail, amino acids 965-986, as shown in Figure

3.3B. However, both clones interacted more favourably with the CaR-tail(923-1078)

construct (++) than the CaR-tail(965-1078) truncation (+), which suggests that residues

923-965 of the CaR-tail contain elements that enhance binding to filamin A. Therefore

the optimal binding region is considered to encompass residues 923-986 of the CaR.

3.2.2.2 Filamin B

A single clone of filamin B, presented in Figure 3.4A, was also identified in the library

screen. The filamin B clone encompassed filamin repeat 21 and showed β-galactosidase

activity equivalent to that of the positive control. This filamin B clone was found to

interact with the same CaR-tail truncations as the two filamin A clones at an intensity

comparable to that of the positive control in β-galactosidase colony lift assays.

3.2.2.3 Testin

Based on the β-galactosidase colony lift assays, all three clones of testin depicted in

Figure 3.5A that were isolated from the yeast two-hybrid library screen were found to

interact very strongly (+++) with the CaR-tail when compared to the positive control.

The region of overlap between the three clones corresponds to 61 amino acids that are

located within the C-terminal half of the protein. This region corresponded to parts of

LIM domains 1 and 2. The results of yeast two-hybrid mapping for testin clone (148-

357) and testin clone (254-419) are shown in Figure 3.5B and reveal that the only

truncation that interacted with testin was the CaR-tail(865-922) construct. In the β-

galactosidase colony lift assay, the strength of the interaction between testin and the

full-length CaR-tail (+++) was equivalent to that of the CaR-tail(865-922) truncation

(+++).

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3.2.2.4 14-3-3 θ

A full-length clone of 14-3-3 θ was isolated from the screen of the haemopoietic cell

line library, as shown in Figure 3.6A. The results from the yeast two-hybrid mapping

studies indicate that 14-3-3 θ was only able to interact with the CaR-tail(865-922)

construct as presented in Figure 3.6B. The comparative potency of the interaction

between 14-3-3 θ and either the entire CaR-tail or the CaR-tail(865-922) truncation

were equivalent, but much greater (+++) than the strength of the interaction of the

positive control.

3.2.2.5 OS-9

As can be seen in Figure 3.7A, the three OS-9 clones isolated from the yeast two-hybrid

screen all contained the region present in OS-9 isoform 1 that is deleted in isoforms 2

and 3. The largest identified OS-9 clone, spanning residues 430-652, also covers a

region of OS-9 that is deleted in isoforms 3 and 4. The two other clones did not extend

into this deleted region. The minimal site of interaction as determined by the overlap of

the three clones spanned 107 amino acids between amino acids 530 and 636. All three

OS-9 clones were observed to have a much stronger interaction (+++) with the CaR-tail

in the yeast two-hybrid system than the positive control interaction. The results of yeast

two-hybrid mapping studies using OS-9 clone (430-652) and OS-9 clone (530-667)

(conducted by Honours student Bernadette Pederson) narrowed the region of the CaR-

tail responsible for the interaction with OS-9 to amino acids 965-986 (Figure 3.7B).

3.2.2.5 Ubc9

The 124 amino acids forming the overlapping region of the three Ubc9 clones presented

diagrammatically in Figure 3.8A correspond to almost 80% of the protein. The strength

of binding between the Ubc9 clones and the CaR-tail was much greater than that of the

positive control as measured by the β-galactosidase colony lift assay. Testing the

capacity of Ubc9 to bind to the CaR-tail truncations using the full length Ubc9 clone

revealed that residues between 965 and 986 of the CaR are critical for the interaction

between the two proteins, as shown in Figure 3.8B.

3.2.2.6 MPc2

A single 122 amino acid clone of MPc2 that contained a His-rich region was isolated

from the yeast two-hybrid library screen, as seen in Figure 3.9A. While the interaction

between the MPc2 clone and the CaR-tail (++) was not as strong as some of the other

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clones, it was still substantially greater than that, of the Hsp90 and CyP40 positive

control. In Figure 3.9B it can be seen that of the CaR-tail truncations cotransformed

with the MPc2 clone, only the CaR-tail(923-1078) and CaR-tail(965-1078) truncations

were found to interact with MPc2. The strength of the interaction between the MPc2

clone was greater with the CaR-tail truncation 923-1078 (++) than its interaction with

the 965-1078 CaR-tail truncation (+). This suggests that there are elements within the

923-965 region that enhance binding of the CaR to MPc2. Therefore the optimal

binding region is considered to encompass residues 923-986 of the CaR.

3.3 Discussion

In the yeast two-hybrid screen of an EMLC.1 mouse pluripotent haemopoietic cell line

library seven proteins that interacted with the intracellular tail of the CaR were revealed.

Although some of the proteins that interact with the CaR-tail share functional

similarities, they represent a diverse collection of proteins. The variety of functions

associated with these CaR binding partners includes those acting as scaffolding proteins

and those involved in the regulation of intracellular signalling, cytoskeletal

organisation, trafficking, degradation, posttranslational modification and transcriptional

repression. The cellular localisation of the proteins that interact with the CaR ranges

from membrane associated to nuclear. In the yeast two-hybrid mapping studies

presented above all seven proteins were shown to require elements contained within

either residues 865-923 or 965-986 of the CaR-tail that were essential for binding.

These two regions of the CaR-tail display the highest level of conservation amongst the

different species (Figure 1.1). This observation is also true of other reported CaR

binding partners for which their site of interaction within the CaR has been mapped.

AMSH interacts with amino acids 865-894 of the CaR, while dorfin binds to CaR

residues 880-900 (Herrera-Vigenor et al. 2006; Huang et al. 2006). An overview of the

properties and functions of the seven interacting protein partners of the CaR-tail

identified in the yeast two-hybrid library screen will be presented below, with possible

links to the CaR emphasised.

3.3.1 Filamins

A number of filamin homologues have been identified, including the three human

isoforms (Stossel et al. 2001). Filamin A was the first member of the filamin family to

be identified when it was isolated from rabbit macrophages in 1975 (Hartwig and

Stossel 1975). Gorlin et al. characterised the human homologue of filamin A 15 years

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later (Gorlin et al. 1990). Filamin B was identified in a yeast two-hybrid screen of a

human bone marrow library that used the intracellular tail of glycoprotein Ibα as bait,

while filamin C was detected in differentiating human skeletal muscle cells (Takafuta et

al. 1998; van der Ven et al. 2000). There is inconsistency in the nomenclature used to

label the three human isoforms of filamin as they have all been known by a series of

different names (Stossel et al. 2001). Filamin A has been known as actin binding protein

(ABP), ABP280, filamin 1, non-muscle filamin, and α-filamin. Filamin B has been

referred to as filamin homolog 1, filamin 3 and β-filamin. Filamin C has also been

called ABP-L, filamin 2 and γ-filamin (van der Flier and Sonnenberg 2001). The three

human isoforms of filamin range from 2602 to 2705 amino acids in length and share

appoximately 70% sequence homology (Popowicz et al. 2006). The key structural

features common to all vertebrate filamins are an actin-binding domain, 24 repeat

sequences and two hinge regions (Stossel et al. 2001). The N-terminal actin-binding

domain of filamin is similar to those present in other actin filament-binding proteins and

is responsible for the interaction between filamin and the F-actin (van der Flier and

Sonnenberg 2001). Beginning after the actin-binding domain the 24 repeats within the

mammalian filamins consist of approximately 96 amino acids that form antiparallel β-

sheets, which overlap to create an overall rod structure (Popowicz et al. 2006). The two

hinge regions between filamin repeats 15-16 and 23-24 are the least conserved parts of

filamin containing only approximately 50% homology between the three human

isoforms (van der Flier and Sonnenberg 2001). It is the final repeat after the second

hinge that is important for the dimerisation of filamin (Stossel et al. 2001).

Over 30 proteins have been found to interact with the vertebrate filamins, with most

interacting with the C-terminal half containing filamin repeats 15-24 (Feng et al. 2005).

The physiological relevance of many of these interactions has yet to be established but

there are cellular processes that have repeatedly been associated with the interaction

between filamin and its binding partners (Popowicz et al. 2006). Filamin has been

shown to act as a scaffolding protein by providing an anchor for membrane associated

proteins to allow for their precise localisation and recruitment of intracellular signalling

components (Stossel et al. 2001). As an actin binding protein, filamin has also been

shown to provide a link between its interacting protein partners and the cytoskeleton,

which facilitates cellular processes involved in cytoskeletal reoganisation (Popowicz et

al. 2006).

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As mentioned in the introduction, the CaR has already been identified as a binding

partner of filamin A and that this interaction is involved in CaR-mediated signalling

cascades (Awata et al. 2001; Hjalm et al. 2001). However, the isolation of a yeast two-

hybrid library clone corresponding to filamin B is the first evidence that the CaR-tail

interacts with other filamin isoforms. Neither of the filamin A clones identified in this

yeast two-hybrid library screen, corresponding to two distinct sites in filamin, overlap

with the previously reported CaR binding domains. Other identified interacting protein

partners that are able to bind to multiple sites within filamin include PKCα and

Forkhead Box C1 (FOXC1) (Berry et al. 2005; Tigges et al. 2003). The interactions

between the CaR-tail and filamin will be further examined in the following chapter.

3.3.2 Testin

Testin, also known as Tes, was originally identified in 1995, when two isoforms of

testin, presumed to be splice variants, were cloned from a mouse testis germ cell library

(Divecha and Charleston 1995). Further testin homologues have since been identified

that exhibit a high degree of conservation between the species, as can be seen in Figure

3.10. The investigation of a fragile site, FRA7G, on chromosomal band 7q31.2, for

genes with a possible role in carcinogenesis, led to the characterisation of the human

homologue of testin (Tatarelli et al. 2000). Human testin mRNA has been detected in an

extensive range of tissue types, with high levels being detected in the thyroid and

pancreas (Tatarelli et al. 2000). The screening of a panel of human tumour cell lines by

Northern analysis with a testin probe revealed that unlike normal tissue there were many

tumour cell lines that did not express testin, which was subsequently shown to be

predominantly due to CpG methylation of the testin gene (Tatarelli et al. 2000). The

lack of testin expression in tumour cell lines led to the hypothesis that testin may

function as a tumour suppressor and in experiments where testin was exogeneously

expressed in the OVCAR5 and HeLa cancer cell lines, a significant reduction in growth

was observed (Tobias et al. 2001). Sarti et al. would later observe that the testin-

negatitive cancer cell lines, T47D and MES-SA, but not the testin-positive MCF-7

cancer cell line, exhibited a reduction in cell growth following the adenoviral

transduction of testin (Sarti et al. 2005). Adenoviral transduction of testin into T47D

and MES-SA cells also impaired tumourigenicity when these cells were inoculated into

nude mice that were treated with carcinogenic agents (Sarti et al. 2005). Both T47D and

MES-SA cells exhibited higher levels of apoptotic markers following the adenoviral

transduction of testin (Sarti et al. 2005). Further evidence of testin’s role as a tumour

87

suppressor was discovered using testin knockout mice in an established model of gastric

cancer where over 81% of mice without testin developed tumours while only 25% of

mice expressing testin developed tumours (Drusco et al. 2005).

The key structural features of testin shown in Figure 3.5A and 3.10 are a prickle,

espinas, testin (PET) domain and three Lin-11, Isl-1, Mec-3 (LIM) domains, which have

led to testin being classified as a member of the PET/LIM domain family, which is a

subgroup of the LIM domain protein family (Gubb et al. 1999; Tatarelli et al. 2000).

The degree of sequence conservation between the LIM domains of PET/LIM domain

family members, testin, prickle, dyxin and LMO6, is higher than when compared to

other members of the LIM domain containing family, such as paxillin and zyxin (Figure

3.11). LIM domains are proposed to be involved in the coordination of protein-protein

interactions and are comprised of two zinc-fingers that fit the following broad

consensus sequence CX2CX16-23HX2CX2CX2CX16-21CX2-3(C/H/D), where X denotes

any amino acid (Zheng and Zhao 2007). Although all of the LIM domains within the

testin fit the LIM domain consensus sequence, there are several members of the

PET/LIM domain family that have a LIM domain 3 that contains an unusually spaced

zinc-finger (Gubb et al. 1999). As the interaction between testin and the CaR was

discovered in a system that utilises the process of DNA-binding it should be noted that

there is no evidence that LIM domains, despite containing zinc-finger DNA binding

motifs, are capable of binding to DNA (Zheng and Zhao 2007).

Although the CaR is the first receptor that has been identified to interact with testin, a

number of other interacting protein partners of testin has already been identified. Two

groups employing different methods identified the known focal adhesion proteins

mammalian enabled (mena), vasodilator-stimulated phosphoprotein (VASP) and zyxin

as being capable of binding to testin (Coutts et al. 2003; Garvalov et al. 2003). In yeast

two-hybrid library screens using full-length human testin as bait, Coutts et al isolated

clones corresponding to zyxin from a human mammary cDNA library and mena, talin,

actin-like 7A and glutamate-receptor-interacting protein 1 from a mouse testis cDNA

library (Coutts et al. 2003). Clones corresponding to testin were also isolated from both

library screens, demonstrating that testin can interact with itself (Coutts et al. 2003).

Garvalov et al. performed pulldown assays on lysates extracted from HeLa cells using

three bacterially produced GST fusion proteins containing the N-terminal half of testin,

the C-terminal half of testin and full-length testin and probed for known focal adhesion

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Human 1 MDLENKVKKMGLGHEQGFGAPCLKCKEKCEGFELHFCRKICRNCKCGQEEHDVL 54 Mouse 1 MDLETKMKKMGLGHEQGFGAPCLKCKENCEGFELHFWRKICRNCKCGQEEHDVL 54 Chicken 1 MDLESKVKKMGLGHEQGFGAPCLKCKDKCEGFELHFWRKICRNCKCGQEEHDVL 54 Dog 1 MELEAKVKKMGLGHEQGFGAPCLKCKEKCEGFELHFWRKICRNCKCGQEEHDVL 54 Horse 1 MDLETKVKKMGLGHGQGFGAPCLKCKEKCEGFELHFWRKICRNCKCGQEEHDVL 54

Human 55 LSNEEDRKVGKLFEDTKYTTLIAKLKSDGIPMYKRNVMILTNPVAAKKNVSINT 108 Mouse 55 LSNEEDRKVGRLFEDTKYTTLIAKLKSDGIPMYKRNVMILTNPVAAKKNVSINT 108 Chicken 55 TSNEEDRKVGKLFEDTKYTTLIAKLKNDGIPMYKRNVMILTNPVPAKKNISINT 108 Dog 55 LSNEEDRKVGKLFEDTKYTTLIAKLKSDGIPMYKRNVMILTNPVAAKKNVSINT 108 Horse 55 LSNEEDRKVGKLFEDTKYTTLIAKLKSDGIPMYKRNVMILTNPVAAKKNISINT 108

PET Domain

Human 109 VTYEWAPPVQNQALARQYMQMLPKEKQPVAGSEEAQHRKKQLAKQLPAHDQDPS 162 Mouse 109 VTYEWAPPVQNQALARQYMQMLPKEKQPVAGSEGAQYRKKQLAKQLPAHDQDPS 162 Chicken 109 VTYEWAPPVQNQTLARQYMQMLPKEKQPVAGSEGAQYRKKQLAKQLPAHDQDPS 162 Dog 109 VTYEWAPPVQNQALARQYMQMLPKEKQPVAGSEGAQYRKKQLAKQLPAHDQDPS 162 Horse 109 VTYEWAPPVQNQALARQYMQMLPKEKQPVAGSEGAQYRKKQLAKQLPAHDQDPS 162

Human 163 KCHELSPREVKEMEQFVKKYKSEALGVGDVKLPCEMDAQGPKQMNIPGGDRSTP 216 Mouse 163 KCHELSPKEVKEMEQFVKKYKSEALGVGDVKFPSEMNAQGDKVHN-PAGNRHAP 215 Chicken 163 KCHELSPNEVKQMEQFVKKYKNEALGVGDVKLPGELETKATDKNNVNSGDRSTS 216 Dog 163 KCHELSPKEVKEMEQFVKKYKSEALGVGDVKLPREMDAQSTNRMYIPGGDRSTA 216 Horse 163 KCHELSPKEVKEMEQFVKKYKNEALGVGDVKLPREMDAQDPNRMCIPGGDRSTT 216

Zinc Finger

Human 217 AAVGAMEDKSAEHKRTQYSCYCCKLSMKEGDPAIYAERAGYDKLWHPACFVCST 270 Mouse 216 AAV-ASKDKSAESKKTQYSCYCCKHTMNEGEPAIYAERAGYDKLWHPACFICST 268 Chicken 217 AAVGAMEDKSADQKASQYSCYRCKLNMKEGDPAVYAERAGYDKLWHPACFVCCT 270 Dog 217 AAVGAMEDKSAEHKRTQYSCYCCKQSMKEGDPAIYAERAGYDKLWHPACFVCST 270 Horse 217 AAVGAKENKLAENKRTQYSCYCCNLSMKEGDPAIYAERAGYDKLWHPACFVCST 270

Zinc Finger Zinc Finger

Human 271 CHELLVDMIYFWKNEKLYCGRHYCDSEKPRCAGCDELIFSNEYTQAENQNWHLK 324 Mouse 269 CGELLVDMIYFWKNGKLYCGRHYCDSEKPRCAGCDELIFSNEYTQAENQNWHLK 322 Chicken 271 CSELLVDMIYFWKNGNLYCGRHYCDSEKPRCAGCDELIFSNEYTQAEGQNWHLK 324 Dog 271 CHELLVDMIYFWKNGKLYCGRHYCDSEKPRCAGCDELIFSNEYTQAENQNWHLK 324 Horse 271 CHELLVDMIYFWKNGKLYCGRHYCDSEKPRCAGCDELIFSNEYTQAENQNWHLK 324

Zinc Finger Zinc-Finger

Overlap

Human 325 HFCCFDCDSILAGEIYVMVNDKPVCKPCYVKNHAVVCQGCHNAIDPEVQRVTYN 378 Mouse 323 HFCCFDCDHILAGKIYVMVTDKPVCKPCYVKNHAVVCQGCHNAIDPEVQRVTYN 376 Chicken 325 HFCCFDCDCVLAGEIYVMVNDKPVCRPCYVKKHAAICQGCHNAIDPEVQRVTYN 378 Dog 323 HFCCFDCDNILAGEIYVMVNDKPVCKPCYVKNHAVVCQGCHNAIDPEVQRVTYN 378 Horse 323 HFCCFDCDSILAGEIYVMVNDKPVCKPCYVKNHAVVCQGCHNAIDPEVQRVTYN 378

Zinc-Finger

Human 379 NFSWHASTECFLCSCCSKCLIGQKFMPVEGMVFCSVECKK-RMS 421 Mouse 377 NFSWHASTECFLCSCCSKCLIGQKFMPVEGMVFCSVECKR-MMS 419 Chicken 379 NFNWHATQECFLCSCCSKCLIGQKFMPVEGMVFCSVECKKKMMS 422 Dog 379 NFSWHASTECFLCSCCSKCLIGQKFMPVEGMVFCSVECKK-MMS 421 Horse 379 NFSWHASTECFLCSCCSKCLIGQKFMPVEGMVFCSVECKK-MMS 421 Figure 3.10: A comparison of the amino acid sequence of testin from different mammalian species. Sequences of the human, mouse, chicken, dog and horse testin homologues have been aligned and conserved residues have been highlighted in black ( X ). The PET domain is indicated with a yellow line (―). The zinc-fingers of LIM domain 1, 2 and 3 are marked with blue (―), green (―) and purple (―) lines respectively. The region of overlap between the three clones identified in the yeast two-library screen is denoted with a red line (―).

LIM Domain 1 Zinc Finger 1 Testin 236 CYCCKLSMKEGDPAIYAERAGYDKLWHPACF 266 Prickle 126 CEQCGLKINGGEVAVFASRAGPGVCWHPSCF 156 Dyxin 243 CELCKGAAPPDSPVVYSDRAGYNKQWHPTCF 273 LMO6 186 CEECGKQIGGGDIAVFASRAGLGACWHPQCF 216 Paxillin 324 CGACKKPIAGQ--VVTAM--G--KTWHPEHF 348 Zyxin 384 CGRCHQPLARAQPAVRA0-LGQ--LFHIACF 410 Zinc Finger 2 Testin 267 VCSTCHELLVDMIYFWKNEKLYCGRH 292 Prickle 157 VCFTCNELLVDLIYFYQDGKIHCGRH 182 Dyxin 274 VCAKCSEPLVDLIYFWKDGAPWCGRH 299 LMO6 287 VCTTCQELLVDLIYFYHVGKVYCGRH 242 Paxillin 349 VCTHCQEEIGSRNFFERDGQPYCEKD 374 Zy xin 411 TCHQCAQQLQGQQFYSLEGAPYCEGC 436

LIM Domain 2 Zinc Finger 1 Testin 301 CAGCDELIFSNEYTQAENQNWHLKHF 326 Prickle 191 CSACDEIIFADECTEAEGRHWHMKHF 216 Dyxin 308 CSGCDEIIFAEDYQRVEDLAWHRKHF 333 LMO6 251 CQACDEIIFSPECTEAEGRHWHMDHF 276 Paxillin 383 CYYCNGPILDKVVT-ALDRTWHPEHF 407 Zyxin 444 CNTCGEPITDRMLR-ATGKAYHPHCF 468 Zinc Finger 2 Testin 327 CCFDCDSILAGEIYVMVNDK-PVCKPC 352 Prickle 217 CCLECETVLGGQRYIMKDGR-PFCCGC 242 Dyxin 334 VCEGCEQLLSGRAYIVTKGQ-LLCPTC 359 LMO6 277 CCFECEASLGGQRYVMRQSR-PHCCAC 302 Paxillin 408 FCAQCGAFFGPEGFHEKDGK-AYCRKD 437 Zyxin 469 TCVVCARPLEGTSFIVDQANRPHCVPD 495 LIM Domain 3 Zinc Finger 1 Testin 361 CQGCHNAIDPEVQRVTYNNFSWHASTECFLCS 392 Prickle 251 CETCGEHIGVDHAQMTYDGQHWHATEACFSCA 282 Dyxin -------------------------------- LMO6 311 CDGCGEHIGLDQGQMAYEGQHWHASDRCFCC- 341 Paxillin 442 CGGCARAI-------LENYISALNTLWHPECF 472 Zyxin 504 CSVCSEPIMPEPGRDETVRVVALDKNFHMKCY 535 Zinc Finger 2 Testin 393 CCSKCLIGQKFMPVEG-------MVFCSVEC 416 Prickle 283 QCKASLLGCPFLPKQG-------QIYCSKTC 306 Dyxin ------------------------------- LMO6 342 -–SRCGRALLGRPFLPRRG----LIFCSRAC 366 Paxillin 473 VCRECFTP----FVNGSFFEHDGQPYCEV-H 492 Zyxin 536 KCEDCGKPLSIEADDNGCFPLDGHVLCRK-C 565 Figure 3.11: Comparison of LIM domains. The human amino acid sequence of the LIM domains of four PET/LIM domain containing proteins (testin, prickle, dyxin and LMO6) and two less related LIM domain containing proteins (paxillin and zyxin) were aligned. Conserved amino acids are highlighted in black ( X ).

proteins with a panel of antibodies (Garvalov et al. 2003). Full-length testin was found

to interact with only mena, VASP and actin. However, the N-terminal half of testin

interacted with actin, α-actinin and paxillin, while the C-terminal half bound to mena,

VASP and zyxin. A more recent yeast two-hybrid screen of a rat kidney library

identified testin and αII-spectrin as interacting partners (Rotter et al. 2005). Of all the

interacting protein partners of testin identified, only the functional relevance of the

interaction between testin and zyxin has been demonstrated experimentally. Thus, zyxin

has been shown to recruit testin to focal adhesions (Garvalov et al. 2003).

To further understand how testin functions within the cell, Coutts et al. generated Rat-1

fibroblasts that stably expressed GFP-testin and discovered that these cells exhibited

increased cell spreading on fibronectin in comparison to wild-type Rat-1 fibroblasts

(Coutts et al. 2003). This observation was supported by findings from experiments

where the cell spreading of chicken embryo fibroblasts was enhanced by the

overexpression of testin, but cell motility was found to be reduced (Griffith et al. 2004).

A later study, examining HeLa cells in which testin expression had been knocked down

by RNA interference, revealed that there was a significant reduction in the level of actin

stress fibre assembly in HeLa cells not expressing testin compared to wild-type HeLa

cells (Griffith et al. 2005). This decrease in actin stress fibres coincided with a reduction

in RhoA activity in testin knockdown HeLa cells compared to wild-type HeLa cells

(Griffith et al. 2005). Emerging evidence that testin has a role in the regulation of cell

morphology suggested that, like prickle, another PET containing protein with three LIM

domains, testin might be involved in neural crest migration and axial elongation in the

early development of Xenopus laevis (Dingwell and Smith 2006). Morphilino

oligonucleotides were used to reduce the expression of testin in Xenopus laevis

embryos, which resulted in embryos developing a foreshortened head and shortened

antero-posterior axis (Dingwell and Smith 2006). As the CaR has also been shown to be

localised at actin stress fibres, associated with the actin cytoskeleton and changes in cell

morphology, the interaction between the receptor and testin may influence these

processes (Bouschet et al. 2007; Davies et al. 2006; Rey et al. 2005). Therefore, the

interaction between the CaR and testin was selected for further examination, the results

of which are presented in Chapter 5.

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3.3.3 14-3-3 θ

In 1991, 14-3-3 θ, alternatively known as 14-3-3 τ, was isolated from T cells and

identified as a member of the 14-3-3 family (Nielsen 1991). The 14-3-3 family of

proteins is expressed in a wide range of tissue types and all eukaryotic organisms

examined, displaying a high degree of conservation both between species and isoforms

(Wang and Shakes 1996). 14-3-3 proteins predominantly exist as either homodimers or

heterodimers formed by two 14-3-3 isoforms binding at their N-terminal α-helices

(Yaffe et al. 1997). In vitro studies examining dimerisation equilibria revealed that 70%

of 14-3-3 θ existed as a dimer and that half of the dimerised 14-3-3 θ was as a

heterodimer (Yang et al. 2006). Experimental evidence has linked 14-3-3 proteins to a

variety of cellular processes including signal transduction, cell cycle regulation,

apoptosis, stress response, cytoskeletal organisation and malignant transformation (van

Hemert et al. 2001). The 14-3-3 family primarily acts in these processes through its

interaction with protein binding partners, of which over 300 have been identified

(Coblitz et al. 2006). The interaction between 14-3-3 proteins and their binding partners

allows 14-3-3 to modify the activity of enzymes, regulate subcellular localisation or act

as a scaffolding protein to promote further protein interactions (van Hemert et al. 2001).

Although 14-3-3 proteins have been found to bind to specific phosphorylated motifs

within many of their interacting partners there are several instances where 14-3-3 binds

to non-phosphorylated proteins (Mackintosh 2004). The CaR contains neither of the two

common 14-3-3 consensus binding sequences, RSXpSXP and RXY/FXpSXP, but does

contain an alternate binding motif, RX1-2SX2-3S (where either serine can be

phosphorylated), which has been shown to interact with 14-3-3 θ (Liu et al. 1997; Yaffe

et al. 1997). The RX1-2SX2-3S binding motif in the CaR spans residues 890-895, which

lies in the region of the CaR-tail that was found to bind to 14-3-3 θ in the yeast two-

hybrid system. The putative 14-3-3 binding site in the CaR is adjacent to a putative ER

retention signal, RXR, located between residues 897-899 (Chang et al. 2007; Shikano et

al. 2005), which suggests that 14-3-3 θ may be involved in trafficking the receptor from

the ER. 14-3-3 proteins have been shown to facilitate the surface trafficking of

membrane proteins by one of three proposed mechanisms: scaffolding, clamping and

masking (Shikano et al. 2006). It is the last that is the most likely to occur as a result of

the interaction between the CaR and 14-3-3 θ with the latter binding to the CaR and

sterically masking its ER retention signal (Shikano et al. 2006).

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Interestingly, both the CaR and 14-3-3 θ have been implicated in diarrhoeal processes

(Hebert et al. 2004; Patel et al. 2006). Infantile diarrhoea caused by enteropathogenic

Escherichia coli results in the production of attaching–effacing lesions on the surface of

intestinal epithelial cells (McNamara and Donnenberg 1998). A major feature of the

attaching–effacing lesions is the formation of pedestals, structures made from

polymerised actin, ezrin, talin and myosin (Goosney et al. 2001). When Patel et al.

examined pedestal formation using a cell line model, HeLa cells infected with

enteropathogenic Escherichia coli, 14-3-3 θ was found to have a role in the cellular

organisation of actin and the formation of pedestals (Patel et al. 2006). With the

emerging evidence of the role of the CaR in actin organisation (Davies et al. 2006) it is

possible the interaction between the CaR and 14-3-3 θ may be involved in the formation

of pedestals.

3.3.4 OS-9

Su et al. performed chromosome microdissection to directly isolate transcripts from a

homogeneously staining region of an osteosarcoma cell line, OsA-CL, cDNA library

(Su et al. 1994). The clone designated OS-9 contained the partial sequence of a gene

that was uncharacterised at the time, but was subsequently named OS-9 (Su et al. 1994).

The human OS-9 gene is made up of 15 exons that can be alternatively spliced to

produce four OS-9 isoforms, schematically represented in Figure 3.7A (Kimura et al.

1998; Wang et al. 2007). Northern analysis of human tissue revealed that OS-9 is

expressed in all 16 tissue types tested and it has been proposed that transcription factor

binding-motifs, common to a number of housekeeping genes, found in the 5' upstream

region of OS-9 are responsible for its ubiquitous expression pattern (Kimura et al. 1997;

Su et al. 1996). Characterisation of the OS-9 yeast homologue, Yos9, revealed that it is

a glycosylated protein containing an ER retention motif (Friedmann et al. 2002). Both

mammalian OS-9 and Yos9 were shown to be ER-associated proteins by confocal

microscopy, although there is conjecture as to whether mammalian OS-9 is on the

lumenal or cytoplasmic side of the ER membrane (Bernasconi et al. 2008; Friedmann et

al. 2002; Litovchick et al. 2002).

There is evidence from functional studies examining Yos9 that it plays a role in the ER-

associated degradation (ERAD) pathway (Bhamidipati et al. 2005; Kim et al. 2005;

Szathmary et al. 2005). Yos9, as part of a complex with the 3-hydroxyl-3-

methylglutaryl-coenzyme A reductase degradation ligase, is able to select aberrant

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glycosylated proteins for proteasomal degradation via the ERAD pathway (Gauss et al.

2006). Recently, mammalian OS-9, as part of a ubiquitin ligase complex containing

Hrd1 and SEL1L, has also been shown to be involved in detecting and delivering

terminally misfolded or unassembled glycosylated proteins for ERAD (Christianson et

al. 2008). Bernasconi et al. discovered that in response to acute ER stress there is an

increase in OS-9 transcription, which correlates with an increase in the degradation of

misfolded proteins (Bernasconi et al. 2008) Currently there are no known endogenous

substrates of mammalian OS-9 or Yos9 (Mueller et al. 2008). As the CaR is a

glycosylated protein that is processed in the ER it is possible that OS-9 may participate

in a quality control surveillance mechanism that ensures that only correctly processed

CaR is released from the ER to be expressed at the cell surface.

It should be noted that several other proteins, N-copine, meprin β, hypoxia-inducible

factor 1α (HIF-1α) and transient receptor potential vanilloid 4 (TRPV4), have been

identified in yeast two-hybrid library screens as protein binding partners of OS-9 and

that no common functional outcome has been proposed for their interaction with OS-9.

It has been suggested that N-copine, which interacts with a similar region of OS-9 as the

CaR, recruits OS-9 to the plasma membrane in a calcium dependent manner (Nakayama

et al. 1999). Like the CaR and N-copine, meprin β was also found to interact with the C-

terminal domain of OS-9, although the site of meprin β binding in OS-9 is not as well

defined (Litovchick et al. 2002). It was hypothesised that OS-9 is involved in the

transportation of meprin β from the ER to the Golgi and that this may be a function that

OS-9 performs with other membrane proteins, including the CaR (Litovchick et al.

2002). This notion is supported by evidence that the yeast OS-9 homologue may also

have a role in the ER to Golgi trafficking of glycosylphosphatidylinositol-anchored

proteins (Friedmann et al. 2002). As part of a complex, OS-9 was found to regulate the

ubiquitination and degradation of HIF-1α (Baek et al. 2005) and may mediate the

ubiquitination of the CaR in a similar fashion. Alternatively, the interaction between the

CaR and OS-9 may be protective against degradation, as OS-9 has been shown to

protect the TPRV4 protein from ubiquitination and subsequent degradation (Wang et al.

2007).

The expression of OS-9 in a variety of tumour cell lines and the association in

differentiation-induced myeloid leukemia cells between decreased OS-9 expression and

apoptosis suggests a role in cell viability (Kimura et al. 1998). Vourvouhaki et al.

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demonstrated that murine FDC-P1 cells overexpressing OS-9 had a greater rate of

survival than wild-type FDC-P1 cells when exposed to three different apoptosis-

inducing conditions, IL-3 deprivation, TNFα treatment and staurosporine treatment

(Vourvouhaki et al. 2007). The role of OS-9 in apoptosis may be linked to its interaction

with the CaR as Zhang et al. has demonstrated in rat cardiac myocytes that exposure to

ischaemia/reperfusion conditions also induced apoptosis and that this induction was

associated with an increase in CaR expression (Zhang et al. 2006). Under hypoxic

conditions, like ischaemia, a decrease in OS-9 mRNA has been observed that led to a

decrease in HIF-1α degradation (Baek et al. 2005). If OS-9 regulates the degradation of

CaR as it does HIF-1α, then it is possible that in cells subjected to ischaemia a decrease

in OS-9 expression may result in a lower rate of CaR degradation leading to the

observed increase in CaR expression and a subsequent increase in apoptosis.

3.3.5 Ubc9

The human homologue of Ubc9, also known as ubiquitin-conjugating enzyme E2I

(UBE2I ), was originally cloned in 1996 and found to share 56% amino acid sequence

identity with the yeast homologue (Wang et al. 1996). A year later the mouse

homologue was cloned and its nucleotide sequence was found to be identical to the

human homologue (Tashiro et al. 1997). Due to its sequence similarities with other

ubiquitin-conjugating enzymes, Ubc9 was originally believed to be involved in

ubiquitination until Johnson and Blobbel demonstrated that Ubc9 was an enzyme

involved in the conjugation of the small ubiquitin-related modifier (SUMO) protein to

other proteins (Johnson and Blobel 1997). The process of conjugating SUMO to its

target protein is referred to as SUMOylation and as outlined in Figure 3.12 is very

similar to ubiquitination (Zhao 2007). The initial step of SUMOylation is the ATP-

dependent activation of SUMO by the E1 activating enzyme, which is a heterodimer

made of Aos1 and Uba2 (Dohmen 2004). SUMO is then transferred to the E2

conjugating enzyme, Ubc9, from where it is subsequently attached to a lysine on the

target protein by SUMO specific E3 ligase (Johnson 2004). In the final stage of

SUMOylation, Ubc9, the E3 ligase and the protein being SUMOylated are all part of a

single complex (Johnson 2004). SUMOylation has been shown to regulate the

functionality of target proteins by altering their intracellular localisation and influencing

their interactions with other proteins (Zhao 2007).

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Ubc9 has been reported as a binding partner for a variety of proteins in over 20 yeast

two-hybrid library screens, with only a few being verified as SUMOylation targets,

while many remain untested (Melchior 2000). While the components involved in

SUMOylation, including Ubc9, are more highly expressed in the nucleus, suggesting

that it is primarily a nuclear process, they are also expressed in the cytoplasm (Melchior

2000; Tashiro et al. 1997; Zhao 2007). Therefore it is possible that the CaR may interact

in vivo with Ubc9 and undergo SUMOylation. There are SUMOylation recognition

sites, ψKXE/D (ψ is any large hydrophobic residue) (Rodriguez et al. 2001; Sampson et

al. 2001), within the second and third intracellular loop of the CaR, spanning residues

707-710 and 803-806, respectively. It is also possible that the interaction between the

CaR and Ubc9 can affect the receptor’s function in a SUMOylation independent

manner. Collec et al. recently reported that the stability of the glycoprotein

Lutheran/basal cell adhesion molecules in MDCK cells is regulated by its interaction

with Ubc9 without SUMOylation (Collec et al. 2007). The identification of Ubc9 as an

interacting partner of the CaR in the yeast two-hybrid screen may also be a result of the

sequence and structural similarities between Ubc9 and one of the ubiquitin-conjugating

enzymes, Ubc7 or Ubc8, that are likely to bind to the CaR (Figure 3.13) (Huang et al.

2006; Tong et al. 1997).

3.3.6 MPc2

MPc2 was originally identified as the mouse homologue of the polycomb protein found

in Xenopus, XPc (Alkema et al. 1997). Like MPc2, the human homologue, hPc2, was

also identified by Southern blot hybridisation using a cDNA probe encompassing part

of the coding region of XPc to screen a cDNA library in 1997 (Satijn et al. 1997). As

part of the polycomb group family of proteins both MPc2 and hPc2 interact with other

polycomb proteins to form a multiprotein complex that is involved in transcriptional

repression (Schwartz and Pirrotta 2007). When the SUMOylation of the transcriptional

corepressor, CtBP, was examined in COS-1 cells it was revealed that hPc2 was an E3

ligase that acted in conjunction with the E2 conjugating enzyme, Ubc9 (Kagey et al.

2003). Although both MPc2 and Ubc9 were identified in the yeast two-hybrid screen of

a haemopoietic cell line library it is unlikely that the two proteins act together to

sumoylate the CaR in vivo because MPc2 and its human homologue are nuclear proteins

that are not exposed to the intracellular tail of the CaR.

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CChhaapptteerr 44 Investigating the Interaction Between the Intracellular Tail of the Calcium-

Sensing Receptor and Filamin A

4.1 Introduction

As discussed in the preceding chapter, two filamin A clones were identified in a yeast

two-hybrid screen of a haematopoietic cell line library. The fragments of filamin A that

these clones represented were distinct and did not overlap with the filamin interaction

domain identified in previous studies as the CaR binding site (Awata et al. 2001; Hjalm

et al. 2001), indicating that CaR may interact with multiple regions of filamin A. Both

the yeast two-hybrid system and pulldown assays have been used to identify other

proteins that recognise multiple binding sites within filamin A, including FOXC1,

integrin β7 and PKCα (Berry et al. 2005; Kiema et al. 2006; Tigges et al. 2003).

FOXC1 is a transcription factor that was shown to bind to filamin A at three different

sites, residues 571-866, 867-1154 and 1779-2284 (Berry et al. 2005). Kiema et al.

demonstrated that the intracellular tail of integrin β7 bound to filamin repeats 19 and 21

(Kiema et al. 2006). PKCα was found to interact with two functionally important

regions of filamin A. These correspond to part of the N-terminal domain, which

contains the actin-binding site and also part of the C-terminal domain, which is the site

of filamin dimerisation (Tigges et al. 2003).

In addition to the two filamin A clones identified in the library screen described in

Chapter 3, a filamin B clone was also found to interact with the intracellular tail of the

CaR. As previously mentioned, the two filamin isoforms are structurally similar and

share approximately 70% homology (Popowicz et al. 2006). It was revealed in tissues

coexpressing filamin A and B that the isoforms could form heterodimers and that this

interaction could possibly compensate for the functional defects in one isoform arising

from mutations (Sheen et al. 2002). However, there are clear differences between

filamin A and B, including their level of expression and tissue distribution (Takafuta et

al. 1998). More importantly, filamin A and B have exhibited different functional

outcomes in response to the same stimuli (Glogauer et al. 1998). For example, in M2

cells, which express filamin B, but not filamin A, mechanical force was found to induce

cell death. In A7 cells however, which express both filamin isoforms, there was no

increase in the rate of cell death, suggesting that filamin A, but not filamin B, protects

98

against mechanical stress (Glogauer et al. 1998). Experiments examining the PKCα

phosphorylation of the filamin isoforms revealed that both filamin A and C could be

phosphorylated by this kinase, whereas filamin B could not (Tigges et al. 2003). This

selective phosphorylation of filamin isoforms has been proposed to be the mechanism

responsible for the functional differences observed between filamin A and B (Tigges et

al. 2003). The differences in filamin A and B are particularly relevant to their

interactions with the CaR because of several experiments performed in M2 and A7 cells

that showed that filamin A was involved in CaR signalling and receptor stabilisation

(Awata et al. 2001; Zhang and Breitwieser 2005). No compensatory effects from filamin

B were observed in these experiments (Awata et al. 2001; Zhang and Breitwieser 2005).

In light of the above information, the following interaction studies will focus on the

interactions between the CaR and the multiple binding sites within filamin A.

4.2 Results

4.2.1 Construction of Filamin A GST-Fusion Proteins for Pulldown Studies

To further investigate the possibility that the intracellular tail of the CaR can interact

directly with more than one binding site within filamin A, pulldown assays were

performed. As the data from Chapter 3 and published reports (Awata et al. 2001; Hjalm

et al. 2001) suggested that the CaR-tail may bind to three distinct regions of filamin,

three GST-fusion proteins containing the corresponding filamin A fragments were

created for direct interaction studies. The GST-fusion protein containing the filamin A

sequence from the smaller of the two filamin A cDNA clones identified in the yeast

two-hybrid screen was designated GST-Fil11 as the sequence primarily encoded filamin

repeat 11. The identified filamin B interacting clone predominantly encoded for filamin

repeat 21, which is highly homologous to the corresponding filamin A sequence (Figure

4.1). As results from the yeast two-hybrid mapping studies suggested that filamin A

residues 1714-2380, which form filamin repeats 16-22, contained a CaR-tail interaction

domain, a GST-fusion construct containing the equivalent filamin A sequence of the

filamin B cDNA library clone was made and called GST-Fil21. The final GST-fusion

generated was GST-FilH, which contained the minimum CaR-tail binding domain

identified by Hjalm et al and provided a positive control for these direct interaction

studies (Hjalm et al. 2001).

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Filamin A 2172 SPSGKTHEAEIVEGENHTYCIRFVPAEMGTHTVSV 2206 Filamin B 2135 SPSGRVTEAEIVPMGKNSHCVRFVPQEMGVHTVSV 2169 Filamin A 2207 KYKGQHVPGSPFQFTVGPLGEGGAHKVRAGGPGLE 2241 Filamin B 2170 KYRGQHVTGSPFQFTVGPLGEGGAHKVRAGGPGLE 2204 Filamin A 2242 RAEAGVPAEFSIWTREAGAGGLAIAVEGPSKAEIS 2276 Filamin B 2205 RGEAGVPAEFSIWTREAGAGGLSIAVEGPSKAEIT 2239 Filamin A 2277 FEDRKDGSCGVAYVVQEPGDYEVSVKFNEEHIPDS 2311 Filamin B 2239 FDDHKNGSCGVSYIAQEPGNYEVSIKFNDEHIPES 2274 Filamin A 2312 PF 2313 Filamin B 2275 PY 2276 Figure 4.1: Alignment of the filamin B fragment that binds to the CaR-tail with its filamin A counterpart. Sequences corresponding to the filamin A equivalent (2172-2313) of a filamin B cDNA library clone aligned with filamin B (2135-2276). Conserved residues are highlighted in black ( X ), while conserved substitutions are highlighted in grey ( X ).

4.2.2 Purification of His-tagged CaR-tail from Insect Cells

Previous direct interaction studies have used bacterially produced His-tagged CaR-tail

(His-CaR-tail) purified using a denaturation/renaturation method (Hjalm et al. 2001).

The denaturation/renaturation technique was employed because purification of CaR-tail

extracted from bacteria by other methods does not produce a soluble protein. However,

there is great variability in recovery at the renaturation stage of this purification method,

with only a small percentage of protein being renatured and correctly folded. To counter

these deficiencies an alternate technique was investigated, the baculoviral expression

system. Outlined below is the first use of a baculovirus expression vector system to

generate His-CaR-tail in Sf21 insect cells. Eukaryotic proteins expressed in insect cells

produced using recombinant baculovirus undergo post-translational modifications

similar to those in mammalian cells and are therefore more representative of the

mammalian protein than a bacterially produced counterpart (Jarvis and Finn 1995).

Aliquots taken at different stages of the purification process were separated on a 15%

SDS-PAGE gel and visualised using Coomassie staining (Figure 4.2A). Lanes 1 and 2

contain the lysate from insect cells expressing the His-CaR-tail before and after being

incubated with nickel-chelate agarose beads, respectively. His-CaR-tail was eluted from

nickel-chelate agarose beads with imidazole and the purified receptor fragment was

concentrated by Centricon microconcentration. The purified His- CaR-tail,

approximately 35 kDa in size, is shown in Figure 4.2A Lane 4. The purified protein was

confirmed to correspond to the His-CaR-tail by Western analysis using an anti-His

antibody (Figure 4.2B).

4.2.3 Pulldown Assays Performed Using His-tagged CaR-tail Purified from Insect cells

His-CaR-tail recovered from Sf21 insect cells was immobilised on nickel-chelate

agarose beads before being incubated with one of the three purified GST-filamin

proteins or GST alone, as a negative control. Bound proteins were eluted with sample

buffer and separated on a 15% SDS-PAGE gel before being transferred to a

nitrocellulose membrane for Western analysis with an anti-GST antibody. Both GST-

Fil11 and GST-Fil21 were found to interact with the His-CaR-tail, with the latter

displaying a more efficient interaction (Figure 4.3). Neither GST-FilH nor GST alone

were found to bind to the His-CaR-tail. Neither the three GST-fusion proteins nor GST

alone were able to bind to the nickel-chelate agarose beads in the absence of the His-

CaR-tail. The lack of binding of GST-FilH was suprising as it had previously been

shown to bind to bacterially produced His-CaR-tail (Hjalm et al. 2001).

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4.2.4 Pulldown Assays Performed Using His-tagged CaR-tail Purified from Bacteria

In order to verify that the GST-FilH construct could bind to the CaR-tail as reported

previously, the pulldown assays were repeated using His-CaR-tail purified from bacteria

as described by Hjalm et al. His-CaR-tail expressed in bacteria was purified on nickel-

chelate agarose beads according to the denaturation/renaturation methodology for use in

in vitro interaction studies. The immobilised His-CaR-tail protein was then exposed to

each of the three purified GST-fusion proteins or GST, as the negative control. Bound

proteins were eluted with sample buffer and separated on a 15% SDS-PAGE gel before

being transferred to a nitrocellulose membrane. Western analysis using an anti-GST

antibody revealed that all three GST-fusion proteins, GST-Fil11, GST-FilH and GST-

Fil21, bound to the His-CaR-tail, while GST did not (Figure 4.4). Again the GST-Fil21

protein interacted more efficiently with the His-CaR-tail than the GST-Fil11 protein. In

the absence of His-CaR-tail, neither the GST-fusion proteins nor GST bound to the

nickel-chelate agarose beads.

4.3 Discussion

There is an extensive collection of proteins that regulate a variety of the GPCR

properties through their interaction with the receptor’s intracellular tail. These proteins

include filamin A (Hall and Lefkowitz 2002). Filamin A has been shown to interact

with a variety of proteins, ranging from transmembrane proteins, like the dopamine

receptors and integrins, to components of signalling cascades, such as RhoA and Rac1

(van der Flier and Sonnenberg 2001). The CaR was identified as a binding partner of

filamin A in 2001 and studies have revealed that their interaction is necessary for certain

CaR-mediated signalling pathways and protects the CaR against degradation (Awata et

al. 2001; Hjalm et al. 2001; Huang et al. 2006; Pi et al. 2002; Zhang and Breitwieser

2005).

Interestingly, several of the experiments examining the functional implications of the

CaR and filamin A interaction were performed in M2 cells, which do not express

filamin A and A7 cells, which are M2 cells stably expressing filamin A. Both M2 and

A7 cells express filamin B. CaR-mediated ERK signalling was found by two groups to

occur in A7 cells, but is abolished in M2 cells (Awata et al. 2001; Zhang and

Breitwieser 2005). An increased level of CaR degradation was detected in M2 cells in

comparison to that observed in A7 cells (Zhang and Breitwieser 2005). Increases in

receptor degradation in the absence of filamin have also been observed in experiments

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examining the relative degradation rates of the calcitonin receptor in M2 and A7 cells

(Seck et al. 2003). These findings indicate that, in relation to at least some functions of

the CaR, filamin B is unable to compensate for a lack of filamin A and that the

interaction between the CaR and filamin B observed in the yeast two-hybrid studies

presented in chapter 3 may regulate functions of the CaR distinct from those involving

filamin A. Examining the direct interaction of the identified filamin B clone with the

CaR using in vitro pulldown assays will be the first step towards understanding what

those functions might be. Following confirmation of interaction, further studies could

examine filamin B could perform a similar function to filamin A by acting as a

scaffolding protein bound to a unique set of accessory proteins.

Published reports define filamin A residues 1566-1719 as the site of CaR interaction

(Awata et al. 2001; Hjalm et al. 2001), but direct interaction studies presented above

show that the CaR is also capable of binding to two other distinct regions of filamin A,

residues 1193-1312 and 2172-2313. In pulldown experiments using baculovirus

expressed His-CaR-tail both the GST-Fil11 and GST-Fil21 constructs, which contained

filamin A amino acids 1193-1312 and 2172-2313 respectively, bound to the CaR-tail,

while GST-FilH, which contained filamin A residues 1534-1719, did not (Figure 4.3).

The region of filamin A contained in the GST-FilH fusion protein had previously been

shown by Hjalm et al. to bind to the CaR-tail in direct interaction pulldown studies

using bacterially derived His-CaR-tail (Hjalm et al. 2001). Therefore, the in vitro

interaction studies were repeated using His-CaR-tail purified from bacteria to verify the

reported binding between the GST-FilH construct and the CaR-tail. All three GST-

fusion proteins were found to bind to the bacterially derived His-CaR-tail (Figure 4.4).

The His-CaR-tail purified from baculovirus and that expressed in bacteria and isolated

by denaturation/renaturation might be folded differently with the baculovirus expressed

protein being incapable of binding to the GST-FilH. To determine if the difference in

purification methods account for these observations it will be necessary to repeat the in

vitro pulldown assays using baculovirus expressed His-CaR-tail purified by the

denaturation/renaturation methodology. Alignment of the three identified CaR

interaction domains, filamin A residues 1193-1312, 1534-1719 and 2172-2313, revealed

a high level of homology in the region boxed in Figure 4.5. It should be noted that these

direct interaction studies observe protein binding under artificial conditions that are not

mimicked within the cell. Therefore, from the data presented here, it can only be

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Filamin A 1193-1312 1193 ----------------------------------- 1213 1534-1719 1534 ---PFKVKVLPTHDASKVKASGPGLNTTGVPASLP 1565 2172-2313 2172 ----------------------------------- 2186 1193-1312 1193 --------------LTIEICSEAGLPAEVYIQDHG 1213 1534-1719 1566 VEFTIDAKDAGEGLLAVQITDPEGKPKKTHIQDNH 1600 2172-2313 2172 --------------------SPSGKTHEAEIVEGE 2186 1193-1312 1214 DGTHTITYIPLCPGAYTVTIKYGGQPVPNFPSKLQ 1248 1534-1719 1601 DGTYTVAYVPDVTGRYTILIKYGGDEIPFSPYRVR 1635 2172-2313 2187 NHTYCIRFVPAEMGTHTVSVKYKGQHVPGSPFQFT 2221 1193-1312 1249 VEPAVDTSGVQCYG--PGIEGQGVFREATTEFSVD 1281 1534-1719 1636 AVPTGDASKCTVTVS-IGGHGLGAGIGPTIQIGEE 1661 2172-2313 2222 VGPLGEGGAHKVRAGGPGLERAEAGVPAEFSIWTR 2256 1193-1312 1282 AR---ALTQTGGP----HVKARVANPSGNLTETYV 1309 1534-1719 1662 TV----ITVDTKAAGKGKVTCTVCTPDGSEVDVDV 1692 2172-2313 2257 EAGAGGLAIAVEGPSKAEISFEDRKDGSCGVAYVV 2291 1193-1312 1310 QDR------------------------ 1312 1534-1719 1693 VENEDGTFDIFYTAPQPGKYVICVRFG 1719 2172-2313 2291 QEPGDYEVSVKFNEEHIPDSPF----- 2313 Figure 4.5: A comparison of the amino acid sequence of the identified CaR-binding sites within human filamin A. Sequences corresponding to the three sites of CaR interaction identified in filamin A have been aligned. Conserved residues highlighted in black ( X ) while conserved substitutions are highlighted in grey ( X ). A region of high homology is boxed in red ( ).

concluded that there are at least three possible sites of CaR interaction within filamin A

that contain a highly homologous region of approximately 40 residues.

Although several proteins that bind to multiple sites within filamin A have been

identified (Berry et al. 2005; Kiema et al. 2006; Tigges et al. 2003), there has been little

speculation as to the purpose of the multiple binding sites. In relation to the CaR, there

are some possible advantages that may arise from interacting with filamin A at multiple

sites. The protective effect that the CaR interaction with filamin A provides against

degradation (Zhang and Breitwieser 2005) may be a result of the stabilisation of CaR

dimers assuming that the intracellular tail of one receptor binds to one binding site

within filamin A while the other binds to a second binding site within filamin A,

effectively clamping the two receptors (Figure 4.6A). The multiple CaR binding sites

within filamin A may also assist in its role as a scaffolding protein. The CaR regulates a

host of signalling cascades that contain proteins that bind to the C-terminal end of

filamin A, including Rho A and JNK kinase (JNKK) (Huang et al. 2006; Pi et al. 2002).

By binding to an interaction domain closer to the C-terminus, the receptor is brought

into close proximity to components of CaR-mediated signalling pathways (Figure

4.6B). However, the CaR also regulates the function of large, transmembrane proteins

like the Kir4.2 channel, which binds to the C-terminus of filamin A (Huang et al. 2007;

Wang et al. 2004). The distance between the CaR and Kir4.2 would be determined by

the size of the two proteins and where they are embedded in the membrane. Under these

circumstances the CaR may need to bind to an interaction domain closer to the N-

terminus allowing filamin A to reach and interact with Kir4.2 (Figure 4.6C).

In conclusion, two novel sites of CaR interaction have been identified and verified in

filamin A that may allow it to more efficiently stabilise the CaR dimer and/or act as a

more versatile scaffolding protein by regulating the binding location of its interacting

partners.

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CChhaapptteerr 55 Investigation of the Interaction Between the Intracellular Tail of the

Calcium-Sensing Receptor and Testin and the Implications for Cell

Function

5.1 Introduction

As discussed in Chapter 3, the CaR is the first receptor to be identified as a binding

partner of testin. In this chapter, the nature of the interaction between the CaR and

testin, as well as the functional implications of the interaction will be examined. The

lack of testin expression in particular tumour cell lines (Tatarelli et al. 2000) has already

been discussed, but there are also several reports that CaR expression is decreased in a

number of tumours (Farnebo et al. 1998; Haven et al. 2004; Kifor et al. 1996). It has

been suggested that although the loss of functional CaR is not sufficent to initiate

carcinogenesis, the disruption of CaR function is likely to contribute to the aberrant

physiology of tumours (Rodland 2004). Experiments examining the downstream effects

of CaR activation in cancer cell lines have revealed that the receptor regulates the

expression of several genes associated with malignancy, including E-cadherin, β-

catenin and the c-myc proto-oncogene (Bhagavathula et al. 2007; Kallay et al. 1997).

Increases in CaR-mediated secretion of PTHrP have also been observed in a variety of

cancer cell lines including those derived from the breast, prostate and testis (Sanders et

al. 2000; Tfelt-Hansen 2008). As there is evidence that both testin and the CaR have a

role in tumourigenesis it is possible that their interaction may also be relevant to this

process.

A possible mechanism by which the interaction between the CaR and testin effect

tumourigenesis is through apoptosis. In testin-negative breast and uterine cancer cell

lines the exogenous expression of testin resulted in tumour reduction and increased

apoptosis (Sarti et al. 2005). Several groups have shown that the stimulation of the CaR

can induce apoptosis in a range of cell types including osteoblasts, cardiac myocytes

and HEK293 cells stably expressing the CaR (Mentaverri et al. 2006; Wu et al. 2005;

Zhang et al. 2006). However, the Sindbis Virus induction of apoptosis in AT-3 prostate

cancer cells, fibroblasts and HEK293 cells stably expressing the CaR was prevented by

CaR activation (Lin et al. 1998). The interaction between the CaR and testin may

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regulate whether CaR stimulation results in either the induction or prevention of

apoptosis.

Another commonality between the CaR and testin is that they have both been shown to

localise at actin stress fibres (Garvalov et al. 2003; Griffith et al. 2004; Hjalm et al.

2001). As mentioned in chapter 3, studies investigating the possible function of testin

have found that it has a role in cytoskeletal processes including actin stress fibre

assembly, cell spreading and cell motility (Coutts et al. 2003; Griffith et al. 2004;

Griffith et al. 2005). In HEK293 cells stably expressing the CaR it was demonstrated

that stimulation of the receptor led to changes in cell morphology and actin stress fibre

reorganisation (Davies et al. 2006). An independent study found that the activation of

the CaR in HEK293 cells stably expressing the receptor induced cell motility and

plasma membrane ruffling, a dynamic process involving the formation and retraction of

cytoplasmic protrusions enriched in filamentous actin (Bouschet et al. 2007).

In light of the evidence of overlap between the biological processes that the CaR and

testin are involved in, it is likely that their interaction, identified in the yeast two-hybrid

system, is physiologically relevant in the mammalian cell. The following chapter will

further examine the CaR and testin relationship and its possible role in cell function.

5.2 Results

5.2.1 Calcium-Sensing Receptor and Testin Interaction Studies

5.2.1.1 Yeast Two-Hybrid Mapping

As outlined in chapter 3, three unique overlapping clones of testin were found to

interact with the intracellular tail of the CaR in a yeast two-hybrid screen of a

haemopoietic cell line library. Analysis of the 61 amino acids present in all three testin

clones, shown in Figure 5.1A, revealed that all three contained a common structural

feature, the second zinc-finger of LIM domain 1. To determine if the zinc-finger motif

was required for binding between the CaR-tail and testin or if the CaR-tail simply

recognised the amino acid sequence of the region per se, the yeast two-hybrid system

was used to investigate binding between the CaR-tail and various mutated testin clone

constructs. Site-directed mutagenesis was performed to substitute an alanine at either

Cys269 or His290 within testin clone 148-357 to interfere with the zinc finger’s

capacity to coordinate metal ions and disrupt its formation. Testin clone 148-357 was

chosen because the overlapping region was contained centrally within this construct.

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Results from β-galactosidase assays displayed in Figure 5.1B indicated that neither

mutant construct was capable of binding to the CaR-tail, while the wild-type testin

construct did interact as observed previously. An alanine scan throughout the loop of

the second zinc-finger was conducted using seven mutant constructs of testin clone 148-

357 to establish the importance of the amino acid sequence to the interaction between

the CaR-tail and testin. Residue Gly270 was omitted from the alanine scan because

there is low conservation of that amino acid amongst species, as seen in Figure 3.10.

Neither a single alanine substitution at residue Met276 nor a tandem alanine substitution

at amino acids Leu288/Tyr289 had an impact on the potency of the interaction between

the CaR-tail and testin clone 148-357. Binding potency between the CaR-tail and testin

clone 148-357 mutants with multiple alanine substitutions at either residues

Ile277/Tyr278/Phe279 or Gly283/Lys284 was less than the strength of the interaction

between the CaR-tail and testin clone 148-357, but equivalent to that of the Hsp90 and

CyP40 positive control. The mutation of amino acids Glu271/Leu272/Leu273,

Val274/Asp275 or Trp280/Lys281/Asn282 to alanines within testin clone 148-357

abolished the interaction between the CaR-tail and testin clone 148-357.

In the previous chapter, residues 865-922 of the CaR were identified as interacting with

testin. To further refine this binding region two more CaR-tail truncation constructs

were created, CaR-tail 865-898 and CaR-tail 899-922, and their ablitiy to interact with

testin investigated in the yeast two-hybrid system. Neither construct was found to

interact with testin clone 148-357 in β-galactosidase assays. This suggests elements

from each of the two constructs are required for testin interaction with the CaR-tail.

5.2.1.2 Cloning of Full-Length Human Testin

To further investigate the interaction between the CaR-tail and testin it was necessary to

obtain a full-length clone of testin. Human testin cDNA was generated by reverse

transcription of RNA extracted by Dr Bryan Ward from the MDA MB231 breast cancer

cell line, which had previously been shown to express testin as detected by Northern

analysis (Tatarelli et al. 2000). The cDNA was subsequently amplified by PCR and

cloned into the pDrive vector and sequenced. Following confirmation that the full-

length human testin clone was error-free it was subcloned into appropriate vectors for

expression in bacteria and mammalian cells.

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5.2.1.3 Direct Interaction Studies

In vitro studies using pulldown assays were proposed to examine the direct interaction

between the CaR-tail and full-length testin. These studies required the purification of

soluble CaR-tail and testin proteins. Bacterially derived, soluble His-tagged CaR-tail

was produced using a denaturation/renaturation method. To express testin as a GST-

fusion protein in bacteria the full-length cDNA of human testin was cloned into the

pGEX4T-1 vector. Expression of GST-testin could be induced by treatment with IPTG

(Figure 5.2A), but no soluble GST-testin could be detected (Figure 5.2B). Soluble GST-

testin could be produced using an alternative purification protocol that included the use

of higher levels of detergent, but this increase in detergent interfered with the release of

the GST-fusion protein from the glutathione beads onto which it had been purified

(Figure 5.2C). An alternative approach to expressing insoluble proteins in a soluble

form has been to create fusion proteins that contain the insoluble protein fused to a

highly soluble protein, such as NusA (Lavallie et al. 1993). A construct containing

NusA fused to human testin was generated, but again no soluble protein was detected

(Figure 5.2D) despite reports of NusA fusion proteins being highly soluble (Harrison

2000). Since we were unable to produce bacterially expressed testin proteins that were

soluble, the approaches to demonstrate direct interaction of testin with the CaR-tail were

not pursued further.

5.2.1.4 Coimmunoprecipitation Studies

Confirmation that the interaction of the CaR and testin occurs in vivo requires the

coexpression of the CaR and testin in a mammalian system. The full-length human

clone of testin was cloned into the mammalian expression vector, pcDNA3/EGFP to

allow the N-terminal EGFP-tagging of testin. The EGFP-testin/pcDNA3 construct was

cotransfected into HEK293 cells with a C-terminal FLAG-tagged CaR/pcDNA3.1

construct used previously by this laboratory (Ward et al. 2004). CaR-FLAG was

immunoprecipitated from the lysates of the cotransfected HEK293 cells with an anti-

FLAG antibody and separated on a 10% SDS-PAGE gel prior to blotting on

nitrocellulose. The Western blot probed with an anti-GFP antibody presented in the

upper panel of Figure 5.3A shows that EGFP-testin was detected in the lane containing

coimmunopreciptates from cells cotransfected with the CaR-FLAG/pcDNA3.1 and

EGFP-testin/pcDNA3 constructs. In the remaining lanes it can be seen that when either

CaR-FLAG or EGFP-testin were expressed alone no EGFP-testin was detected. The

reciprocal experiment is shown in the upper panel of Figure 5.3B where EGFP-testin

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was immunoprecipitated with an anti-GFP antibody from the lysates of the

cotransfected HEK293 cells and run on a 7.5% SDS-PAGE gel prior to western analysis

with an anti-FLAG antibody. Coimmunoprecipitation of CaR-FLAG is observed when

both CaR-FLAG and EGFP-testin are coexpressed in HEK293 cells.

5.2.2 Colocalisation of Testin and the Calcium-Sensing Receptor

Confocal microscopy was employed to investigate the cellular distribution of the CaR

and testin and to determine the degree to which the two proteins colocalise. HEK293

cells cotransfected with the following plasmid combinations: CaR/pcDNA3.1 and

EGFP-testin/pcDNA3, CaR/pcDNA3.1 and EGFP/pcDNA3 empty vector or pcDNA3.1

empty vector and EGFP-testin/pcDNA3, were stained with an anti-FLAG antibody and

a goat anti-mouse secondary antibody conjugated to Alexa Fluor-546 to detect the CaR-

FLAG, while EGFP-testin was detected by the fluorescence emitted from the EGFP tag.

In the left column of images in Figure 5.4 the CaR (red) can be observed at both the cell

surface and within the cytoplasm in HEK293 cells expressing CaR-FLAG (A and D).

EGFP-testin (green) in panels B and H of Figure 5.4, is also present at the cell surface

and in the cytoplasm. The distribution of EGFP (green) throughout the cell, seen in

panel E of Figure 5.4, suggests that the EGFP tag does not interfere with the targeting of

testin to specific subcellular locations. Colocalisation of the CaR-FLAG and EGFP-

testin can be observed either at or near the cell surface in Panel C of Figure 5.4.

5.2.3 The Effects of Testin on Calcium-Sensing Receptor Activated ERK Signalling

As previously mentioned, both the CaR and testin have been shown to be involved in

the process of apoptosis (Sarti et al. 2005; Sun et al. 2006). While it has been

demonstrated that the CaR is able to induce apoptosis via the ERK pathway, the

mechanism by which testin promotes apoptosis is unknown (Sarti et al. 2005; Sun et al.

2006). As testin was found to bind to the membrane proximal region of the CaR

intracellular tail, a region implicated in the receptor’s transduction of the ERK

signalling pathway (Zhang and Breitwieser 2005), the influence of the interaction

between the CaR and testin on ERK signalling was investigated. HEK293 cells stably

expressing the CaR that had been transfected with either EGFP-testin/pcDNA3 or

EGFP/pcDNA3 empty vector were dosed with a range of extracellular calcium

concentrations and the effect on ERK phosphorylation examined by Western blot

analysis using a phosphospecific anti-ERK antibody. Figure 5.5A presents a Western

blot representative of triplicate experiments that show that increasing the concentration

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of extracellular calcium increases the level of phosphorylated ERK produced in

HEK293-CaR cells. However, when comparing HEK293-CaR cells either with or

without EGFP-testin that have been treated with the same amount of extracellular

calcium, there is no significant difference in the level of ERK phosphorylation as

determined by densitometry. This result is depicted graphically in Figure 5.5B.

Although the overexpression of testin does not influence CaR-mediated ERK

phosphorylation in response to extracellular calcium the possibility remained that the

interaction between the CaR and testin may impact on the receptor’s ability to be

allosterically modulated, for example by amino acids (Mun et al. 2004). A

representative Western blot of triplicate experiments is presented in Figure 5.6A and

shows that in the presence of 10 mM phenylalanine there is still a dose dependent

increase in ERK activity in response to increasing concentrations of extracellular

calcium. Densitometric analysis did not reveal any significant difference in the level of

ERK phosphorylation detected in EGFP-testin expressing HEK293-CaR cells compared

with those expressing EGFP empty vector alone, as represented in Figure 5.6B.

5.2.4 The Effects of Testin on Calcium-Sensing Receptor-Mediated Rho Signalling

Transient intracellular calcium oscillations generated by amino acid stimulation of the

CaR were shown to require an intact actin cytoskeleton and functional Rho kinase (Rey

et al. 2005). However, stimulation of the CaR with extracellular calcium produces

sinusoidal intracellular calcium oscillations that are unaffected by disruption to the actin

cytoskeleton, but still require functional Rho kinase (Rey et al. 2005). Activation of the

CaR in HEK293 cells stably expressing the receptor with either extracellular cations or

amino acids also revealed differential signalling in response to the alternate stimuli

(Davies et al. 2006). Treatment of HEK293 cells stably expressing the CaR with either

calcium or magnesium resulted in activation of the Rho kinase that led to actin stress

fibre assembly and morphological changes, while treatment with amino acids failed to

activate processes related to increased Rho activity (Davies et al. 2006). As there is

evidence suggesting a role for both the CaR and testin in changes to the cytoskeletal

structure of cells it was hypothesised that testin may have an effect on CaR-mediated

changes to cell morphology by influencing the receptor’s ability to activate Rho

signalling. Pi et al. has previously demonstrated that treatment of HEK293-CaR cells

with increasing levels of extracellular calcium revealed a dose-dependent increase in

Rho activity as measured using an SRE-luciferase reporter construct (Pi et al. 2002).

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Using the same SRE-luciferase reporter construct, generously supplied by Professor

Jeffrey Pessin from SUNY at Stony Brook, it was found that when HEK293-CaR cells

transfected with EGFP-testin were treated with elevated levels of extracellular calcium,

the Rho activity detected was significantly greater than that observed in HEK293-CaR

cells not transfected with EGFP-testin at the same concentrations of extracellular

calcium, as shown in Figure 5.7A. As evidence that the increase in Rho activity was due

to the overexpression of testin enhancing CaR-mediated Rho signalling and not the

overexpression of testin being solely responsible for the increase in Rho activity,

HEK293 cells, which do not endogenously express the CaR, were transfected with

EGFP-testin or EGFP and exposed to 5 mM of extracellular calcium. There was no

increase in Rho activity above basal observed in HEK293 cells either expressing EGFP-

testin or EGFP upon stimulation with 5 mM extracellular calcium (Figure 5.7B),

indicating that the CaR is required for the overexpression of testin to impact on Rho

signalling.

5.2.5 The Calcium-Sensing Receptor Regulates Changes in Cell Morphology

In order to examine the influence that the interaction between the CaR and testin may

have on the morphology and cytoskeletal organisation of cells, it was necessary to first

replicate the findings of Davies et al, which showed that the CaR had a role in

regulating these processes (Davies et al. 2006). HEK293 cells stably expressing the CaR

were incubated for 3 hr in serum-free DMEM containing a range of extracellular

magnesium concentrations: 0.8 mM, 2.8 mM, 5.8 mM and 8.8 mM. In Figure 5.8 a

graded decrease in the number of cellular extensions formed correlated with an increase

in the extracellular magnesium concentration to which HEK293-CaR cells were

exposed, with virtually no extensions detected at 8.8 mM extracellular magnesium.

Phalloidin-Alexa Fluor-568 was used to detect the formation of actin stress fibres in

HEK293-CaR cells incubated in serum-free DMEM for 3 hr in the presence of

extracellular magnesium at a range of concentrations which included 0.8 mM, 5.8 mM

and 8.8 mM. There are no detectable actin stress fibres in HEK293-CaR cells treated

with 0.8 mM extracellular magnesium, as seen in Figure 5.9A, but in HEK293-CaR

cells incubated with higher concentrations of extracellular magnesium, 5.8 mM and 8.8

mM, there is evidence of actin stress fibre formation, as shown in Figures 5.9C and E,

respectively. These results are consistent with those observed by Davies et al. 2006. As

paxillin phosphorylated Tyr118 is a marker of focal adhesions, the HEK293-CaR cells

described above were also stained with an anti-phospho-paxillin-(Y118) antibody

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followed by an Alexa Fluor-488 rabbit anti-mouse secondary antibody to detect focal

adhesions, which are anchorage points of actin stress fibres (Pellegrin and Mellor 2007).

When HEK293-CaR cells are stimulated with increasing concentrations of extracellular

magnesium, 0.8 mM, 5.8 mM and 8.8 mM, the focal adhesions become more defined at

points at the cell surface, as seen in Figure 5.9B, D and F, respectively.

5.2.6 The Impact of Testin Knockdown on HEK293 Cells Stably Expressing the

Calcium-Sensing Receptor

Having shown that the overexpression of testin accentuates CaR-mediated Rho

signalling in HEK293 cells stably expressing the receptor in section 5.2.5, the effects of

removing testin from HEK293-CaR cells was investigated using shRNA knockdown

technology, specifically the OligoEngine pSUPER RNAi system. The HEK293-CaR

cells used to this point were unsuitable for this process as they were already under G418

selection and the generation of stable testin knockdowns using the OligoEngine

pSUPER RNAi system required the cells to be G418 treated to select for those

expressing the pSUPERIOR.retro.neo+gfp vector. Therefore HEK293-CaR cells that

were under hygromycin selection were used. Incorporation of the

pSUPERIOR.retro.neo+gfp vector into HEK293-CaR cells following viral-mediated

infection resulted in HEK293-CaR cells that also stably expressed EGFP and shRNA-

containing sequences homologous to testin. HEK293-CaR cells that had incorporated

the testin knockdown shRNA were selected by flow cytometry based on their

expression of EGFP. In the initial collection of testin knockdown HEK293-CaR stable

cells it was found that 12.9% of cells sorted contained EGFP. These cells were further

cultured and subjected to a second round of cell sorting to produce a culture in which

98.7% of cells were found to express EGFP. Proteins isolated in lysates from HEK293-

CaR cells and HEK293-CaR stables expressing testin knockdown shRNA were

separated on a 10% SDS-PAGE gel and transferred to nitrocellulose. An anti-testin

antibody was used to detect endogenous testin in both lysates by Western Blot analysis

and showed that efficient knockdown of testin has occurred in cells treated with testin-

targeting shRNA (Figure 5.10).

Results from triplicate experiments examining Rho kinase activity in testin knockdown

HEK293-CaR cells are shown in Figure 5.11. Following an 8 hr incubation with 0.5

mM (basal level) extracellular calcium the level of Rho kinase activity measured in

testin knockdown HEK293-CaR cells using an SRE-luciferase reporter assay was

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approximately half of that observed in the wild-type HEK293-CaR cell line. However,

upon stimulation of the CaR with 5 mM extracellular calcium there was no significant

difference observed between wild-type and testin knockdown HEK293-CaR cells, as

both exhibited an increase in Rho kinase activity approximately 15-fold greater than the

activity observed in control cells at 0.5 mM extracellular calcium.

Testin knockdown HEK293-CaR cells were examined under serum-starved conditions

to determine if they undergo the same changes in morphology and cytoskeletal structure

as wild-type HEK293-CaR cells in response to the same degree of magnesium

stimulation. Following 3 hr of serum starvation in the presence of 5.8 mM extracellular

magnesium, both wild-type HEK293-CaR cells and testin knockdown HEK293-CaR

cells exhibited similar morphologies as shown in Figure 5.12C and D, respectively.

However, unlike the HEK293-CaR cells grown for 3 hr under serum-free conditions

with an extracellular magnesium concentration of 0.8 mM (Figure 5.12A), testin

knockdown HEK293-CaR cells (Figure 5.12B) showed no signs of stellation, with a

morphology resembling that observed in both serum-starved HEK293-CaR cells and

testin knockdown cells treated with 5.8 mM extracellular magnesium (Figure 5.12C and

D). There were also differences in the cytoskeletal organisation between the wild-type

HEK293-CaR cells and testin knockdown cells, observed following serum starvation for

3 hr at an extracellular magnesium concentration of 0.8 mM. As previously observed,

HEK293-CaR cells showed no signs of actin stress fibre organisation when serum

starved in the presence of 0.8 mM extracellular magnesium, but testin knockdown cells

did exhibit actin stress fibre formation under these conditions, as seen in Figure 5.13A

and B, respectively. Punctate staining, indicating phosphorylated paxillin, was observed

throughout wild-type HEK293-CaR cells treated for 3 hr with 0.8 mM extracellular

magnesium in serum-free media (Figure 5.14A). However, in testin knockdown cells

there is a dramatic decrease in staining corresponding to phospho-paxillin-(Y118) in the

cytoplasm and the staining is concentrated at points at the cell surface representing focal

adhesions, circled in Figure 5.14B. In the presence of 5.8 mM extracellular magnesium

both wild-type HEK293-CaR cells (Figure 5.13C and Figure 5.14C) and testin

knockdown cells (Figure 5.13D and Figure 5.14D) form actin stress fibres and focal

adhesions. The cytoskeletal structure of serum-starved testin knockdown cells in the

presence of 0.8 mM extracellular magnesium (Figure 5.14B) resembles that of

magnesium stimulated HEK293-CaR cells (Figure 5.14D). Table 5.1 summarises the

results of the testin knockdown studies presented in this section.

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5.3 Discussion

5.3.1 The Calcium-Sensing Receptor and Testin Interaction

The results from a yeast two-hybrid library screen presented in Chapter 3 showed that

the CaR interacted with the LIM domain focal adhesion protein testin. This is the first

described instance of testin interacting with a membrane-bound receptor. Studies

conducted in yeast indicated that the interaction between the CaR and testin required the

second zinc finger of the first LIM domain to be intact, with several residues within the

zinc finger being essential for interaction. The site of testin binding was mapped, using

the yeast two-hybrid system, to the membrane proximal region of the CaR-tail. Direct

interaction studies using pulldown assays could not be performed due to the insolubility

of testin fusion proteins, but the interaction between testin and the CaR was confirmed

in coimmunoprecipitation experiments using lysates from mammalian cells

cotransfected with expression plasmids for both proteins. Confocal microscopy studies

revealed that both the CaR and testin colocalised either at or near the cell surface. As

testin bound to a region of the CaR shown to be important for CaR-mediated signalling,

the role of the CaR and testin interaction was examined in relation to two signalling

pathways. The overexpression of testin did not alter the level of CaR-mediated ERK

phosphorylation, but was found to enhance the level of CaR-induced Rho kinase

activity. As the CaR had been recently shown to regulate cell morphology and

cytoskeletal structure via the Rho pathway, the effect of testin knockdown in relation to

these processes was examined. Interestingly, preliminary analysis has shown that the

cell morphology and cytoskeletal structure observed in non-stimulated testin

knockdown HEK293-CaR cells, mimicked the effect seen in CaR agonist stimulated

HEK293-CaR cells.

5.3.2 Sites of Interaction Between the Calcium-Sensing Receptor and Testin Identified

in the Yeast Two-Hybrid System

Testin contains LIM domains, which are protein-protein binding motifs comprised of

two zinc-fingers (Zheng and Zhao 2007). Testin also contains a PET domain, a

characteristic that is shared by the other members of a subfamily of the LIM domain

containing family, including prickle and dyxin (Bekman and Henrique 2002). Yeast

two-hybrid studies presented in Chapter 3 indicated that only the second zinc-finger of

LIM domain 1 was required for binding with the CaR-tail. A comparison between the

amino acid sequences of the LIM domains of the PET/LIM family revealed that the

second zinc-finger of LIM domain 1 displayed the highest degree of conservation

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throughout the family (Figure 3.11). The zinc-finger structure was demonstrated to be

critical for the interaction between the CaR-tail and testin as this interaction was lost

when the zinc-finger was disrupted by mutation of either one of the cysteines or

histidines that coordinate the zinc ion. The results of an alanine scan through the zinc-

finger revealed that binding between the CaR-tail and testin was unaltered upon

substitution of Met276 with alanine, suggesting that this residue is not involved in the

interaction between the CaR and testin. Simultaneous substitution of Leu285 and

Tyr286 also had no influence on binding, indicating that the large hydrophobic residues

located at the end of the second zinc-finger do not participate in CaR recognition.

Alanine scans through the remaining segments of the second zinc finger eliminated

binding between the receptor and testin consistent with a disruption of the structural

integrity of the zinc finger motif or loss of a binding recognition site. Further mapping

studies are necessary to identify specific amino acids essential for CaR-testin

interaction. Since residues within the second zinc finger, corresponding to the putative

CaR interaction domain are highly conserved throughout mammalian species (Figure

3.10) and with other members of the PET/LIM domain family (Figure 3.11), it is

possible that other family members may also interact with the CaR. Further dissection

of the established interaction domain for testin within the membrane proximal region of

the CaR, corresponding to residues 865-922, failed to identify a more specific contact

domain, suggesting that multiple elements might exist within this 57-residue region or

that the individual fragments used in the study disrupted the recognition site.

5.3.3 Calcium-Sensing Receptor and Testin Interaction Studies

Having demonstrated that the CaR and testin interacted within yeast, further studies

were aimed to establish a direct interaction between the two proteins and whether this

occurred in mammalian cells. The insolubility of testin was a problem for the direct

interaction pulldown studies that, despite a number of alternate approaches, could not be

overcome. It was not possible to perform the reciprocal experiment as GST-CaR was

also found to be insoluble (data not shown). Without information from in vitro

pulldown studies it can not be determined if the CaR and testin bind directly or through

an intermediary protein as part of a complex. A combination of coimmunoprecipitation

and colocalisation studies revealed that the interaction between the CaR and testin

occurred in a mammalian system (Figures 5.3 and 5.4). The colocalisation of the CaR

and testin at the cell membrane also meant that the interaction between the two proteins

might regulate the signalling cascades initiated by the CaR in response to extracellular

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stimuli. In order to strengthen the evidence of CaR and testin colocalisation at the cell

membrane, future studies utilising cell surface biotinylation of the receptor could be

performed. Briefly, these experiments would be performed in HEK293 cells

cotransfected with CaR-FLAG and EGFP-testin. Biotinylation would be used to label

all cell surface proteins prior to whole cell lysis. EGFP-testin would then be

immunoprecipitated from the lysates with an anti-GFP antibody and separated on a

SDS-PAGE gel before being transferred to nitrocellulose. Streptavidin would then be

used to detect biotinylated CaR, if it coimmunoprecipitates with EGFP-testin,

demonstrating that the two proteins interact at the cell surface. As other cell surface

proteins to which testin binds may also coimmunoprecipitate, the identity biotinylated

CaR should be confirmed using the FLAG antibody.

5.3.4 The Effects of Testin Binding on Calcium-Sensing Receptor Regulated Signalling

5.3.4.1 Calcium-Sensing Receptor-Mediated ERK Phosphorylation is Unaffected by

Testin Overexpression

There was no obvious candidate CaR-mediated signalling pathway to examine in

relation to the interaction between CaR and testin, as testin has not been previously

associated with a receptor. However, testin was first identified as a tumour suppressor,

an attribute that has recently been associated with the CaR, suggesting possible overlap

between the mechanisms by which the two proteins act (Bhagavathula et al. 2005;

Tatarelli et al. 2000). Sarti et al. showed that the adenoviral transduction of testin into

the T47D and MES-SA cancer cell lines impaired tumourigenicity and increased

apoptosis (Sarti et al. 2005). The CaR has been shown to increase apoptosis in response

to agonist stimulation via the ERK pathway (Sun et al. 2006). The yeast two-hybrid

mapping studies indicated that the membrane proximal region of the CaR-tail contained

the testin interaction domain, a region previously demonstrated to be essential to CaR-

mediated ERK signalling (Zhang and Breitwieser 2005). Experiments using an ERK

assay revealed that overexpression of testin did not alter the level of CaR-mediated

ERK activity in response to either extracellular calcium alone or in the presence of the

amino acid, phenylalanine (Figures 5.5 and 5.6). These findings suggest that testin

neither promotes CaR-induced ERK phosphorylation nor interferes with the interaction

between the CaR and components of the ERK cascade, despite binding to the membrane

proximal region of the CaR-tail critical for CaR-mediated ERK signalling.

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5.3.4.2 Testin Accentuates Calcium-Sensing Receptor-Mediated Rho Kinase Activity

By examining the cellular localisation and binding partners of testin, Coutts et al., found

that the role of this protein in normal tissue was linked to cell spreading and motility

(Coutts et al. 2003). Further studies confirmed the involvement of testin involvement in

a range of cytoskeletal processes including actin stress fibre assembly (Coutts et al.

2003; Griffith et al. 2004; Griffith et al. 2005). Recently, the CaR has also been shown

to have a role in actin stress fibre reorganisation and cell morphology, which was

revealed to be dependent on Rho kinase signalling (Davies et al. 2006). The knockdown

of testin in HeLa cells resulted in a reduction of both actin stress fibre assembly and

Rho A activity, an upstream component of the Rho kinase signalling cascade (Griffith et

al. 2005). Using a SRE-luciferase reporter assay, CaR-mediated Rho kinase activation

was found to be enhanced in HEK293-CaR cells overexpressing testin (Figure 5.7).

shRNA technology was used to knockdown testin in HEK293-CaR cells, which were

then studied to determine the effects of CaR stimulation on Rho kinase activity in the

absence of testin. The basal level of Rho kinase activity detected in testin knockdown

HEK293-CaR cells was significantly lower than that of wild-type HEK293-CaR cells

(Figure 5.11), which is consistent with the decrease in Rho A activity observed in testin

knockdown HeLa cells (Griffith et al. 2005). However, an equivalent level of Rho

kinase activity was detected in testin knockdown HEK293-CaR cells as in HEK293-

CaR cells in response to elevated (5 mM) extracellular calcium (Figure 5.11). Taken

together, these results suggest that, while testin is not essential for CaR-mediated Rho

kinase activation, the interaction of testin with the CaR contributes to increased activity

of this signalling pathway.

5.3.5 The Relationship Between Cell Morphology and the Calcium-Sensing Receptor’s

Interaction with Testin

As previously mentioned, both the CaR and testin have been found to be involved in the

regulation of a series of related processes including cell motility, actin stress fibre

reorganisation and cell morphology (Coutts et al. 2003; Griffith et al. 2004; Griffith et

al. 2005)). In addition to the cytoskeletal processes regulated by the CaR already

described by others (Bouschet et al. 2007; Davies et al. 2006), the current study shows

that stimulation of the CaR in HEK293-CaR cells results in an increase in the formation

of peripheral focal adhesions over that seen in unstimulated cells (0.8 mM Mg2+)

(Figure 5.9B, D and F). Since focal adhesions are anchorage points for actin stress fibre

assembly this would be predicted from the studies of Davies et al, which shows that

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agonist stimulation of HEK293-CaR cells increases actin stress fibre formation (Davies

et al. 2006). However, the studies by Davies et al. and those in this thesis are in contrast

to those of Griffith et al. conducted in HeLa cells where it was shown that peripheral

focal adhesions and actin stress fibre networks were quite extensive even under non-

stimulatory conditions. This difference in basal levels of focal adhesion formation and

actin stress fibre assembly between the two cell lines, HeLa and HEK293-CaR cells

underscores the possibility of cell-type specific differences in cytoskeletal structure

impacting on potential testin modulation of CaR-mediated cytoskeletal changes.

CaR-mediated changes in cell morphology and actin stress fibre assembly require Rho

kinase activation (Davies et al. 2006) and RNAi knockdown of testin at basal agonist

levels reduces Rho A activity with concurrent loss of actin stress fibre assembly

(Griffith et al. 2005). Paradoxically, results presented in Chapter 5 show that, although,

testin overexpression increases Rho kinase activity and testin knockdown reduces it, the

reduction of Rho activity associated with knockdown of testin at basal agonist levels did

not result in the absence of actin stress fibre formation and cell extensions, which might

have been predicted from the studies of Davies et al. with HEK293-CaR cells.

However, these potentially interesting but preliminary results can only be considered

further once certain confirmatory and control studies have been undertaken. Towards

this end, it will be necessary to determine if the increase in stress fibre assembly

observed in the testin knockdown HEK293-CaR cells is specifically CaR-mediated, by

generating and comparing testin knockdown HEK293 cells without the CaR.

Furthermore, the observations made with testin knockdown HEK293-CaR cells with

respect to Rho kinase activation (Figure 5.11), cell morphology (Figure 5.12), actin

stress fibre assembly (Figure 5.13) and focal adhesion formation (Figure 5.14) should

also be evaluated against mock shRNA treated cells, as opposed to the wild-type,

untreated HEK293-CaR cells used in these experiments.

The methodologies used to examine the relationship between Rho signalling and the

CaR-induced cellular alterations differ between the studies presented here and those of

Davies et al in that a downstream effect of Rho kinase activity was examined, while

Davies et al used inhibitors of Rho A and Rho kinase. The drawback of using inhibitors

is that this does not just target CaR-mediated Rho signalling, but inhibits all Rho

signalling throughout the entire cell. However, measuring an outcome of Rho

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signalling, such as SRE-luciferase activity, is limiting because only a single downstream

event is measured and does not account for Rho kinase activity leading to alternate

outcomes. Therefore, despite a decrease in Rho kinase activity being detected via the

SRE-luciferase assay there remains the possibility that CaR-induced Rho signalling has

been diverted to favour another specific response. A proposed mechanism by which

CaR-mediated Rho signalling may be diverted is through interactions between the

intracellular tail of the CaR and specific binding partners, which will be discussed in

further detail in the following chapter. There is an alternative method to measure Rho

activity more directly that is based on the principle that activated Rho exists in a GTP-

bound state, while inactive Rho is bound to GDP (Machesky and Hall, 1996). By using

a GST fusion protein that contains a Rho binding domain motif that specifically binds to

the active, GTP-bound Rho it is possible to perform a pulldown assay using glutathione

beads to detect only active Rho (Ren 1999). Further experiments measuring activated

Rho or inhibiting Rho A and the Rho kinase in CaR-stimulated testin knockdown

HEK293-CaR cells will determine the necessity of the Rho pathway in the observed

morphological and cytoskeletal changes.

In conclusion, testin has been identified as a novel binding partner of the CaR

intracellular tail that was found to enhance CaR-mediated Rho kinase signalling and

may play a role in the receptor’s regulation of cell morphology and cytoskeletal

structure.

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CChhaapptteerr 66 General Discussion

6.1 The Calcium-Sensing Receptor

The CaR was cloned and characterised in an attempt to identify a protein capable of

responding to changes in extracellular calcium and has been shown to be an integral part

of calcium homeostasis (Brown and MacLeod 2001). However, later studies would

reveal that the CaR, unlike most receptors, was able to be activated by a diverse range

of stimuli (Breitwieser et al. 2004). Although extracellular calcium is still considered

the primary physiological agonist of the CaR, the list of additional endogenous stimuli

includes other multivalent cations, amino acids, polyamines, polypetides, as well as

changes in ionic strength and pH (Riccardi 2002). Due to the important physiological

and pathophysiological role of the CaR, a great deal of research has been focused on

examining exogenous CaR agonists that can be used therapeutically to modulate the

receptor’s activity and includes additional multivalent cations and polyamines, as well

as pharmacological agents such as aminoglycoside antibiotics, calcimimetics and

calcilytics (Riccardi 2002). In addition to being able to recognise a multitude of

different agonists, the CaR is also able to initiate a host of different signalling cascades,

including the PLC, MAPK and Rho signalling pathways (Ward 2004). In turn, this array

of intracellular signals is transformed into precise biological outcomes, which include

proliferation, apoptosis, differentiation and gene expression (Brown and MacLeod

2001). Many of the cellular processes regulated by the CaR occur in a tissue-specific

manner and there are even instances where CaR activation has been shown to have

opposing effects on cellular processes in different cells (Huang and Miller 2007). A

simplistic interpretation of the CaR’s translation of extracellular signals into signalling

pathways that result in a variety of biological responses is depicted in Figure 6.1. The

translation of extracellular signals into intracellular signals by the CaR is likely to be

coordinated by several mechanisms working in concert to ensure the correct outcome,

including ligand specific conformational changes of the receptor and receptor

phosphorylation. Another proposed mechanism that may determine the response of the

CaR in a tissue-specific manner may be mediated through the participation of protein

binding partners of the receptor.

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6.2 Interacting Protein Partners of the Calcium-Sensing Receptor

A number of proteins have been found to interact with the CaR, with several being

identified in yeast two-hybrid library screens using the intracellular tail of the CaR as

bait (Awata et al. 2001; Hjalm et al. 2001; Huang and Miller 2007; Huang et al. 2007).

The yeast two-hybrid libraries used in these studies were all generated from tissues

involved in whole body calcium homeostasis. Several years ago, the Ratajczak group

used the CaR-tail as bait to screen a commercially produced brain library to identify

proteins that are involved in processes unrelated to calcium homeostasis. Not a single

interacting protein was isolated from this library screen. This thesis presents results of a

subsequent yeast two-hybrid screen performed by the Ratajczak group of an EMLC.1

mouse pluripotent haemopoietic cell line library using the CaR-tail as bait that

identified several novel CaR binding partners. The seven proteins that were found to

interact with the CaR-tail in this screen, filamin A, filamin B, testin, 14-3-3 θ, OS-9,

Ubc9 and MPc2, are a diverse group of proteins that have been shown to be associated

with a wide variety of cellular functions. Yeast two-hybrid mapping studies revealed

that all seven proteins recognised essential binding elements present within either

residues 865-922 or 965-986 of the CaR-tail. Other reported CaR binding partners with

interaction sites mapped within the CaR-tail, have also been found to bind to either of

these two regions. Within the CaR-tail these two putative contact domains are the most

highly conserved between mammalian CaRs (Figure 1.1). This high level of

conservation suggests that the binding elements recognised in these regions have been

conserved throughout evolution. Additional evidence that these two regions of the CaR-

tail are critical for protein interactions may be obtained in further mapping studies of

other CaR-tail binding partners.

6.3 Interacting Protein Partners of the Calcium-Sensing Receptor Regulate its

Function

While the seven interacting partners of the CaR-tail identified in the yeast two-hyrid

library screen are a diverse group, several have been shown to be associated with

common functional processes. There are several basic mechanisms that determine

whether these accessory proteins can regulate the function of the CaR. In order to

influence the activity of the CaR, the interacting protein partners must obviously be

expressed in the same cell and at the same subcellular location. Gene expression

provides a level of control in cells by determining which binding partners are expressed,

when they are expressed and the level of expression. However, in cells that express both

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the CaR and an interacting protein, the trafficking and subsequent localisation of the

binding partner will dictate whether an interaction between the two proteins can occur.

For instance, the subcellular localisation of testin is dependent on it being in the correct

conformational state to allow binding to zyxin, for its recruitment to specific subcellular

localisations, namely, focal adhesions (Garvalov et al. 2003). Therefore, the interaction

between the CaR and testin is dependent on testin’s recruitment by zyxin to a

subcellular location where the receptor and testin can colocalise. Results presented in

Chapter 5, show that in HEK293-CaR stable cells, stimulation of the CaR with

extracellular magnesium results in the formation of focal adhesions (Figure 5.9). Further

colocalisation studies will need to be performed to establish whether CaR activation

results in greater expression of both the CaR and testin in these newly formed focal

adhesions.

As mentioned, there is some overlap in the biological processes with which some of the

CaR binding partners are associated, which suggests another method by which their

influence on the CaR can be regulated. CaR interacting proteins that promote cellular

processes leading to a common outcome may act together as a complex to regulate CaR

function. The CaR binding partners, filamin A and β-arrestins, were found to form a

complex involved in actin cytoskeletal changes regulated by the activity of other

receptors (Scott et al. 2006). Alternatively, the CaR binding partners may compete for

sites of interaction and therefore the receptor’s function would be regulated by its

affinity for the various accessory proteins. Considering the observed trend for CaR

interacting proteins to bind to only two regions of the intracellular tail, it is very likely

that there is strong competition for these sites.

An outline of CaR processes that are proposed to be regulated by the receptor’s

interaction with accessory proteins is presented below.

6.3.1 The Effect of Interacting protein partners on Calcium-Sensing Receptor

Dimerisation

Dimerisation has been shown to be important for the function of the CaR, which is

predominantly expressed at the cell surface as a homodimer (Bai et al. 1998).

Speculation concerning the roles of multiple CaR binding sites in filamin A that were

identified in Chapter 4, has led to the proposal that if the intracellular tail of each CaR

in a homodimer binds to a distinct site within filamin A, then filamin A may act as a

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clamp to stabilise the CaR homodimer (Figure 4.6A). While there is no direct evidence

to support this theory, the stabilisation of the CaR homodimer in this way could explain

how the interaction between the CaR and filamin A protects the receptor against

degradation perhaps by preventing receptor internalisation (Zhang and Breitwieser

2005).

6.3.2 The Regulation of Calcium-Sensing Receptor Trafficking by Interacting Proteins

Yeast two-hybrid mapping studies presented in Chapter 3 reveal that 14-3-3 θ binds to a

region of the CaR-tail that contains a 14-3-3 consensus binding sequence that lies

adjacent to a potential ER retention motif. The facilitation of the surface trafficking of

membrane proteins by the masking of ER retention motifs has been proposed as a

function of 14-3-3 proteins (Shikano et al. 2006). Once it has been established that the

ER retention signal is specifically able to retain CaR in the ER, the hypothesis that the

masking of the ER retention signal by 14-3-3 θ is required for trafficking of the CaR to

the cell membrane could be tested by examining the trafficking of mutant CaR

constructs containing deletions or mutations of the 14-3-3 binding motif. Alternatively

the movement of CaR from the ER following the knockdown of 14-3-3 θ could be

examined.

OS-9, which was shown to bind to residues 965-987 of the CaR-tail in Chapter 3, has

also been associated with the trafficking of the membrane protein, meprin β from the

ER to the Golgi (Litovchick et al. 2002), whereas Wang et al. has demonstrated that

OS-9 impedes the release of TRPV4 from the ER (Wang et al. 2007). Through its

interaction with OS-9, the CaR may be involved in either of these processes.

6.3.3 The Regulation of Calcium-Sensing Receptor Degradation by Interacting Proteins

OS-9 has also been associated with the degradation of proteins (Baek et al. 2005;

Mueller et al. 2008; Wang et al. 2007). As part of a ubiquitin ligase complex, OS-9 is

involved in the detection and delivery of terminally misfolded or unassembled

glycosylated proteins for degradation by the ERAD pathway (Christianson et al. 2008).

As the CaR is a glycosylated protein that is processed in the ER it is possible that

misfolded CaR is targeted for degradation by OS-9 via the ERAD pathway. Huang et al.

demonstrated that the dorfin-mediated degradation of immature CaR occurred as part of

a protein complex including the valosin-containing protein, suggesting that the observed

degradation was also via the ERAD pathway (Huang et al. 2006b). The binding sites of

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OS-9 and dorfin are distinct allowing for the possibility that they act in unison to target

the CaR for degradation. Coimmunoprecipitation studies could be helpful in revealing

whether OS-9 form part of a complex with the CaR and dorfin suggesting a role for OS-

9 in targeting CaR for ubiquitin mediated degradation. On the other hand, Christianson

et al. demonstrated that OS-9 in conjunction with the Hsp90 paralogue, GRP94, targets

aberrantly folded glycoproteins in the ER lumen via the Hrd1/Sel1 complex for

ubiquitination and proteasomal degradation. The possibility that the CaR represents a

target for OS-9 in this context could also be examined by appropriate

coimmunoprecipitation and subcellular localisation studies.

Experiments examining the oxygen-dependent ubiquitination and degradation of HIF-

1α found that this process was regulated by OS-9 (Baek et al. 2005). In contrast, OS-9

was shown to protect TPRV4 from ubiquitination and degradation (Wang et al. 2007).

Considering the disparity between the observed effects that interaction with OS-9 has

on degradation it is unclear what role, if any, OS-9 plays in CaR degradation. However,

yeast two-hybrid mapping studies have revealed that the site of OS-9 interaction in the

CaR overlaps that of filamin A binding. Since the interaction between the CaR and

filamin A protects the CaR against degradation (Zhang and Breitwieser 2005). It is

possible that filamin A, by competing with OS-9 for binding sites within the CaR-tail, is

able to prevent OS-9 from targeting the receptor for degradation.

6.3.4 Calcium-Sensing Receptor-Mediated Intracellular Signalling is Directed by

Interacting Proteins

As mentioned, the CaR can activate a wide variety of signalling pathways in a cell

specific manner (Ward 2004). For example, in fibroblasts, stimulation of the CaR

initiates the JNK cascade, but this does not occur in the MC3T3-E1 osteoblastic cell

line, (Ogata et al. 2006; Yamaguchi et al. 2000). Recently, a comparison between an

immortalised murine mammary cell line, Comma-D, and mammary epithelial cells

revealed that in the former cAMP production was increased by CaR activation but

inhibited in the latter (Mamillapalli et al. 2008). The difference in CaR signalling was

attributed to a difference in G protein coupling between the CaR in Comma-D cells,

which coupled to Gαs and the CaR in mammary epithelial cells, which coupled to Gαi

(Mamillapalli et al. 2008). This change in G protein coupling was also observed with

the β2 adrenoreceptor, where the change was attributed to the recruitment of PDE4

cAMP phosphodiesterase to the receptor by β-arrestins (Baillie et al. 2003). As β-

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arrestins also interact with the CaR, it is possible that they lead to G protein switching

associated with the CaR. In other members of the GPCR family C, the intracellular

loops have been shown to be crucial for G protein-coupling and Chang et al.

demonstrated that the second and third intracellular loops are important for CaR

signalling (Chang et al. 2000; Pin et al. 1994). Post-translational modifications of these

loops may determine which G proteins bind to the CaR. Both the second and third

intracellular loops of the CaR contain SUMOylation sites that are recognised by a CaR

binding partner identified in Chapter 3, Ubc9. SUMOylation of the tumour necrosis

factor receptors was shown to inhibit their apoptotic signalling (Okura et al. 1996). To

determine if Ubc9 is involved in the selection of G proteins that bind to the CaR, the

possibility of SUMOylation of the CaR must first be investigated.

Scaffold proteins of GPCRs associate with multiple signalling components, linking

them to the receptors. Both filamin and 14-3-3 proteins have both been characterised as

scaffolding proteins (Hall and Lefkowitz 2002). The interaction between the CaR and a

14-3-3 protein was first discovered as part of this study but the interaction between the

CaR and filamin A has already been examined in a number of studies. In relation to

signalling, the interaction with filamin A has been shown to be important for the CaR

activation of the ERK, JNK and Rho pathways (Awata et al. 2001; Hjalm et al. 2001;

Huang et al. 2006a; Pi et al. 2002; Rey et al. 2005). The signalling components JNKK,

from the JNK pathway and Rho A, from the Rho pathway, have been shown to bind to

residues within the filamin A repeats 21-23 and 24, respectively (Marti et al. 1997; Ohta

et al. 2006). Having identified multiple sites of CaR interaction within filamin A it was

proposed that the precise binding site of filamin A to which the CaR may bind could

dictate which signalling components are accessible and therefore, able to be activated.

The role of testin in CaR-mediated signalling was examined in Chapter 5. There was no

observed impact on CaR-induced ERK phosphorylation observed in HEK293-CaR cells

expressing testin. However, the overexpression of testin in HEK293-CaR cells was

found to enhance the induction of Rho signalling by the CaR as measured by the SRE-

luciferase assay. The knockdown of testin in HEK293-CaR cells resulted in a decrease

in Rho kinase activity at basal levels of CaR agonist. However, the Rho activity

detected in both HEK293-CaR cells and testin knockdown HEK293-CaR cells

following stimulation with extracellular calcium was not significantly different. This

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suggests that although testin is not essential for CaR-mediated Rho kinase activity, it

can act to enhance the response.

6.2.5 The Role of the Calcium-Sensing Receptor and its Binding Partners in Cell

Morphology and Organisation of the Cytoskeleton

Studies have demonstrated that the CaR is involved in changes to cell morphology and

rearrangement of the actin cytoskeleton (Bouschet et al. 2007; Davies et al. 2006;

Procino et al. 2004). Stimulation of the CaR resulted in an increase in forskolin-induced

actin stress fibre assemby that was associated with a decrease in cAMP accumulation in

a cell line derived from kidney cortical collecting duct cells (Procino et al. 2004).

Swaney et al. showed in fibroblasts that increases in cAMP accumulation inhibit the

forskolin-mediated formation of focal adhesions and actin stress fibres (Swaney et al.

2006). CaR stimulation by divalent cations and calcimimetics, but not amino acids,

induced cell morphological changes and actin stress fibre assembly that was linked to

Rho kinase activation in HEK293-CaR cells (Davies et al. 2006). In addition to

replicating the findings of Davies et al. in Chapter 5, it was revealed that stimulation of

the CaR in HEK293-CaR cells with extracellular magnesium also induced an increase

in focal adhesion formation (Figure 5.9), as might be predicted since focal adhesions are

anchorage points of actin stress fibres.

Recently, CaR activity was shown to induce plasma membrane ruffling, a process that

involves the formation and retraction of protrusions enriched with filamentous actin,

which required the interaction with a complex containing β-arrestin-1 (Bouschet et al.

2007). Several of the CaR binding partners have also been associated with changes in

cell morphology and cytoskeletal organisation, including filamin A, 14-3-3 proteins and

testin (Birkenfeld et al. 2003; Griffith et al. 2005; Ohta et al. 2006; Vardouli et al.

2005). As a significant portion of the identified CaR interacting proteins are associated

with alterations of cell morphology and cytoskeletal structure, the coordination of their

binding to the CaR-tail may modulate the CaR’s regulation of these processes.

Studies examining the possible relationship between CaR-mediated changes in cell

morphology and cytoskeletal structure and the receptor’s interaction with testin were

conducted using testin knockdown HEK293-CaR cells. Preliminary results have shown

that the cell morphology and cytoskeletal structure observed in non-stimulated testin

knockdown HEK293-CaR cells, mimicked the effect seen in CaR agonist stimulated

146

HEK293-CaR cells. This is potentially an interesting finding but needs to be confirmed

with additional control studies.

6.4 Future Studies

6.4.1 Filamin A

The data presented in Chapter 4 indicates that the CaR can bind to three distinct sites

within filamin A, which contain a highly homologous region of 40 amino acids. Further

mapping studies would be required to more precisely delineate the CaR sites of

interaction within this 40 residue region and/or potentially other regions.

Coimmunprecipitation experiments with a filamin A construct with these three CaR

interacting regions deleted might reveal if this collective loss can be compensated for by

additional regions in filamin A. The hypothesis that filamin A acts as a clamp to

stabilise the CaR homodimer, could be tested by expressing in M2 cells (which do not

express endogenous filamin A) mutant filamin A constructs in which all but one of the

CaR binding sites have been disrupted. It would be predicted that the remaining CaR

binding site should still be sufficient for interaction between a single CaR and filamin A

but without the capacity to bind to two CaRs, the proposed stabilising effect should be

lost. To examine this, Western analysis could be performed to determine if there was

diminished CaR expression in M2 cells cotransfected with the CaR and mutant filamin

A in comparison to the expression of CaR in M2 cells cotransfected with CaR and wild-

type filamin A. The functional importance of the novel CaR-filamin interaction sites on

CaR-mediated processes such as ERK activation could also be examined following the

further delineation of the binding sites in both filamin A, as described above, and the

CaR. If the novel filamin binding domains interact at differential sites within the 923-

986 region of the CaR, then the use of specific filamin-TAT peptides (Hjalm et al.

2001) could be used to block interaction sites and demonstrate the roles of these novel

sites of interaction have in relation to CaR-mediated processes.

6.4.2 Filamin B

In the yeast two-hybrid studies presented in Chapter 3, filamin B was also shown to

bind to the CaR-tail, albeit with a lower binding affinity. Filamin A and B share

approximately 70% homology and are structurally similar proteins (Popowicz et al.

2006). However, several experiments performed in M2 cells that express filamin B but

not filamin A, revealed a role for filamin A in CaR signalling and degradation that was

apparently not compensated for by filamin B (Awata et al. 2001; Zhang and Breitwieser

147

2005). A better understanding of the possible relationship between filamin B and the

CaR requires verification of their interaction by pulldown studies as described in

Chapter 4. Further studies, following the confirmation of the interaction, could examine

the affect of filamin B overexpression and knockdown on various CaR signalling

pathways, including those known to be influenced by filamin A, such as the ERK and

Rho pathways.

6.4.3 Testin

In order to obtain more definitive data from the studies examining CaR-mediated

cellular changes, the methodology will be expanded to include the collection of a

greater number of images of treated and untreated cells. With the increase in number of

cell images it will be possible to use computer software (e.g. Adobe Photoshop) to

perform morphometric analysis on the cells and generate quantitative and statistically

relevant data.

Initial studies performed using testin knockdown HEK293-CaR cells revealed that, in

the presence of basal levels of CaR agonist, these cells exhibited cell morphology and a

cytoskeletal structure that mimicked that observed in stimulated HEK293-CaR cells.

Confirmatory and control studies are necessary to place the significance of these

preliminary results in proper context. To accomplish this, it will be necessary to

determine if the increase in actin stress fibre assembly and focal adhesion formation

observed in the testin knockdown HEK293-CaR cells is specifically CaR-mediated, by

generating and comparing them to testin knockdown CaR-negative HEK293 cells. In

addition, the observed effects of shRA testin knockdown in HEK293-CaR cells must be

evaluated against mock shRNA treated HEK293-CaR cells. To elucidate whether Rho

signalling is necessary for the morphological and cytoskeletal changes observed in

testin knockdown HEK293-CaR cells, experiments inhibiting Rho A and the Rho kinase

must be performed.

The apparent contrasting effects of testin knockdown on actin stress fibre assembly in

HEK293-CaR cells and HeLa cells may well be a cell specific effect, as HEK293-CaR

cells do not contain stress actin fibres at basal levels of CaR agonist but HeLa cells do.

As the knockdown of testin in HeLa cells results in the loss of actin stress fibre

formation it would be interesting to see if CaR agonist stimulation of testin knockdown

HeLa cells overexpressing the CaR would restore actin stress fibre assembly. Also of

148

interest is the cell morphology and cytoskeletal structure present in embryonic

fibroblasts in testin knockout mice compared to embryonic fibroblasts from normal

mice, a resource that has recently been made available. The morphological and

cytokeletal characteristics of the testin negative mouse embryonic fibroblasts could then

be examined in relation to the overexpression and stimulation of the CaR.

Aside from the organisation of the cytoskeleton, other processes where the function of

the CaR and testin overlap include cell motility and cell adhesion. In chicken embryo

fibroblasts the overexpression of testin resulted in a reduction in cell motility (Griffith et

al. 2004). Experiments performed in Boyden’s chambers, have recently revealed that

increasing the concentration of extracellular calcium triggered the migration of MDA

MB 231 cells via the ERK and PLC pathways (Mentaverri et al. 2007). If testin plays a

role in CaR-mediated cell motility, then the overexpression of testin in MDA MB 231

cells should inhibit CaR-induced cell migration. This hypothesis could be tested by

repeating the experiments using the Boyden’s chamber and observing the effects of

testin knockdown and overexpression on CaR-mediated MDA MB 231 cell motility.

Comparative studies with another breast cancer cell line, T47D, which does not express

endogenous testin, could also be highly instructive.

While the effects of testin interaction on CaR-mediated ERK and Rho signalling have

been examined, there are other CaR-induced pathways that may be influenced by the

binding of testin, including the PLC and inhibitory cAMP pathways. Investigating the

impact of the interaction between the CaR and testin on additional signalling pathways

will provide further insight into how the multiple accessory proteins that bind to the

intracellular tail of the CaR regulate the signalling of the receptor.

6.5 Conclusions

Regulation of the CaR’s response to its multitude of stimuli in a cell specific manner is

a key element of the receptor’s role in the body. Like other GPCRs, an important

mechanism for this regulation relate to the accessory proteins that bind to its

intracellular tail. Six novel binding partners of the CaR-tail were identified as part of

this study using the yeast two-hybrid system. The identified CaR interacting proteins

have been associated with a diverse range of functions and additional investigations into

their relationship with the CaR will provide further insight into the functionality of the

receptor. Combining the data from yeast two-hybrid mapping studies presented in this

149

thesis and the known interaction domains of previously identified CaR binding partners,

has revealed two regions of the CaR intracellular tail, 865-922 and 965-986, that appear

to be essential for the interaction of accessory proteins.

Filamin A was the seventh CaR interacting protein identified in the yeast two-hybrid

library screen. Previous studies of the interaction between filamin A and the CaR

suggest that filamin A acts as a scaffolding protein that recruits accessory proteins to the

intracellular tail in order facilitate CaR-mediated signalling. Two additional sites of

CaR interaction within filamin A that may allow it to stabilise the CaR dimer and/or act

as a more versatile scaffolding protein were identified in this study.

The relationship between a novel CaR binding partner, testin, was selected for further

investigation based on the overlap in biological function between the two proteins. The

interaction with testin, while not essential, was found to enhance CaR-mediated Rho

signalling. Preliminary data indicates that testin may be involved in the CaR’s

regulation of changes to cell morphology and cytoskeletal structure.

150

148

((HHaavveenn eett aall.. 22000044;; KKiiffoorr eett aall.. 11999966;; LLaavvaalllliiee eett aall.. 11999933;; LLiinn eett aall.. 11999988;; MMaarrttii eett aall.. 11999977;; MMeennttaavveerrrrii eett aall.. 22000077;; OOggaattaa eett aall.. 22000066;; OOhhttaa eett aall.. 22000066;; OOkkuurraa eett aall.. 11999966;; PPiinn eett aall.. 11999944;;

PPrroocciinnoo eett aall.. 22000044;; SSccootttt eett aall.. 22000066;; SSwwaanneeyy eett aall.. 22000066;; VVaarrddoouullii eett aall.. 22000055)) ((AArrddeesshhiirrppoouurr eett aall.. 22000066;; AAssllaannoovvaa eett aall.. 22000066;; AAwwaattaa eett aall.. 22000011;; BBaa aanndd FFrriieeddmmaann 22000044;; BBaaii 22000044;; BBaaii eett aall.. 11999977;; BBaaii eett aall.. 11999966;; BBaaii eett aall.. 11999988aa;; BBaaii eett aall.. 11999999;; BBaaii eett aall.. 11999988bb;; BBaannddyyooppaaddhhyyaayy eett aall.. 22000077;; BBiikkllee eett aall.. 11999966;; BBoocckkaaeerrtt aanndd PPiinn 11999999;; BBoosseell eett aall.. 22000033;; BBoouusscchheett eett aall.. 22000055;; BBrraauunneerr--OOssbboorrnnee eett aall.. 11999999;; BBrraauunneerr--OOssbboorrnnee eett aall.. 22000077;; BBrroowwnn eett aall.. 11998877;; BBrroowwnn eett aall.. 11999933;; BBrroowwnn aanndd HHeebbeerrtt 11999977;; BBrroowwnn eett aall.. 11999944;; BBrroowwnn 11999999;; BBrroowwnn 22000000;; BBrroowwnn eett aall.. 11999911aa;; BBrroowwnn eett aall.. 11999911bb;; BBrroowwnn aanndd MMaaccLLeeoodd 22000011;; BBuucchhaann eett aall.. 22000011;; BBuutttteerrss eett aall.. 11999977;; CCaarraaffoollii 22000033;; CCaarree eett aall.. 11996666;; CCaarrlliinngg eett aall.. 22000000;; CCaarrrriilllloo--LLooppeezz eett aall.. 22000088;; CChhaakkrraabbaarrttyy eett aall.. 22000033;; CChhaanngg eett aall.. 22000011;; CChhaanngg aanndd SShhoobbaacckk 22000044;; CChhaanngg eett aall.. 22000000;; CChhaanngg eett aall.. 11999988;; CChhaanngg eett aall.. 22000077;; CChhaanngg eett aall.. 11999999;; CChhaattttooppaaddhhyyaayy eett aall.. 11999988aa;; CChhaattttooppaaddhhyyaayy eett aall.. 22000000;; CChhaattttooppaaddhhyyaayy eett aall.. 11999977;; CChhaattttooppaaddhhyyaayy eett aall.. 22000044;; CChhaattttooppaaddhhyyaayy eett aall.. 11999988bb;; CChhaattttooppaaddhhyyaayy eett aall.. 11999999;; CChheenn eett aall.. 11998899;; CChheenn aanndd GGooooddmmaann 22000044;; CChheenngg eett aall.. 11999977;; CChheenngg eett aall.. 11999988;; CChheenngg eett aall.. 22000044;; CChheenngg eett aall.. 22000022;; CChhiikkaattssuu eett aall.. 22000000;; CCiimmaa eett aall.. 11999977;; CCoonniiggrraavvee eett aall.. 22000000;; DDaauubb eett aall.. 11999966;; DDaavviieess eett aall.. 22000077;; DDeetthhlleeffsseenn eett aall.. 11999988;; DDeeWWiirree eett aall.. 22000077;; DDiiaazz eett aall.. 11999977;; DDuuffnneerr eett aall.. 22000055))((DDvvoorraakk eett aall.. 22000044;; EEmmaannuueell eett aall.. 11999966;; EEvveenneeppooeell 22000088;; FFaann eett aall.. 11999977;; FFaann eett aall.. 11999988;; FFaarrnneebboo eett aall.. 11999988;; FFeerrrreeiirraa aanndd BBaaiillllyy 11999988;; FFeerrrreeiirraa eett aall.. 11999988;; FFeerrrryy eett aall.. 22000000;; FFrreeddrriikkssssoonn eett aall.. 22000033;; GGaammaa aanndd BBrreeiittwwiieesseerr 11999988;; GGaammaa eett aall.. 22000011;; GGaarrrreetttt eett aall.. 11999955;; GGoollddssmmiitthh eett aall.. 11999999;; HHaammmmeerrllaanndd eett aall.. 11999999;; HHaannddllooggtteenn eett aall.. 22000011;; HHaauuaacchhee 22000011;; HHaauuaacchhee eett aall.. 22000000;; HHeeaatthh eett aall.. 11999966;; HHeebbeerrtt eett aall.. 22000044;; HHeebbeerrtt eett aall.. 11999966;; HHeennddyy eett aall.. 22000000;; HHeerrrreerraa--VViiggeennoorr eett aall.. 22000066;; HHjjaallmm eett aall.. 22000011;; HHoobbssoonn eett aall.. 22000000;; HHoobbssoonn eett aall.. 22000033;; HHooffeerr aanndd BBrroowwnn 22000033;; HHooffff eett aall.. 11999999;; HHuu eett aall.. 22000000;; HHuu eett aall.. 22000066;; HHuu eett aall.. 22000055;; HHuu eett aall.. 22000022;; HHuu aanndd SSppiieeggeell 22000033;; HHuu aanndd SSppiieeggeell 22000077;; HHuuaanngg aanndd MMiilllleerr 22000077;; HHuuaanngg eett aall.. 22000077aa;; HHuuaanngg eett aall.. 22000044;; HHuuaanngg eett aall.. 22000066aa;; HHuuaanngg eett aall.. 22000066bb;; HHuuaanngg eett aall.. 22000077bb;; JJaanniicciicc eett aall.. 11999955aa;; JJaanniicciicc eett aall.. 11999955bb;; JJiiaanngg eett aall.. 22000022;; JJuussttiinniicchh eett aall.. 22000088))((KKaallllaayy eett aall.. 11999977;; KKaammeeddaa eett aall.. 11999988;; KKiiffoorr aanndd BBrroowwnn 11998888;; KKiiffoorr eett aall.. 11999977;; KKiiffoorr eett aall.. 11999988;; KKiiffoorr eett aall.. 22000033;; KKiiffoorr eett aall.. 22000011;; KKiirrcchhhhooffff aanndd GGeeiibbeell 22000066;; KKoommuuvveess eett aall.. 22000022;; KKuunniisshhiimmaa eett aall.. 22000000;; LLaattrroonniiccoo eett aall.. 11999988;; LLii eett aall.. 22000055;; LLiieennhhaarrddtt eett aall.. 22000000;; LLooppeezz--BBaarrnneeoo aanndd AArrmmssttrroonngg 11998833;; LLoorreennzz eett aall.. 22000077;; LLoorreettzz 22000088;; LLoorreettzz eett aall.. 22000044;; LLoouurrddeell eett aall.. 22000022;; MMaaccLLeeoodd eett aall.. 22000044;; MMaaiittii eett aall.. 22000088;; MMaallaarrkkeeyy eett aall.. 11999955;; MMaammiillllaappaallllii eett aall.. 22000088;; MMccCCuulllloouugghh eett aall.. 22000044;; MMccLLaarrnnoonn eett aall.. 22000022;; MMccNNeeiill eett aall.. 11999988;; MMeennttaavveerrrrii eett aall.. 22000066;; MMiieeddlliicchh eett aall.. 22000044;; MMoorrffiiss eett aall.. 22000033;; MMoottooyyaammaa aanndd FFrriieeddmmaann 22000022;; MMuunn eett aall.. 22000055;; MMuutthhaalliiff eett aall.. 11999966;; NNeemmeetthh eett aall.. 22000011;; NNeemmeetthh eett aall.. 11999988;; NNeemmeetthh aanndd CCaarraaffoollii 11999900;; NNiiwwaa eett aall.. 22000011;; OOddaa eett aall.. 22000000;; OOggaattaa eett aall.. 22000066;; PPaaccee eett aall.. 11999999;; PPaarrmmeennttiieerr eett aall.. 22000022;; PPeeaarrccee eett aall.. 11999966;; PPeeiirriiss eett aall.. 22000077;; PPeettrreell eett aall.. 22000044))((PPii eett aall.. 22000055;; PPii eett aall.. 22000022;; PPiiddaasshheevvaa eett aall.. 22000066;; PPiinn eett aall.. 22000033;; PPoollllaakk eett aall.. 11999933;; PPrreennzzeell eett aall.. 11999999;; PPrroocciinnoo eett aall.. 22000044;; PPuurrkkiissss aanndd BBooaarrddeerr 11999922;; QQuuaarrlleess 22000033;; QQuuiinnnn eett aall.. 22000044;; QQuuiinnnn eett aall.. 11999988;; QQuuiinnnn eett aall.. 11999977;; RRaacckkee aanndd NNeemmeetthh 11999933aa;; RRaacckkee aanndd NNeemmeetthh 11999933bb;; RRaayy eett aall.. 22000077;; RRaayy eett aall.. 11999988;; RRaayy eett aall.. 11999977;; RRaayy eett aall.. 22000044;; RRaayy eett aall.. 11999999;; RReeyy eett aall.. 22000055;; RReeyyeess--CCrruuzz eett aall.. 22000011;; RReeyyeess--IIbbaarrrraa eett aall.. 22000077;; RRiiccccaarrddii 22000022;; RRiiccccaarrddii eett aall.. 11999988;; RRiiccccaarrddii eett aall.. 11999955;; RRooddllaanndd 22000044;; RRooggeerrss eett aall.. 11999977;; RRuuaatt eett aall.. 11999955;; RRuuaatt eett aall.. 11999966;; RRuutttteenn eett aall.. 11999999;; SSaannddeerrss eett aall.. 22000000;; SShheerrwwoooodd eett aall.. 11996666;; SShhoobbaacckk eett aall.. 11998833;; SSiillvvee eett aall.. 22000055;; SSkkeellllyy aanndd FFrraannkklliinn 22000077;; SSuuppaattttaappoonnee eett aall.. 11998888;; TTffeelltt--HHaannsseenn aanndd BBrroowwnn 22000055;; TTffeelltt--HHaannsseenn eett aall.. 22000033;; TThhaakkkkeerr 22000044;; TTrriivveeddii eett aall.. 22000088;; TTuu eett aall.. 22000088;; TTuurrkksseenn aanndd TTrrooyy 22000033;; VVaannHHoouutteenn eett aall.. 22000044;; VVaassssiilleevv eett aall.. 11999977;; VViizzaarrdd eett aall.. 22000088))((AAddaammss eett aall.. 22000066;; AAllkkeemmaa eett aall.. 11999977;; BBaaeekk eett aall.. 22000055;; BBeerrnnaassccoonnii eett aall.. 22000088;; BBeerrrryy eett aall.. 22000055;; BBhhaammiiddiippaattii eett aall.. 22000055;; BBoocckkaaeerrtt eett aall.. 22000033;; BBoocckkaaeerrtt eett aall.. 22000044;; BBoouusscchheett eett aall.. 22000077;; CChhrriissttiiaannssoonn eett aall.. 22000088;; CCoobblliittzz eett aall.. 22000066;; CCoolllleecc eett aall.. 22000077;; CCoouuttttss eett aall.. 22000033;; DDaavviieess eett aall.. 22000066;; DDiinnggwweellll aanndd SSmmiitthh 22000066;; DDiivveecchhaa aanndd

149

CChhaarrlleessttoonn 11999955;; DDoohhmmeenn 22000044;; DDrruueekkee 22000066;; DDrruussccoo eett aall.. 22000055;; FFeenngg eett aall.. 22000055;; FFiieellddss aanndd SSoonngg 11998899;; FFrriieeddmmaannnn eett aall.. 22000022;; WWaanngg eett aall.. 11999966aa;; WWaarrdd eett aall.. 22000044;; WWaarrdd eett aall.. 22000066;; WWaarrdd 22000044;; WWaarrdd eett aall.. 11999988;; WWaasshhbbuurrnn eett aall.. 22000000aa;; WWaasshhbbuurrnn eett aall.. 22000000bb;; WWeessss 11999977;; WWoojjcciikkiieewwiicczz 22000044;; YYaammaagguucchhii eett aall.. 11999988;; YYaammaagguucchhii eett aall.. 22000000;; YYaanngg eett aall.. 11996677;; YYaannoo eett aall.. 22000044aa;; YYaannoo eett aall.. 22000044bb;; YYee eett aall.. 11999977aa;; YYee eett aall.. 11999977bb;; YYuu eett aall.. 22000044;; ZZhhaanngg aanndd BBrreeiittwwiieesseerr 22000055;; ZZhhaanngg eett aall.. 22000022;; ZZhhaanngg eett aall.. 22000011;; ZZhhaaoo eett aall.. 11999999))((CCaarrrreelllloo eett aall.. 11999999;; GGaarrvvaalloovv eett aall.. 22000033;; GGaauussss eett aall.. 22000066;; GGoorrlliinn eett aall.. 11999900;; GGrriiffffiitthh eett aall.. 22000044;; GGrriiffffiitthh eett aall.. 22000055;; GGuubbbb eett aall.. 11999999;; HHaarrttwwiigg aanndd SSttoosssseell 11997755;; KKaaggeeyy eett aall.. 22000033;; KKiimm eett aall.. 22000055;; KKiimmuurraa eett aall.. 11999977;; KKiimmuurraa eett aall.. 11999988;; LLiittoovvcchhiicckk eett aall.. 22000022;; LLiiuu eett aall.. 11999977;; MMaacckkiinnttoosshh 22000044;; MMccNNaammaarraa aanndd DDoonnnneennbbeerrgg 11999988;; MMeellcchhiioorr 22000000;; MMuueelllleerr eett aall.. 22000088;; NNaakkaayyaammaa eett aall.. 11999999;; NNiieellsseenn 11999911;; PPaatteell eett aall.. 22000066;; PPooppoowwiicczz eett aall.. 22000066;; RRooddrriigguueezz eett aall.. 22000011;; RRootttteerr eett aall.. 22000055;; SSaammppssoonn eett aall.. 22000011;; SSaarrttii eett aall.. 22000055;; SSaattiijjnn eett aall.. 11999977;; SScchhwwaarrttzz aanndd PPiirrrroottttaa 22000077;; SShhiikkaannoo eett aall.. 22000055;; SShhiikkaannoo eett aall.. 22000066;; SSttoosssseell eett aall.. 22000011;; SSuu eett aall.. 11999966;; SSuu eett aall.. 11999944;; SSzzaatthhmmaarryy eett aall.. 22000055;; TTaakkaaffuuttaa eett aall.. 11999988;; TTaasshhiirroo eett aall.. 11999977;; TTaattaarreellllii eett aall.. 22000000;; TToobbiiaass eett aall.. 22000011;; TToonngg eett aall.. 11999977;; vvaann ddeerr FFlliieerr aanndd SSoonnnneennbbeerrgg 22000011;; vvaann ddeerr VVeenn eett aall.. 22000000;; vvaann HHeemmeerrtt eett aall.. 22000011;; VVoouurrvvoouuhhaakkii eett aall.. 22000077;; WWaanngg aanndd SShhaakkeess 11999966;; WWaanngg eett aall.. 11999966bb;; WWaanngg eett aall.. 22000077;; YYaaffffee eett aall.. 11999977;; YYaanngg eett aall.. 22000066;; ZZhhaaoo 22000077;; ZZhheenngg aanndd ZZhhaaoo 22000077))((GGoooossnneeyy eett aall.. 22000011;; HHoouussee eett aall.. 11999977;; HHuunntteerr eett aall.. 22000055;; IInngglleeyy eett aall.. 22000000;; JJoohhnnssoonn 22000044;; JJoohhnnssoonn aanndd BBlloobbeell 11999977;; LLiimm eett aall.. 22000022;; TTssaaii eett aall.. 11999944;; vvaann HHeemmeerrtt eett aall.. 22000011;; WWaanngg eett aall.. 11999966bb;; ZZhhaanngg eett aall.. 22000066)) ((BBaaiilllliiee eett aall.. 22000033;; BBeekkmmaann aanndd HHeennrriiqquuee 22000022;; BBhhaaggaavvaatthhuullaa eett aall.. 22000077;; BBhhaaggaavvaatthhuullaa eett aall.. 22000055;; BBiirrkkeennffeelldd eett aall.. 22000033;; BBrreeiittwwiieesseerr eett aall.. 22000044;; CCoouuttttss eett aall.. 22000033;; GGllooggaauueerr eett aall.. 11999988;; HHaallll aanndd LLeeffkkoowwiittzz 22000022;; KKiieemmaa eett aall.. 22000066;; MMaacchheesskkyy aanndd HHaallll 11999966;; MMuunn eett aall.. 22000044;; PPeelllleeggrriinn aanndd MMeelllloorr 22000077;; SSeecckk eett aall.. 22000033;; SShheeeenn eett aall.. 22000022;; SSuunn eett aall.. 22000066;; TTaakkaaffuuttaa eett aall.. 11999988;; TTffeelltt--HHaannsseenn 22000088;; TTiiggggeess eett aall.. 22000033;; WWaanngg eett aall.. 22000044;; WWuu eett aall.. 22000055))

((RReenn eett aall.. 11999999))

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Appendix 1: Oligonucleotides

Primer Sequence

CaRCTF 5' GCC ATA TGT CCC GCA ACA CC 3'

CaRCTR 5' CGT CGA CTT ATG AAT TCA CTA CG 3'

CaR923F 5' GCC ATA TGC CAT TCC CAC AGC C 3'

CaR965F 5' GCC ATA TGA AGG TCA TCT TTG G 3'

CaR987F 5' GCC ATA TGA TGG CCC ACG GGA ATT C 3'

CaR997R 5' CGT CGA CTT AGG AGT TCT GGT GCG 3'

TesF 5' GGA TCC ATG GAC CTG GAA AAC AAA GTG 3'

TesR 5' GAG CTC CTA AGA CAT CCT CTT CTT ACA TTC 3'

Tes476F 5' GTG GGC TCC TCC TGT CCA G 3'

M13F 5' GTT GTA AAA CGA CGG CCA GT 3'

923SalIF 5' CGT CGA CCT CCA TTC CCA CAG CCC 3'

965SalIF 5' CGT CGA CCT AAG GTC ATC TTT GGC 3'

987SalIF 5' CGT CGA CCT ATG GCC CAC GGG AAT TC 3'

CaRT898R 5' CGT CGA CCT ACC GCT TGC GGG AGA CGT TGC 3'

CaRT899F 5' CGT CGA CCT TCC AGC AGC CTT GGA GGC 3'

Fil36F 5' CGA ATT CCT GAC CAT TGA GAT CTG CTC GG 3'

Fil36R 5' GCT CGA GCT AAC GGT CCT GAA CGT AGG TCT CCG 3'

FilBF 5' CGG ATC CAG CCC ATC GGG CAA GAC CCA TG 3'

FilBR 5' GCT CGA GCT AGA AGG GGC TGT CGG GAA TGT G 3'

Tes(91-109)F 5' GGA TTC GAA CTG CAC TTC T 3'

Tes(91-109)R 5' AGA AGT GCA GTT CGA ATC C 3'

Tes(C271A)F 5' CTG CAG CAC CGC TGG TGA ACT CTG GTC GAC 3'

Tes(C271A)R 5' GTC GAC CAG GAG TTC ACC AGC GGT GCT GCA G 3'

Tes(H292A)F 5' GAA GCT GTA CTG TGG CAG AGC TTA CTG TGA CAG TGA

G 3'

Tes(H292A)R 5' CTC ACT GTC ACA GTA AGC TCT GCC ACA GTA CAG CTT

C 3'

Tes(ELL)F 5' GTT TTA TCT GCA GCA CCT GTG GTG CAG CCG CGG TCG

AC 3'

Tes(ELL)R 5' GTC GAC CGC GGC TGC ACC ACA GGT GCT GCA GAT AAA

AC 3'

172

Tes(VD)F 5' GCA CCT GTG GTG AAC TCC TGG CCG CCA TGA TTT ACT

TC 3'

Tes(VD)R 5' GAA GTA AAT CAT GGC GGC CAG GAG TTC ACC ACA GGT

GC 3'

Tes(M)F 5' GAA CTC CTG GTC GAC GCG ATT TAC TTC TGG AAG AAT

G 3'

Tes(M)R 5' CAT TCT TCC AGA AGT AAA TCG CGT CGA CCA GGA GTT

C 3'

Tes(IYF)F 5' GGT CGA CAT GGC TGC CGC CTG GAA GAA TGG GAA GCT

G 3'

Tes(IYF)R 5' CAG CTT CCC ATT CTT CCA GGC GGC AGC CAT GTC GAC

C 3'

Tes(WKN)F 5' CAT GAT TTA CTT CGC GGC GGC TGG GAA GCT GTA CTG 3'

Tes(WKN)R 5' CAG TAC AGC TTC CCA GCC GCC GCG AAG TAA ATC ATG 3'

Tes(GK)F 5' CTT CTG GAA GAA TGC GGC GCT GTA CTG TGG CAG 3'

Tes(GK)R 5' CTG CCA CAG TAC AGC GCC GCA TTC TTC CAG AAG 3'

Tes(LY)F 5' GAT TTA CTT CTG GAA GAA TGG GAA GGC GGC CTG TGG

CAG 3'

Tes(LY)R 5' CTG CCA CAG GCC GCC TTC CCA TTC TTC CAG AAG TAA

ATC 3'

VP16-2 5' GAG TTT GAG CAG ATG TTT ACC G 3'

173

Appendix 2: Anitbodies and Western Blotting Conditions

Antibody Blocking

Conditions (Buffer/Incubation)

Primary Antibody

Conditions (Dilution -

Buffer - Incubation)

First Washing

Conditions

Secondary Antibody



Final Washing

Conditions

FLAG Antibody

3% skim milk powder in TBS

30 minutes at room temperature

1 in 10,000 FLAG

Antibody 3% Skim

Milk Powder in TBS

30 minutes at room

temperature

3 times in TBST for 5 minutes

1 in 10,000 goat anti-

mouse 3% Skim

Milk Powder in TBS

30 minutes at room

temperature


ERK Antibody

5% skim milk powder in PBST 1 hour at room

temperature

1 in 10,000 ERK

antibody 5% Skim

Milk Powder in PBST 1 hour at

room temperature

3 times in PBST for 5 minutes

1 in 10,000 Goat Anti-

Rabbit 5% Skim


Room Temperature


P-ERK Antibody

5% skim milk powder in PBST 1 hour at room

temperature

1 in 2,000 P-ERK

antibody 5% Skim


room temperature



Rabbit 5% Skim


Room Temperature


GFP Antibody

5% skim milk powder in TBS 1

hour at room temperature

1 in 1,000 EGFP

Antibody 3% Skim

Milk Powder in TBS

30 minutes at room

temperature



Rabbit 5% Skim

Milk Powder in TBST 1 hour at

Room Temperature


Testin Antibody



1 in 5,000 Tesin

antibody 5% Skim

Milk Powder in TBS

1 hour at room

temperature



Rabbit 5% Skim

Milk Powder in TBS

1 hour at Room

Temperature


His Antibody

5% skim milk powder in PBS 1


1 in 5,000 His antibody

5% Skim Milk Powder

in PBS 1 hour at

room temperature



Mouse 5% Skim

Milk Powder in PBS

1 hour at Room

Temperature


174

Antibody Blocking

Conditions (Buffer/Incubation)

Primary Antibody



First Washing

Conditions

Secondary Antibody



Final Washing

Conditions

α-Tubulin



1 in 5,000 α-Tubulin

Antibody 3% Skim

Milk Powder in TBS

1 hour at room

temperature


1 in 10,000 goat anti-

mouse 3% Skim

Milk Powder in TBS

1 hour at room

temperature

3 times in TBST for

10 minutes

175

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Proteins Associated with the Intracellular Signalling Tail of the ...€¦ · Proteins Associated with the Intracellular Signalling Tail of the Calcium-Sensing Receptor and Their