Characterization of Novel Glucagon Receptor Interactors

that Modify Receptor Activity

by

Sean Froese

A thesis submitted in conformity with the requirements for the degree of

Master of Science

Department of Physiology

University of Toronto

© Copyright by Sean Froese 2015

ii

Characterization of Novel Glucagon Receptor Interactors that

Modify Receptor Activity

Sean Froese

Master of Science

Department of Physiology

University of Toronto

2015

Abstract

Glucagon helps maintain blood glucose homeostasis by stimulating gluconeogenesis and

glycogenolysis. Elevated glucagon levels have been reported in type 2 diabetics, and may be in

part responsible for their abnormally high blood glucose. A comprehensive understanding of

glucagon receptor regulators is currently unavailable. Members of my lab performed a mass

spectrometry screen to detect glucagon receptor interactors, 5 of which were validated to be true

interactors by co-immunoprecipitation and Western blotting. The project outlined in this thesis

began with the functional characterization of these interactors. Two interactors, low-density

lipoprotein receptor (LDLR) and transmembrane emp24 domain trafficking protein 2 (TMED2)

significantly enhanced glucagon stimulated glucose production, while tyrosine 3-

monooxygenase/tryptophan 5-monooxygenase activation protein, beta (YWHAB) lowered

glucagon stimulated glucose production in primary hepatocytes. Because of YWHAB’s ability to

lower glucose production, the underlying mechanism was explored and evidence suggests

YWHAB enhances endocytosis of the glucagon receptor into the cell following glucagon

stimulation.

iii

Acknowledgments

This thesis would not have been possible on my own. The support and guidance from everyone in

the lab, my family, and my friends, has been invaluable throughout these past two years.

I would like to thank first and foremost Dr. Wheeler for taking a chance on an inexperienced

undergraduate two and a half years ago, for his guidance and insights, and his constant patience

during my time in the lab. I have learned more about science, and myself, in these past two years

than any other time and my life, and this is in no small part due to Dr. Wheeler.

Thank you to my committee members, Drs. Adeli, Brubaker and Heximer. Your patience with

planning committee meetings and willingness to accommodate my schedule was incredibly

appreciated. More importantly, your insights and challenges were highly motivating and incredibly

valuable to my education.

I would like to thank all of my co-authors, particularly Junfeng Han and Ming Zhang whose work

formed the basis for this thesis. I would also like to thank my fellow lab members for all of their

amazing work their constant helpful advice and proofing. Special mention to Kacey Prentice and

Andrea Eversley for listening to me vent for hours on end. Your scientific insight and friendship

has been vital for my education, and my sanity.

I would like to thank my girlfriend Sabrina Parrotta for her unwavering support, her constant

encouragement, and her understanding when I can’t go to the movies because my cells need to be

split.

Last, I would like to thank my family, especially my parents Patricia and Daryl Froese for their

encouragement, support, and dedication to providing me with an education many would envy.

Most say they would do anything for their children, my parents have proved it.

iv

Table of Contents

Abstract……………………………………………………………………………………..........ii

Acknowledgments……………………………………………………………………………….iii

Table of Contents………………………………………………………………………………..iv

List of Figures………………………………………………………………………………......vii

Abbreviations………………………………………………………...…………...…………......ix

Chapter 1: General Introduction

1.1 Glucagon

1.1.1 Blood glucose homeostasis………………………………………………………………1

1.1.2 Glucagon expression and processing……………………………………………………2

1.1.3 Secretion…………………………………………………………………………………2

1.1.4 The effects of glucagon

1.1.4.1 Stimulatory effects…………………………………………………………………...5

1.1.4.2 Inhibitory effects……………………………………………………………………..6

1.2 Glucagon Receptor

1.2.1 Regulation of receptor expression………………………………………………………..6

1.2.2 Structure………………………………………………………………………………….7

1.2.3 Signalling

1.2.3.1 Activation and transduction…………………………………………………………..8

1.2.3.2 Glucagon receptor regulation………………………………………………………....9

1.3 Type 2 Diabetes and the Role of Glucagon

1.3.1 Pathogenic glucagon secretion………………………………………………………….11

1.3.2 Glucagon receptor antagonists………………………………………………………….12

1.4 Receptor Interactomes

1.4.1 Effects of accessory proteins on G-protein coupled receptors…………………………..13

1.4.2 Experimental approaches to receptor interactome identification

1.4.2.1 Membrane yeast two hybrid system…………………………………………………15

1.4.2.2 Fluorescence resonance energy transfer……………………………………………..16

v

1.4.2.3 Mass spectrometry…………………………………………………………………..16

1.5 Rationale and Hypothesis………………………………………………………………….…17

Chapter 2: Screening and Characterization of Glucagon Receptor Interactors

2.1 Introduction…………………………………………………………………………..………21

2.2 Materials and Methods

2.2.1 Animals and cell culture…………………………………………………………………22

2.2.2 Isolation of primary mouse hepatocytes…………………………………………………22

2.2.3 Plasmid preparation and transfection……………………………………………………23

2.2.4 Western blotting………………………………………………………………………....23

2.2.5 Glucose production assay………………………………………………………………..23

2.2.6 cAMP assay……………………………………………………………………………..23

2.2.7 Quantitative real-time PCR……………………………………………………………...24

2.2.8 Statistics and Bioinformatics…………………………………………………………….24

2.3 Results

2.3.1 Glucagon receptor interactors affect glucose production

in primary mouse hepatocytes………………………………………………………………….25

2.3.2 Changes to cAMP accumulation mediated by select glucagon receptor interactors

2.3.2.1 Primary mouse hepatocytes………………………………………………………….27

2.3.2.2 CHO and HepG2-GCGR cells………………………………………………………28

2.3.4 Expression of key gluconeogenic genes in primary mouse hepatocytes with glucagon

receptor interactor over expression……………………………………………………………..29

2.3.5 YWHAB does not alter cellular proliferation……………………………………………30

2.3.6 YWHAB overexpression decreases cell surface expression

of GCGR in HepG2-GCGR cells……………………………………………………………….31

2.3.7 siRNA knockdown of YWHAB enhances cAMP production

and decreases GCGR gene expression………………………………………………………….32

Chapter 3: Discussion

3.1 Summary

3.1.1 Effects of select glucagon receptor interactors on receptor function…………………….35

vi

3.1.2 Effects of interactor overexpression on expression of

key gluconeogenic genes……….................................................................................................36

3.1.3 Potential mechanism of YWHAB mediate reduction

in cAMP production……………………………………………………………………………37

Chapter 4: Future Direction and Conclusions

4.1 Future Directions…………………………………………………………………………..…39

4.2 Conclusions…………………………………………………………………………………..40

References……………………………………………………………………………………….41

vii

List of Figures

Figure 1. Diagram of proglucagon products that result from tissue specific processing

……………….………………………………………………………………………………….....2

Figure 2. Diagram of the glucagon secretory pathway in pancreatic α-cells……………….……...3

Figure 3. Crystal structure of the human glucagon receptor………………….…………….……...7

Figure 4. Schematic of the glucagon receptor signalling pathway in hepatocytes………….……...8

Figure 5. Diagram of the fate of internalized glucagon

receptor…………......…………….……………………………………………………………...10

Figure 6. GLP-1R interactome identified using the MYTH screening system……………..…….17

Figure 7. GLP-1R interactome identified using the affinity purification

tandem mass spectrometry approach……………….………………………………...……….….18

Figure 8. Co-immunoprecipitation validation of GCGR accessory protein interaction.……...….19

Figure 9. Glucose production in hepatocytes overexpressing interactors of interest……………..26

Figure 10. cAMP production in hepatocytes overexpressing interactors in

interest………………………………………………………………………………......….…….27

Figure 11. cAMP production in two cells lines overexpressing interactors of interest……..…….28

Figure 12. mRNA expression of gluconeogenic genes in interactor overexpressing

hepatocytes……………………………………………………………………………………….30

Figure 13. Cell proliferation in CHO cells overexpressing YWHAB……………...…...….…….31

Figure 14. Cell surface expression of the GCGR in YWHAB overexpressing

viii

HepG2-GCGR cells……………………………………………………………………………...32

Figure 15. mRNA expression of endogenous YWHAB and GCGR in primary hepatocytes…….33

Figure 16. cAMP production in hepatocytes following siRNA knockdown of YWHAB….…….33

Figure 17. mRNA expression of GCGR in primary hepatocytes following siRNA knockdown of

YWHAB…………………………………………………………………………………………34

Figure 18. Schematic of the proposed mechanism of YWHAB’s inhibition of

glucose production……………………………………………………………………...….…….38

ix

Abbreviations

CAMK Ca2+/calmodulin-dependant protein kinase

CAV1 Caveolin 1

cAMP cyclic adenosine monophosphate

CHO Chinese hamster ovary

CREB cAMP-response element binding protein

DMEM Dulbecco's Modified Eagle Medium

FRET Fluorescence resonance energy transfer

G-6-P Glucose-6-phosphate

G6Pase Glucose-6-phosphatase

GABA Gamma-amminobutyric acid

GALK1 Galactokinase 1

GASP G-protein-coupled receptor associated sorting protein

GCGR Glucagon receptor

GFP Green fluorescent protein

GLP-1/2 Glucagon-like peptide 1/2

GLP-1R Glucagon-like peptide-1 receptor

GPCR G-protein-coupled receptor

GRK G-protein-coupled receptor kinase

IBMX 3-isobutyl-1-methylxanthine

Katp ATP regulated potassium channel

LDLR Low density lipoprotein receptor

MPGF Major proglucagon fragment

MYTH Membrane yeast two-hybrid

PC1/3 Prohormone convertase 1/3

PC2 Prohormone convertase 2

PEPCK Phosphoenolpyruvate carboxykinase

PKA Protein kinase A

PLC Phospholipase C

RAMP Receptor activity-modifying protein

x

RGS Regulators of G-protein-coupled receptors

T2D Type 2 Diabetes

TMED2 Transmembrane Emp24 Domain Trafficking Protein 2

YWHAB Tyrosine 3-Monooxygenase/Tryptophan 5-Monooxygenase Activation Protein,

Beta

1

Chapter 1 General Introduction 1.1 Glucagon

1.1.1 Blood glucose homeostasis

The regulation of glucose metabolism and maintenance of normal blood glucose levels,

euglycemia, primarily involves the concerted action of hormones secreted from the pancreas,

insulin and glucagon, but also in part by hormones such as glucagon-like peptide-11. Euglycemia

is critical for healthy function, and disrupted blood glucose homeostasis leads to a number of

complications such as nephropathy and neuropathy2. In addition, neural control of the pancreas

(through branches of the vagus and splanchnic nerve) and liver (through the splanchnic nerve)

affect glucose metabolism3.

Ordinarily, an increase in blood glucose leads to an increase in glucose uptake into β-cells,

which is metabolised, creating ATP and ultimately leading to the influx of calcium causing

secretion of insulin4. Insulin, secreted from pancreatic β-cells lowers blood glucose through a

simultaneous stimulation of glucose uptake in skeletal muscle and adipose tissue, and a

suppression of glucose production in the liver5. On the other hand, the counterregulatory pancreatic

hormone glucagon increases blood glucose levels by stimulating gluconeogenesis and

glycogenolysis, and preventing the storage of glucose as glycogen6. In type 2 diabetes (T2D),

insulin resistance and β-cell dysfunction lead to a dysregulation of blood glucose homeostasis7.

Typically, normal glucose tolerant, non-diabetic individuals rely on glucagon to increase blood

glucose concentrations during periods of fasting, but this response is supressed following post-

prandial increases in blood glucose7. However, this suppression of blood glucose elevation is

absent in type 2 diabetics following a meal7. Complications related to T2D will then develop due

to the high levels of circulating glucose. The development of T2D is the result of a combination

of lifestyle factors (including lack of exercise and high fat diets) and genetics (possessing a strong

hereditary component7, 8).

2

1.1.2 Glucagon expression and processing

The preproglucagon gene encodes a large 180 amino acid preproprotein that serves as a

precursor to a number of hormones produced from proglucagon9. The functional products of this

proprotein differ between tissues (represented schematically in Figure 1.) due to the presence of

tissue specific prohormone

convertases (PCs)10. Glucagon,

produced in the α-cells of

pancreatic islets, is produced

upon posttranslational processing

of its proglucagon precursor by

prohormone convertase 2 (PC2)6.

This 29 amino acid peptide

hormone protects against

hypoglycemia during fasting,

raising plasma glucose levels

through its stimulation of gluconeogenesis and glycogenolysis11. In addition to glucagon, the major

proglucagon fragment (MPGF) is also derived from posttranslational processing of proglucagon

in α-cells11. In the intestines, proglucagon is processed by prohormone convertase 1/3 (PC1/PC3)

to produce glucagon-like peptide-1 and -2 (GLP-1, GLP-2)11. GLP-1 can also be released from the

MPGF10,12, however this full length N-terminally extended GLP-1 (1-37) and GLP-1 (1-36amide)

peptide have limited biological activity10,13. On the other hand, truncated GLP-1(7-37) directly

processed from proglucagon in intestinal L cells by PC1/3 (31 amino acids versus 37 for the full

length inactive GLP-1) is a potent potentiator of glucose-dependant insulin secretion10,13.

1.1.3 Secretion

Like insulin, glucagon secretion is primarily regulated through blood glucose levels that

lead to changes in intracellular ATP production14. Circulating glucose is brought into the α-cells

by facilitated transport through the GLUT 1 transporter. During periods of low blood glucose,

decreased ATP/ADP ratios keep potassium channels (KATP) open, leading to opening of T-type

Figure 1. Schematic diagram of some of the products that result

from tissue specific processing of the proglucagon precursor. This

tissue specific processing is the result of prohormone convertases

that differ between tissues.

3

calcium channels

when the

membrane is at a

potential of

approximately -60

mV15. Ca2+ then

enters the cell

causing

depolarization of

the membrane and

opening of voltage

gated Na+ channels.

Efflux of Na+

causes further

depolarization and opening of L-type calcium channels leading to sustained influx of Ca2+ into the

cell which ultimately lead to glucagon granule exocytosis. During periods of high blood glucose,

increased levels of glucose in the cell elevates the ATP/ADP ratio leading to closure of KATP

channel preventing the opening of T-type calcium channels and preventing the rest of the glucagon

exocytosis cascade from proceeding.

In addition to glucose sensing, glucagon secretion is also regulated in part by fatty

acids16,17,18 and amino acids19,20,21. Early studies involving the effects of fatty acids on glucagon

secretion concluded that fatty acids inhibited glucagon secretion, it has more recently been shown

that many fatty acids may actually stimulate glucagon secretion by pancreatic α-cells. Chain

length, spatial configuration and saturation all determine the action of specific fatty acids on

glucagon secretion16. While the action of many fatty acids at the cellular level remain unknown,

palmitate has been studied at the cellular level and was found to increase exocytosis of glucagon

from α-cells by increasing entry of calcium ions through L-type channels18. Furthermore, some

amino acids can also act as stimulators of glucagon secretion19,20,21. Glycine, for example, is able

to stimulate glucagon secretion by binding to the glycine receptor on the α-cell plasma membrane

leading to an influx of Ca2+ and thus secretion of glucagon20,22.

Figure 2. Schematic outline of the glucagon secretion pathway in pancreatic α-

cells in the presence and absence of glucose.

4

The proximity of α-cells to a number of other endocrine cells within pancreatic islets leads

to dynamic regulation of glucagon secretion via paracrine action. Insulin action on α-cells is one

of the most important paracrine signalling mechanisms which inhibits glucagon secretion14; in fact,

insulin has been shown to be essential for α-cell function23,24. In mouse α-cells, binding of insulin

to its receptor and subsequent activation of the insulin receptor-PIK3 pathway increases the

sensitivity of KATP channels to ATP25. In rats, insulin was also shown to inhibit glucagon secretion

by increasing KATP channel activity26. Taken together, this evidence suggests insulin regulates

glucose stimulated glucagon secretion in α-cells via changes in cell membrane potential.

Furthermore, insulin induces translocation of the gamma-aminobutyric acid (GABA) receptor A

in α-cells to the cell membrane14,27. Pancreatic β-cells contain glutamic acid decarboxylase (GAD),

the enzyme responsible for the synthesis of GABA, and therefore also possess high levels of

GABA27. With the increased level of GABA receptor A on the surface of α-cells, GABA can bind

to and activate the receptor, leading to membrane hyperpolarization and an inhibition of glucagon

secretion due to a decrease in intracellular Ca2+ 27. Last, somatostatin, secreted from δ-cells in the

islets of Langerhans, inhibits glucagon secretion from α-cells14,28. Activation of somatostatin

receptor 2 (SSTR2) in α-cells leads to efflux of K+ ions, hyperpolarizing the membrane and

inhibiting glucagon secretion. Additionally, activation of SSTR2 inhibits cAMP production in α-

cells, thereby reducing PKA-dependant secretion14,28,29,30.

In addition to the paracrine factors outlined above, the hormones GLP-1 and gastric

inhibitory polypeptide (GIP), are important endocrine regulators of glucagon secretion14. GLP-1,

produced from proglucagon in the intestinal L-cells, inhibits glucagon secretion from pancreatic

islets11,31. It is known that GLP-1 inhibits glucagon secretion in murine species and humans32,33,

however the exact mechanism through which this inhibition is achieved still remains

controversial11. While the GLP-1R is expressed in both pancreatic β-cells and δ-cells, most α-cells

do not express GLP-1R11,32,33. Although increased glucose stimulated insulin secretion due to

GLP-1 stimulation on β-cells could certainly explain some of the inhibitory effects of GLP-1 on

α-cell glucagon secretion, glucagon secretion is still inhibited in the absence of insulin, such as in

Type 1 diabetic patients11,34,35. Instead, GLP-1’s inhibition of glucagon secretion may be the result

of its stimulation of somatostatin which also inhibits glucagon secretion as outlined above11. While

GLP-1 supresses the post-prandial glucagon response by decreasing glucagon secretion, GIP,

another incretin hormone similarly released by the gut in response to a meal, enhances the post-

5

prandial glucagon response by stimulating glucagon secretion from α-cells36,37,38. Upon activation

of GIP receptors on the cell membrane of α-cells by GIP, increases in cAMP production leads to

increased PKA-dependant exocytosis and therefore an increase in glucagon exocytosis36,37,38.

Finally, glucagon has an autocrine function in pancreatic α-cells, enhancing its own

secretion14,39. Following exocytosis of glucagon from α-cells, glucagon can bind to the glucagon

receptor, stimulating the production of cAMP, and increasing PKA-dependant exocytosis of more

glucagon14,39.

1.1.4 The effects of glucagon

Glucagon’s role as a counter regulatory hormone to insulin involves the combined action

of stimulating glucose production and release of glucose from glycogen stores, and inhibiting

glucose breakdown and storage40.

1.1.4.1 Stimulatory effects

Glucagon enhances glycogenolysis in the liver to release stored glucose. Upon stimulation

of the glucagon receptor by glucagon, an increase in cAMP production leads to an activation of

protein kinase A (PKA), which phosphorylates and activates glycogen phosphorylase kinase,

which in turn phosphorylates and activates glycogen phosphorylase41,42. Once active, glycogen

phosphorylase releases single glucose-1-phosphate (G-1-P) molecules from glycogen, which are

converted first to G-6-P and then to glucose by the enzymes phosphoglucomutase and glucose-6-

phosphatase (G6Pase), respectively41,42. Once the phosphate group has been removed, glucose can

leave the liver through GLUT2 and enter the blood stream43.

In addition to enhancing the release of glucose stores, glucagon also increases

gluconeogenesis in the liver40. The entry and rate-limiting step of gluconeogenesis is regulated by

the enzyme phosphoenolpyruvate carboxykinase (PEPCK), which converts oxaloacetate into

phosphoenolpyruvate44. Glucagon stimulation leads to a downstream increase in PEPCK

expression and ultimately an increase in gluconeogenesis44,45. Activation of PKA leads to

phosphorylation of cAMP-response element binding protein (CREB). Phosphorylated CREB can

then bind to cAMP response element regions on the promotor of a gene, recruitment of CREB

6

binding protein (CBP) and an increase in transcription46,47. In the context of enhancing release of

stored glucose, phosphorylated CREB can bind to the promotor of the transcription co-factor PGC-

1, increasing its transcription and ultimately leading to an increase in PEPCK transcription46,47,48.

1.1.4.2 Inhibitory effects

While glucagon effectively raises blood glucose through the stimulation of glucose

producing pathways, the profound effects are also due in part to a simultaneous inhibition of

glucose storage. Glycogen synthase, an enzyme responsible for incorporating glucose molecules

into growing glycogen chains, is inhibited by glucagon49. Recall that when glucagon binds to its

receptor, PKA is activated. While PKA has a stimulatory effect on glycogen phosphorylase, PKA

inhibits glycogen synthase, preventing the storage of glucose in glycogen and increasing the

availability of free glucose50,51. In addition to inhibiting storage of glucose via inactivation of

glycogen synthase, glucagon stimulation also inhibits the breakdown of glucose by inhibiting

activity of phosphofructokinase-1 a key enzyme involved in the glycolytic pathway52,53.

1.2 Glucagon Receptor

1.2.1 Regulation of receptor expression

Glucagon exerts its effects upon binding to its cognate receptor, the glucagon receptor

(GCGR). GCGR is found abundantly in multiple tissues including liver, pancreas, brain and

heart6,54, and in lower levels in the adrenal glands, thyroid and skeletal muscle6,54. Expression of

the receptor is mediated by its own ligand: increased GCGR activity following glucagon binding

increases cAMP production and cAMP binds cAMP responsive elements on the GCGR gene

promotor to down-regulate GCGR transcription55,56,57. In fact, any stimulus that leads to an

increase in the production of the second messenger cAMP can lead to suppression of GCGR

transcription55,56,57. In addition to glucagon, artificial stimulation of the GCGR with 3-isobutyl-1-

methylxanthine (IBMX) (a phosphodiesterase inhibitor) and forskolin (an organic compound that

activates adenylyl cylcase), both raise cAMP production in hepatocytes and thus lower expression

of the GCGR57. Conversely, the hormone somatostatin which lowers the production of cAMP,

leads to an increase in the mRNA expression of GCGR57,58. Both mouse and human GCGR genes

contain two promotor regions that are regulated differentially. While GCGR transcription is

7

supressed by cAMP, transcription cofactor PGC-1α prevents the cAMP mediated suppression of

the receptor, providing fine tuning of GCGR expression55.

1.2.2 Structure

The GCGR is a member of the class B, or secretin, G-protein-coupled receptor (GPCR)

family. The GCGR is a seven transmembrane domain protein, 477 amino acids in length and

approximately 55 kDa in mass6,59. The N-terminal end of the GCGR lies outside of the cell at the

end of helix I, a region known as the “stalk” which maintains the extracellular domains orientation

and is also involved in

glucagon binding59.

Extracellular domains of

the GCGR are widely

spaced, forming a deep

ligand-binding pocket59.

Aspartic acid 63, tyrosine

65 and lysine 98 stabilize

the extra-cellular domain

of the GCGR59. The

intracellular C-terminal

end of the GCGR is rich in

serine residues which may

be phosphorylated to

begin the uncoupling

process, discussed further

in section 1.2.3.259,60.

Upon binding of glucagon

to the receptor binding

pocket, the stalk region

captures glucagon and

guides the N-terminus of

glucagon into the proper

Figure 3. Structure of the human GCGR determined through x-ray

crystallography with binding domain in purple, transmembrane spanning

region in blue and glucagon in green. (Siu et al., 2013)

8

orientation within the binding pocket, while tryptophan 36 provides a hydrophobic interaction site

for the C-terminus of glucagon59.

As seen in Figure 3. the extracellular stalk region of the GCGR (in purple) partially

occludes the binding pocket, orienting glucagon (in green) within the pocket and securing it within.

Additionally the stalk can be seen providing structural support to the rest of the extracellular

domain. Following binding of glucagon to the glucagon receptor, conformational changes in the

receptor occur leading to the second messenger cascade which will be discussed in section 1.2.3.

1.2.3 Signaling

1.2.3.1 Activation and transduction

The GCGR is a member of the class B G-protein-coupled receptor family, sharing this

classification with other important receptors involved in blood glucose regulation such the GLP-1

receptor and GIP receptor6,61. As a member of the class B G-protein-coupled receptor family, many

of the early steps of the GCGR signalling cascade mirror that of other class B GPCRs. Figure 4.

outlines schematically the glucagon signalling cascade. Glucagon is a potent hormone able to

rapidly increase blood glucose through simultaneous activation of Gαs and Gq signalling

Figure 4. Schematic overview of the primary glucagon-glucagon receptor signalling cascade. A

simultaneous stimulation of glucose production and inhibition of glucose storage and breakdown leads

profound increase in blood glucose.

9

pathways14,40,63,64,65. Upon binding of glucagon to the GCGR, conformational changes in the

GCGR allow the receptor to interact directly with the G-protein complex40,62. This interaction leads

to a subsequent change in the Gαs-subunit’s conformation allowing for the exchange of GDP for

GTP40,62. This exchange leads to dissociation of the α-subunit from the βγ dimer and activation of

adenylyl cyclase leading to the conversion of ATP to cyclic AMP (cAMP)40,62. Increases in

intracellular cAMP lead to activation of protein kinase A (PKA) which leading to a number of

effects as outlined in section 1.1.3. First, active PKA will phosphorylate phosphorylase kinase,

which in turn phosphorylates and activates glycogen phosphorylase increasing breakdown of

glycogen to release glucagon14,40. Second, PKA phosphorylates the cAMP response element

binding protein (CREB) ultimately leading to an increase in gluconeogenic gene expression14,40,46.

While signalling through the Gαs-cAMP cascade causes a profound increase in free glucose,

activation of a Gq-related signalling cascade leads to further changes in blood glucose.

In addition to Gαs, dissociation of Gq from the GCGR leads to an increase in inositol

trisphosphate production and a subsequent release of Ca2+ 14,40,63,64. Increased cytosolic Ca2+ leads

to activation of Ca2+/calmodulin-dependant protein kinase (CAMK). Activation of these kinases

leads to further reduction in glycolysis and glycogenesis via activation of the FOXO1 transcription

factor thereby increasing free glucose65. Furthermore activation of CAMK in addition to PKA

leads to an increase in CREB activity, further enhancing expression of gluconeogenic genes65,66.

These two independent GCGR signalling pathways, involving activation of both Gas and Gq

cascades, allow for a rapid and profound increase in blood glucose upon glucagon stimulation.

1.2.3.2 Glucagon receptor regulation

GCGR, like most GPCRs67,68,69, is subject to a number of regulatory processes that prevent

overstimulation of the receptor and an exaggerated response to glucagon70,71. Regulation of GCGR

signalling is accomplished through desensitization and uncoupling of the receptor67,68,69,70 followed

by internalization where it may be degraded or recycled back to the cell surface72,73. Following

binding of glucagon to the GCGR, serine residues, essential for desensitization of the receptor, are

phosphorylated71,73. Phosphorylation of serine residues is achieved through G-protein-coupled

receptor kinases (GRKs), causing recruitment of β-arrestin 1 and 2 which uncouple the receptor

from its associated G-proteins72,74. Additionally, other kinase, in particular PKC phosphorylate the

GCGR leading to enhanced recruitment of β-arrestins which can occur even without activation of

10

the GCGR by glucagon73,75,76. Thus, GCGRs are tightly regulated through both homologous

(ligand-mediated) and heterologous (ligand-independent) desensitization. Following uncoupling

of the receptor from the G-protein complex, β-arrestins also target the receptor for internalization

in clathrin-coated pits where they are internalized in endosomes73,77. Internalized GCGR is then

either recycled to the cell surface, or degraded72,73. Recycling of internalized GCGR to the cell

surface is critical for resensitizing cells to glucagon, since it would take significantly longer to

synthesize new GCGR72. Recycling of internalized GCGR is accomplished through multiple

recycling pathways involving both Rab4 and Rab11 positive endosomes72,78. Rab4 endosomes are

responsible for recycling of GCGR in early endosomes that remain close to the cells surface, while

Rab11 endosomes are involved in recycling of GCGR (and other GPCRs) from the trans-Golgi

network79,80. GCGR destined to be degraded involves sorting signals on the C-terminus tail of the

GCGR, like other GPCRs, which are ubiquitinated and targeted for degradation81. The exact

mechanism through which GCGR is sorted for either recycling or degradation is still poorly

understood72. The fates of stimulated glucagon receptor are represented schematically in Figure 5.

Figure 5. Overview of the two fates of internalized glucagon receptor following glucagon stimulation.

Recycling (left) allows for rapid resensitization of cells to glucagon without the need to synthesize new

receptor. Chronic stimulation of the receptor leads to degradation of the receptor (right).

11

Short term stimulation (30 minutes) of the GCGR with glucagon leads to recycling of the receptor

to the cells surface, while long term (5 hours) stimulation with glucagon targets the receptor for

degradation72. The observation that prolonged exposure of ligand causes a shift from recycling to

degradation has also been demonstrated for other GPCRs82. Recently, G-protein-coupled receptor

associated sorting proteins (GASPs) have been shown to be critical for the degradation of GPCRs,

where they recognize specific GASP binding motifs on the receptor and target it for degradation83.

Furthermore, GASPs have been shown to associate with Gαs, and depleted of Gαs was shown to

significantly reduce degradation of GPCRs, demonstrating a potential combined role for GASP

and Gαs in targeting GCGR for degradation as opposed to recycling84.

1.3 Type 2 Diabetes and the Role of Glucagon

1.3.1 Pathogenic glucagon secretion

The role of glucagon in the development of type 2 diabetes (T2D) has been known for

decades85,86,87. In type 2 diabetics, an overabundance of glucagon, known as hyperglucagonemia,

is the result of dysfunction in both β-cells and α-cells. Normally, an increase in insulin secretion

in response to high blood glucose results in (among other things) a decrease in glucagon secretion.

As discussed in section 1.1.3, insulin as a paracrine factor plays a key role in inhibiting glucagon

secretion in α-cells by increasing sensitivity of KATP channels and causing downstream

hyperpolarization of the cellular membrane preventing the influx of calcium23,24,25,26. In the early

stages of developing T2D, increased demand for insulin due to insulin resistance is met through a

compensatory increase in β-cell mass and insulin secretion, resulting in hyperinsulinemia during

early disease progression88. This increase in β-cell mass provides a stable compensatory

mechanism to combat chronically high blood glucose for a period of time88. In later T2D, in

addition to an increased resistance to insulin, insulin secretion from β-cells is also reduced88. With

constant challenge of insulin secretion, an increase in β-cell mass is no longer an adequate

compensatory mechanism88. With time, decompensation occurs along with β-cell exhaustion,

eventually leading to a decrease in β-cell mass and insulin secretion88,89. Thus in later T2D, insulin

secretion is diminished and paracrine regulation of glucagon secretion is greatly reduced which in

part explains the hyperglucagonemia present in type 2 diabetics. While decreased insulin secretion

12

in type 2 diabetics certainly contributes to hyperglucagonemia23,24,25,26,90, dysfunction in α-cells

has also been shown to be a significant contributor.

In addition to the direct role of β-cells in the development of T2D, α-cells have also been

shown to be responsible for the development of the disease. In fasted type 2 diabetics, elevated

blood glucagon levels have been observed in addition to impaired suppression of glucagon

secretion in response to glucose91. It has been previously reported that obese non-diabetics and

obese glucose-intolerant individuals already had an observed impairment in glucose-induced

glucagon suppression92. Since this impairment was noted despite the presence of hyperinsulinemia,

it was hypothesized that these individuals’ α-cells had become resistant to insulin’s paracrine

action in reducing glucagon secretion91,92,93. This resistance to insulin-induced glucagon

suppression in α-cells has been defined as paracrinopathy91,94. In addition to paracrinopathy,

dedifferentiation may play a role in the development of hyperglucagonemia91,95. Increased demand

for insulin in T2D and the resultant stress to β-cells has been shown to lead to dedifferentiation of

these β-cells to progenitor cells which begin to produce and secrete glucagon, further increasing

the inappropriate glucagon secretion91,95.

Thus, in T2D the absence of insulin is in itself not solely responsible for hyperglycemia.

Rather, hyperglucagonemia resulting from the lack of insulin suppression of glucagon secretion,

dysfunction in α-cells, and dedifferentiation of β-cells to glucagon secreting progenitors all

contribute to chronically elevated blood glucose. Thus, as opposed to the more “insulin-centric”

model of T2D in the past, development of the disease involves a complex dysregulation of both

insulin and glucagon. Initially, hyperinsulinemia as a compensatory mechanism leads to resistance

of insulin’s glucagon supressing effects in α-cells. In later T2D, there is a reduction in β-cell mass

and insulin secretion, dedifferentiation of β-cells to glucagon producing cells, and a lack of tonal

regulation of α-cell glucagon secretion.

1.3.2 Glucagon receptor antagonists

Potential avenues of T2D treatment typical fall within two developmental approaches:

increasing insulin secretion, biosynthesis and or sensitivity, or reducing glucagon section,

biosynthesis and or sensitivity. Defendants of the glucagon-focused approaches to the treatment

of T2D91,96 cite the fact that hyperglucagonemia is present in all poorly controlled diabetes, known

glucagon suppressors such as somatostatin eliminate inappropriate glucose production in total

13

insulin deficiency and, incredibly, glucagon-receptor knockout mice do not develop diabetes even

with complete β-cell destruction91,96,. Experiments involving glucagon receptor knockout mice

provide compelling support for the notion that glucagon may be at the center of diabetes, as

inducible diabetic mice with a whole-body glucagon receptor knockout have been shown to be

protected from diabetes97. Following destruction of β-cells with streptozotocin, GCGR+/+ mice

became hyperglycemic were sacrificed after 6 weeks. GCGR-/- mice on the other hand were

characterized by a complete absence of the manifestations of diabetes97. Even with destruction of

β-cells, these GCGR knockout mice had normal fasting glucose and oral glucose tolerance tests97.

The nature of the current project has focused on the glucagon receptor itself, therefore

discussion of the glucagon-centric approaches to the treatment of T2D will focus specifically on

regulation of the glucagon receptor itself. Some of the earliest GCGR antagonists (as far back as

1986) were peptides98,99,100. Despite their ability to bind to the GCGR and partially prevent the

activation of the cAMP-PKA pathway, these peptide antagonists were unable to lower plasma

glucose levels in diabetic rats99. Years later, Qureshi et al. identified a non-peptide antagonist of

the GCGR, Cpd 1101. Cpd 1 was found to prevent glucagon-mediated glycogenolysis, and blocked

the increase in blood glucose in mice following intraperitoneal injection of glucagon101. The small

molecule skyrin, a fungal bisanthroquinone, was found to bind to the GCGR and prevent glucagon-

induced activation of the cAMP-PKA pathway102.

While there have been numerous attempts to develop a clinically significant glucagon

receptor antagonist for the treatment of T2D, difficulties have arisen due to a lack of specificity of

the antagonist, failure to induce meaningful and long-term suppression of blood glucose levels in

diabetic animals, or lack of further investigation of the clinical impact. The development of an

effective GCGR antagonist for the treatment of T2D could be made easier with a deeper

understanding of GCGR function and how its activity is regulated in vivo.

1.4 Receptor Interactomes

1.4.1 Effects of accessory proteins on g-protein couple receptors

While the signalling pathway of many class B G-protein coupled receptors follow similar

cascades (i.e. activation of adenylyl cyclase and PKA), GPCRs have been shown to be involved

in protein-protein interactions with accessory proteins that can dramatically alter their function and

14

activity103. These protein-protein interactions can produce profound changes in not only ligand

binding or the downstream response to ligands, but also in receptor localization103,104. GPCRs may

be involved in single protein-protein interactions with an accessory protein,105 or complex

interacting networks (receptor interactomes) involving multiple accessory proteins that bind to the

receptor and to one another105.

Discussed in section, 1.2.3.2, β-arrestins and GRKs are one such example of GPCR

accessory proteins and their role in uncoupling GPCRs and endocytosis are critical to preventing

overstimulation of GPCRs106. Many other families of GPCR accessory proteins have been

identified which alter GPCRs in a variety of ways. Another family of GPCR accessory proteins,

known as receptor activity-modifying proteins (RAMPs), modify the cell surface expression and

pharmacology of certain GPCRs altering their trafficking and the strength of the response to

receptor ligand106,107. On the other hand, regulators of G-protein signalling (RGS) proteins have

been shown to negatively regulate G-proteins, turning off G-protein-coupled receptors106,108.

Another family, GPCR-associated sorting proteins (GASPs), are responsible for the removal of

GPCRs from the cell surface following ligand stimulation106,109,110.

Thus, while strategies to target GPCRs in the treatment of diabetes are promising

approaches, the presence of accessory proteins and interacting networks complicates the

development of these treatment options. Development of GCGR antagonists for the treatment of

T2D could be improved with a complete understanding of the GCGR interactome.

1.4.2 Experimental approaches to receptor interactome identification

A new experimental paradigm involving hypothesis generating studies has developed with

advances in high-throughput screening tools, leading to the development of many “omic” sub-

fields within biology. Omics refers to a full and complete set of all relevant biological molecules

in a field of study, such as genomics which explores the complete set of genes in an organism (the

genome), or metabolomics, which explorers the complete metabolic profile of a cell, tissue or

organism (the metabolome). Since GPCRs have been shown to be involved in a number of complex

interactions with accessory proteins, interactomics was developed which aims to reveal the

complete set of interacting proteins for a particular receptor (the interactome). The nature of

interactomics demands high-throughput screens of receptor interactors to make experiments cost

15

and time efficient. A number of a screening methods exist to this end, and a list of some of the

more common screening methods will be summarized below.

1.4.3.1 Membrane yeast two-hybrid system

The membrane yeast two-hybrid system (MYTH) takes advantage of the fact that in vivo,

the ubiquitin protein is fragmented into two moieties, a C-terminal fragment (Cub) and an N-

terminal fragment (Nub) that spontaneously re-associate with each other. However, by mutating

isoleucine 13 on the Nub fragment to glycine (NubG), this spontaneous re-association is prevented.

With these fragments, a bait protein (such as a GPCR), is fused to the Cub fragment, while a library

of prey proteins are fused to the NubG fragment. In addition, an artificial transcription factor is

fused to the Cub fragment. If a protein-protein interaction exists between the bait (receptor) and

prey (library) proteins, their physical interaction brings the two ubiquitin fragments into close

enough proximity to form pseudo-ubiquitin. This pseudo-ubiquitin protein can then be recognized

by deubiquitinating enzymes, releasing the transcription factor and leading to activation of a suite

of reporter genes that can be used to assess the interaction between the bait and prey. The MYTH

approach has been successfully employed by a number of groups to identify protein

interactomes111,112,113. Recently, our lab has successfully employed the MYTH system to screen

for interactors of the glucagon-like peptide-1 receptor (GLP-1R) revealing 38 novel GLP-1R

interacting candidates114.

While the MYTH system allows for high throughput screens of receptor interactomes, there

are significant limitations to this approach. 1) Interactions between bait and prey proteins must be

direct interactions in order to be detected. Indirect interactions in which another protein mediates

the interaction between bait and prey may not bring the ubiquitin fragments into close enough

proximity to fuse, preventing detection. 2) Screens are performed in a yeast vector using human

bait and prey proteins which may require other intracellular machinery and factors to properly

interact which may greatly differ in amino acid sequence and structure such as PKA and PLC 3)

Screening of receptor interactomes must be performed in the unliganded state. Since many

previously identified receptor interactors are responsible for desensitization or alteration of

receptor activity following agonist binding, the lack of receptor stimulation in the MYTH screen

means some potential receptor interactors may be missed.

16

1.4.3.2 Fluorescence resonance energy transfer

The florescence resonance energy transfer system (FRET) for detecting in vivo protein-

protein interactions is similar in principle to the MYTH system115. In FRET, two proteins of

interest are labelled with a donor molecule or an acceptor fluorophores, which can be a dye,

inorganic ion or fluorescent protein115,116. When excited, the donor molecule will transfer energy

to the acceptor molecule when in close proximity leading to an increase in the acceptor’s

fluorescent signal116. In MYTH, a protein-protein interaction allowed the ubiquitin fragments to

be in close enough proximity to re-associate. In FRET, binding of one protein to another brings

the conjugated donor and acceptor molecules in close enough proximity for the energy transfer

between the donor and acceptor to occur116. Unlike MYTH, liganded experiments are possible

with FRET screens allowing differences in protein interactions in the unliganded vs liganded state

to be determined.

While FRET has significant advantages over MYTH, it still possesses some of the

limitations inherent in the MYTH system. Because the FRET system requires conjugation of

fluorophores to two proteins of interest, a library of potential protein interactors with the protein

of interest must be constructed, which limits the potential high-throughput screening applications.

Like MYTH, constructing a library of potential interactors means each protein must be pre-

selected for inclusion in the screen, which may lead to missing potential protein-protein

interactions that were not predicted.

1.4.3.3 Mass spectrometry

Mass spectrometry has applications in both chemistry and biology for its ability to identify

unknown substances117. In biological applications of mass spectrometry, peptides are digested with

proteases and separated by various characteristics, such as polarity, using chromatography and

then ionized117. Since peptides were fragmented with proteases and separated, each fragment is

given a unique charge-to-mass ratio which can be plotted and used to identify the parent peptide

the fragment belongs to. In the study of receptor interactomes, the receptor of interest may be

stimulated with ligand, purified and fragmented with proteases. Following separation of the

fragments, ionization and detection, the data can be plotted as a mass spectrum, which plots the

17

intensity of fragments vs the charge-to-mass ratio. Since the receptor was purified from other

proteins prior to mass spectrometry, proteins identified other than the receptor itself may be

potential receptor interactors which can be further validated. Compared to MYTH and FRET, mass

spectrometry is an incredibly robust high throughput screening method for identifying receptor

interactors in both the liganded and unliganded state. Potential interactors are not required to be

labelled, drastically enhancing the number of novel interactors that may be detected and allowing

for hypothesis generating research. One of the first successful use of mass spectrometry to study

the interactome of a GPCR came from Daulet et al., who revealed the interactome of two melatonin

receptors and found 38 receptor interactors between the two118. Recently, our lab successfully

employed affinity-purification and mass spectrometry to study the interactome of the glucagon-

like peptide 1 receptor, and revealed 99 potential receptor interactors between two cell lines119.

1.5 Rationale and Hypothesis

The Wheeler labs first high-throughput

screen to identify receptor interactomes involved

the use of the membrane yeast two hybrid system

to screen for GLP-1 receptor interactors. Using a

human fetal liver cDNA prey library and the GLP-

1R as bait, Huang et al. were able to identify 38

potential GLP-1R interactors, seen in Figure 6, 36

of which were validated with co-

immunoprecipitation114. Functional

characterization of the interaction between these

novel interactors and the GLP-1R revealed a

number of significant alterations to cAMP

production114.

While the initial MYTH screen was

successful in identifying a number of novel GLP-

1R interactors (many of which were able to alter

Figure 6. Interactome of the GLP-1R as

identified by Huang et al. using the membrane

yeast two hybrid system (MYTH). In total, 38

potential GLP-1R interactors were identified

from the screen. (Huang et al., 2014)

18

receptor signalling), the limitations of MYTH, as outlined above, prompted the Wheeler lab to

explore more robust screening approaches for the identification of GPCR interactors. As a follow-

up study, Zhang et al. performed another screen of the GLP-1R, this time using affinity purification

and tandem mass spectrometry119. Following the GLP-1R mass spectrometry screen, Zhang et al.

identified 99 potential GLP-1R interactors within two cell lines119, as seen in Figure 7. One

interactor, PGRMC1, was shown to significantly enhance GLP-1R signalling and represents a

potential target for future therapeutics. The success of the mass spectrometry screen prompted the

Wheeler lab to explore other GPCR interactomes.

Figure 7. Interactome of the GLP-1R as identified by Zhang et al. using affinity purification and tandem

mass spectrometry. In total, 99 potential GLP-1R interactors were identified from the screen between two

cell lines, Min6 and CHO. (Zhang et al., 2014)

19

The role of glucagon in the development of T2D has made targeting the glucagon receptor

an attractive option for the treatment of the disease. A greater understanding regarding the

glucagon receptor’s interactome may improve the development of glucagon receptor antagonists.

Many members of the class B G-protein coupled receptor family have been shown to be involved

in many complex interactions with accessory proteins that dramatically alter their activity and

expression. Thus, it is reasonable to expect that there are a number of unknown glucagon receptor

interactors.

To this end, our lab has used affinity purification and tandem mass spectrometry to identify

a number of potential GCGR interactors. In total, 33 potential glucagon receptor interactors were

identified from the screen of human GCGR transfected in Chinese hamster ovary (CHO) cells in

both the unliganded and liganded state. After selecting 8 of these potential interactors (Figure 8.)

based on their previously reported functional roles in cell signalling and molecular transport, and

their known localization (on the cell surface), 5 of these potential interactors were validated with

co-immunoprecipitation: CAV1,

GALK1, LDLR, TMED2 and

YWHAB. These 5 validated

interactors were therefore selected for

functional and mechanistic studies in

this thesis.

Based on the previously

reported involvement of accessory

proteins in the regulation of other

class B GPCRs, it was hypothesized

that some or all of these interactors

will significantly alter the activity and

expression of the GCGR. The

interaction between the GCGR and

these accessory proteins will explain

in part the difficulties faced in the

generation of GCGR antagonists and

Figure 8. Validation of the interaction between GCGR and 8

potential interactors using co-immunoprecipitation.

YWHAE, TMED10 and YWHAQ were excluded due to

absence in the eluate or aspecific binding to the affinity gel,

respectively. (Han et al., 2015)

20

further reveal the methods in which fine tuning of the glucagon’s effects in the liver are achieved.

It was further hypothesized that altering the expression of these interactors may reveal their

therapeutic potential.

21

Chapter 2

High Throughput Screening and Characterization of Glucagon Receptor Interactors

The following chapter is based in part on work published in PloS One:

Han, J., Zhang, M., Froese, S., Dai, F. F., Robitaille, M., Bhattacharjee, A., Huang, X., Jia, W.,

Angers, S., Wheeler, M.B. & Wei, L. (2015). The Identification of Novel Protein-Protein

Interactions in Liver that Affect Glucagon Receptor Activity. PloS one, 10 e0129226 (2015).

Conceived and designed the experiments: MBW SA LW WJ. Performed the experiments: JH MZ

SF AB XH MR. Analyzed the data: JH MZ SF MR. Contributed reagents/materials/analysis

tools: SA. Wrote the paper: JH SF FD MB LW.

Glucose production, cAMP production, gene expression, cell proliferation, cell surface

expression and siRNA knockdown experiments were conducted by SF

2.1 Introduction

Glucagon is responsible for maintaining normal blood glucose during periods of fasting

through a simultaneous promotion of both glycogenolysis and gluconeogenesis. Synthesized in the

pancreatic α-cells in the islets of Langerhans, glucagon’s role in mediating blood glucose begins

upon binding to the glucagon receptor. The GCGR belongs to the class B G-protein-coupled

receptor superfamily, increasing glucose production primarily through the Gs alpha-cAMP-PKA

pathway as well as Gq and phospholipase C (PLC) pathway6. Following binding of glucagon to

the GCGR, the Gs alpha subunit is released from the G protein complex and activates adenylate

cyclase, leading to a downstream increase in cAMP levels, which in turn leads to increased

activation of PKA120. Furthermore, disassociation of Gq from the G protein complex leads to the

activation of PLC, causing increases in intracellular calcium121,122. Glucagon has been found to be

elevated in type 2 diabetics85, and disruption of glucagon’s activity improves hyperglycemia in

obese mice123. Because of these observations, GCGR antagonists represent a potential avenue of

diabetes treatment. A number of GCGR antagonists have been investigated, such as the fungal

bisanthroquinone skyrin102, or Cpd-1101. These antagonists fail due to their poor potency or a lack

of specificity. The development of GCGR antagonists would be greatly aided by a complete,

comprehensive understanding of each factor involved in regulating GCGR function.

22

G-protein coupled receptor interactors, or accessory proteins, have been the subject of a

great deal of study in recent years in an effort to reveal the diverse function and regulatory

mechanisms of the receptors. Many accessory proteins, such as the GABAB receptor accessory

proteins KCTDs, and GLP-1 receptor accessory protein beta-arrestin 1, dramatically alter receptor

function and are critical components of receptor activity124,125. Many GPCR interactomes have

been discovered in recent years yet the interactome of the GCGR remains unknown. Several

studies have employed affinity purification and mass spectrometry for high-throughput screening

of other GPCRs126,127,128. The Wheeler lab has recently revealed a number of novel GLP-1R

interactors in CHO and MIN6 cells expressing the GLP-1R using a similar affinity purification

mass and spectrometry method revealing 99 potential interactors, one of which significantly

augments GLP-1 stimulated insulin secretion119. In a more recent study, using this method the

Wheeler lab revealed 33 potential GCGR interactors, 8 of which were selected for further

functional and mechanistic study following validation of the interaction.

2.2 Materials and Methods

2.2.1 Animals and cell culture

Mice ages 8-12 weeks of age of C57BL/6 background were used for experiments. All

experiments have been approved by the Animal Care Committee (University of Toronto). Animals

were handled according to the Canadian Council of Animal Care guidelines. HepG2-GCGR cells,

the stable human glucagon receptor expressing human liver carcinoma cells, generated for this

study were cultured with high glucose Dulbecco’s Modified Eagle Medium (DMEM) with 10%

fetal bovine serum as well as 1% penicillin-streptomycin. Chinese hamster ovary (CHO) cells were

cultured under the same conditions and were passaged approximately every 4 days. Transient

transfections in primary hepatocytes as well as the two cell lines were performed using

Lipofectamine 2000 following the manufacturer’s instructions (Invitrogen, Carlsbad, California).

2.2.2 Isolation of primary mouse hepatocytes

Mice were fasted overnight and primary hepatocytes were isolated and cultured as

described previously129. Briefly, primary hepatocytes were isolated using collagenase IV (Sigma,

Canada) perfusion. Cells were seeded using DMEM supplemented with 1 g/L glucose, 10 M/L

sodium lactate, 0.01 μM/L dexamethasone, 5 mM/L HEPES, and 2 mM/L L-Glutamine.

23

2.2.3 Plasmid preparation and transfection

cDNA of the human GCGR (c-terminal Flag-tagged) and GCGR interactors (c-terminal

HA-tagged) were constructed in the pcDNA3.1 vector. Plasmids were purified using the Midi-

Prep kit according to the manufacturer’s protocol (Qiagen, Toronto, Canada).

2.2.4 Western blotting

Total cell lysate was collected using protein lysis buffer (10% glycerol, 50 mM Hepes, 150

mM NaCl, 2 mM EDTA, 0.25% n-dodecyl-b-d-maltoside, and complete protease inhibitor mixture

(Roche)). Anti-Flag antibody (1:1000 dilution; Sigma-Aldrich, United States), anti-GLUT2

antibody (1:1000 dilution; EMD Millipore, United States) and HRP-conjugated mouse secondary

antibody were used for detection of protein. The membranes were developed with the ECL

advance kit (GE Healthcare) and imaged using the Kodak ImageStation 4000 Pro (Care stream

Health Inc, Rochester, New York).

2.2.5 Glucose production assay

Primary mouse hepatocytes (2×105 cells per well in twelve-well plates) were first serum

starved overnight prior to stimulation. Following serum starving, primary hepatocytes were

preincubated with glucose-free DMEM without phenol red for 2 hours. Cells were then washed

with PBS and stimulated with adenylate cyclase activator, forskolin (10 μM/L), or glucagon (100

nM/L) in glucose-free DMEM without phenol red for 4 h. Culture media was collected for

measurement of glucose concentration using the Glucose (GO) assay kit (Sigma, Canada) and

readings were normalized to total protein content using the Bradford assay.

2.2.6 cAMP assay

Intracellular cAMP content was measured in primary hepatocytes as described

previously130. In brief, cells were washed with cold PBS and harvested using 80% ethanol. Lysates

were centrifuged and the supernatant was collected and lyophilized using a SpeedVac. The pellet

was resuspended in cAMP assay buffer (0.05 mM/L sodium acetate (pH 6.2) and 0.01% sodium

azide) and measured using an intracellular cAMP ELISA kit (Biomedical Technologies Inc, US).

For cAMP measurements in CHO and HepG2-GCGR cells, the Cisbio cAMP cell-based assay kit

was used according to the manufacturer’s instructions.

24

2.2.7 Quantitative real-time PCR

Total RNA from primary hepatocytes was extracted using an RNA-easy kit (Qiagen,

Canada) and cDNA was generated by SuperScript II enzyme (Invitrogen, Canada). mRNA

expression was analyzed by qPCR using Power SYBR Green PCR master mix according to the

manufacturer’s instructions (Applied Biosystems, Carlsbad, California) and ViiA 7 Real-Time

PCR System (Life-Technology, Canada). Data was normalized to β-actin expression. Primers for

PCR were designed using the Primer3 software program. Relative gene expression was estimated

by the standard curve method.

2.2.8 Statistics and Bioinformatics

The data are presented as the mean ± SE. Student’s t-test was used to measure the mean

difference for measurements of glucose production and cAMP in primary hepatocytes. One-way

ANOVA was used to measure the mean difference for cAMP production in CHO and HepG2-

GCGR cells and differences were considered statistically significant at p < 0.05.

25

2.3 Results

2.3.1 Glucagon receptor interactors affect glucose production in primary mouse

hepatocytes

The glucagon receptor is expressed in many tissues but it is found most abundantly in the

liver. In response to low blood glucose, glucagon is secreted from the pancreas and binds to the

glucagon receptor leading to glucose production in the liver through a simultaneous stimulation of

both glycogenolysis and gluconeogenesis. To determine the functional role that the identified

GCGR interactors have as GCGR accessory protein, glucose production was assessed in primary

mouse hepatocytes. Prior to over expression of the 5 GCGR interactors, the transfection efficiency

of lipofectamine 2000 in primary mouse hepatocytes was tested using overexpression of a green

fluorescent protein (GFP) plasmid. The transfection efficiency of GFP was confirmed to be

upwards of 70% (Figure. 9A). In pcDNA3.1 (empty vector) transfected primary hepatocytes,

glucose production in response to glucagon was shown to be dose-dependent (Figure. 9B),

indicating that isolated and transfected hepatocytes were healthy and the GCGR signalling

pathway was functional. Next, the five interactors validated by Co-IP and Western blot (Figure.

7) were transfected into primary hepatocytes and the EC50 concentration of 100 nM glucagon was

selected for treatment to examine glucose production. GCGR accessory proteins CAV1 and

GALK1 were both found to increase glucose production at the basal (without glucagon treatment)

concentration, however no changes to glucose production were found in the presence of glucagon

(118.87±9.4%, p<0.05, N = 6 and 120.03±13.0%, p<0.05, N = 6 respectively) (Figure. 9C). On

the other hand overexpression of two interactors (LDLR and TMED2) had no effect at the basal

level of glucagon, but were found to enhance glucagon-stimulated glucose production significantly

(128.97±12.6%, p<0.01, N = 6 and 131.15±10.3%, p<0.01, N = 6 respectively). Overexpression

of the fifth interactor, YWHAB, conversely reduced glucagon-induced glucose production

26

significantly (65.59±4.6%, p<0.01, N = 6) (Figure. 9D) while having no effect on glucose

production at the basal glucagon concentration. GCGR interactors CAV1 and GALK1 were found

to increase glucose production under basal conditions, but prevented glucose production in the

presence of glucagon which suggests they may block receptor function. While LDLR, TMED2

Figure 9. A. Expression of green fluorescent protein in primary hepatocytes, approximately 70%

transfection efficiency. B. Isolated hepatocytes transfected with pcDNA3.1 control produce glucose in

response to glucagon indicating healthy function of the cells. C. Glucose production in primary

hepatocytes with over expression of interactors that affect basal glucose production. D. Glucose

production in primary hepatocytes with over expression of interactors that affect glucagon stimulated

glucose production. Hepatocytes isolated by Han and Zhang, glucose production assays performed by

Zhang and Froese. * = p<0.05, ** = p<0.01. (Han et al., 2015)

27

and YWHAB were selected for further characterization, future studies should characterize the

effects of CAV1 and GALK on GCGR function.

2.3.2 Changes to cAMP accumulation mediated by select glucagon receptor

interactors

Prior to the activation of protein kinases responsible for increased glucose production,

GCGR activation in response to glucagon leads to activation of adenylyl cyclase, an increase in

cAMP production and activation of PKA. To determine whether the effects of LDLR, TMED2

and YWHAB were the result of regulation of the cAMP pathway, the effects that overexpression

of these interactors had on cAMP production in response to glucagon was assessed in isolated

primary mouse hepatocytes, and two cell lines: CHO and HepG2-GCGR.

2.3.2.1 Primary mouse hepatocytes

The effects of GCGR interactors on cAMP production in primary hepatocytes was assessed

using 1.0 nM glucagon, as determined from the EC50 in Figure. 10A. Overexpression of LDLR

and TMED2 significantly increased glucagon-induced cAMP accumulation to 127.74±2.1%

Figure 10. A. Glucagon-cAMP dose-response curve in primary hepatocytes demonstrates isolated

hepatocytes signalling pathway remains intact following isolation. B. cAMP production in primary

hepatocytes overexpressing GCGR accessory proteins of interest. LDLR and TMED2 significantly

increase cAMP production above control, while YWHAB significantly inhibits cAMP production. cAMP

assay performed by Froese. ** = p<0.01. (Han et al., 2015)

28

(p<0.01, N = 6) and to 80.4±2.13% (p<0.05, N = 6) respectively as anticipated based on the results

of the glucose production experiments (Figure. 10B). YWHAB significantly attenuated cAMP

production in response to glucagon by 57.24±7.9% (p<0.01, N = 6, Figure. 10B). Previous studies

have demonstrated that increased cAMP production is the key factor of GCGR mediated glucose

production6. Thus, the increase in cAMP production with LDLR and TMED2 overexpression, and

the decrease in cAMP production with YWHAB overexpression is a plausible explanation for the

changes to glucose production seen in Figure. 9.

2.3.2.2 CHO and HepG2-GCGR cells

Consistent with the cAMP production in primary hepatocytes, YWHAB overexpression

significantly decreased 1.0 nM glucagon induced cAMP accumulation in both CHO co-transfected

with GCGR and HepG2-GCGR cells (p<0.05, N = 3, Figure. 11A and 11B). In CHO cells,

overexpression of LDLR and TMED2 led to a significant increase in glucagon induced cAMP

production (Figure. 11A). Curiously, in HepG2-GCGR cells overexpressing LDLR and TMED2,

Figure 11. cAMP production in A CHO cells and B HepG2-GCGR cells overexpressing GCGR accessory

proteins of interest. Only YWHAB showed a consistent effect on cAMP production between cell lines,

that is, an inhibition of cAMP production. LDLR and TMED2 increase cAMP production in CHO cells

only. C. No change in forskolin stimulated cAMP production was found between YWHAB over

expressing CHO cells and control indicating YWHAB’s is not a general adenylyl cyclase inhibitor. cAMP

assay performed by Froese. * = p<0.05, ** = p<0.01. (Han et al., 2015)

29

no significant difference in cAMP production was found. To test whether the suppression of cAMP

production in cells overexpressing YWHAB was due to an overall suppression of adenylyl cyclase,

or the glucagon receptor itself, YWHAB overexpressing CHO cells were stimulated with

forskolin. Stimulation with forskolin did not affect cAMP production in CHO cells expressing the

GCGR (Figure. 11C), indicating YWHAB’s suppressive effects are mediated via action upstream

of adenylyl cyclase, likely the GCGR.

2.3.3 Expression of key gluconeogenic genes in primary mouse hepatocytes with

glucagon receptor interactor overexpression

Gluconeogenesis is in part responsible for the increased glucose production in the liver

following glucagon stimulation. Increased cAMP production leads to an increase in PKA activity,

enhancing gluconeogenesis131. Since LDLR, TMED2 and YWHAB were all shown to alter cAMP

production in primary mouse hepatocytes and two cells lines, changes in expression of key

gluconeogenic genes was assessed using qPCR. Two enzymes,

phosphoenolpyruvatecarboxykinase (PEPCK) and glucose-6-phosphatase (G6Pase) are critical

regulators of the rate limiting steps in gluconeogenesis and GCGR-mediated glucose

production132. One of the earliest steps in gluconeogenesis is the conversion of oxaloacetate to

phosphoenolpyruvate, catalyzed by PEPCK, and the final step of gluconeogenesis is the hydrolysis

of glucose-6-phosphate to glucose and inorganic phosphate, catalyzed by G6Pase. Following

overexpression of LDLR, TMED2, and YWHAB in primary hepatocytes, cells were treated with

or without glucagon, and prepared for qPCR. In the basal glucagon condition, LDLR, TMED2 and

YWHAB overexpression had no effect on gene expression of either PEPCK or G6Pase (Figure

12A and 12B). However, when stimulated with glucagon, expression of PEPCK and G6Pase in

LDLR- overexpressing hepatocytes was significantly increased to 76.52±2.3% and 108±1.7%

respectively (p<0.01, N = 6, Figure. 12A 12B). TMED2 was also shown to upregulate G6Pase (but

not PEPCK) gene expression, significantly to 114.15±3.5% (p<0.01, N = 6) following glucagon

stimulation (Figure 12B). Since LDLR and TMED2 over-expression led to increased expression

of gluconeogenic genes following stimulation with glucagon, it is possible that LDLR and TMED2

may be involved in enhancing gluconeogenesis in the liver. On the other hand, overexpression of

YWHAB lead to a significant reduction in PEPCK and G6Pase gene expression following

30

glucagon stimulation to

52.31±1.4% (p<0.05, N = 6)

and 41.61±0.8% (p<0.01, N =

6) respectively (Figure 11A

and 11B). Thus, as opposed to

LDLR and TMED2, YWHAB

may be involved in supressing

gluconeogenesis in the liver

following glucagon

stimulation.

In the context of a

potential treatment for TD2,

the ability of YWHAB to

supress glucagon-stimulated

cAMP and glucose production

as well as gluconeogenic gene

expression is a valuable

finding. For this reason,

YWHAB was selected as the

GCGR accessory protein of

primary interest, and

conducted further studies to

elucidate the mechanism

through which these effects are

accomplished.

2.3.4 YWHAB does not alter cellular proliferation

YWHAB has been found previously to be involved in proliferative pathways where it

interacts with RAF kinases and enhances their activity133,134. To determine if YWHAB’s effects

on cellular proliferation, the XTT Cell Proliferation Assay Kit was used. Following overexpression

Figure 12. mRNA expression of A PEPCK and B G6Pase in primary

hepatocytes transfected with GCGR interactors of interest. LDLR

significantly increased expression of both genes, while TMED2 only

increased expression of G6Pase. YWHAB overexpression led to a

significant decrease in expression of both gluconeogenic genes.

qPCR performed by Froese, Han, and Zhang. * = p<0.05, ** = p<0.01

(Han et al., 2015)

31

of YWHAB in CHO cells and

recovery for 24 hours, cells

XTT reagent was added and

absorbance was measured

every hour for 4 hours.

Overexpression of empty

vector, YWHAB, GCGR or

YWHAB and GCGR co-

transfection had no significant

effect on cellular proliferation

in CHO cells. Treatment of

cells overnight with 100 mM

hydrogen peroxide lead to a

significant decrease in cell

proliferation (not shown).

These results further support

the notion that YWHAB’s ability to lower gluconeogenesis is due specifically to its interaction

with the GCGR, and not on targets downstream of the receptor.

2.3.5 YWHAB overexpression decreases cell surface expression of GCGR in

HepG2-GCGR cells

Thus far, the mechanism through which YWHAB suppresses gluconeogenesis was not

found to be the result of inhibition of adenylyl cyclase (Figure. 11C), or changes to cellular

proliferation (Figure. 13). Since YWHAB physically interacts with the GCGR, as determined by

the initial AP-MS screen and Co-immunoprecipitation validation, it was hypothesised that

YWHAB may affect expression of the GCGR at the cell surface. To investigate this further, cell

surface isolation experiments were performed in HepG2-GCGR cells the GCGR was probed

following stimulation with glucagon for 15 minutes60, the time required for maximal endocytosis

of the glucagon receptor following stimulation by glucagon. Following stimulation with glucagon

and isolation of cell surface proteins, cell surface expression of the GCGR was found to be

Figure 13. Cellular proliferation in CHO cells under a variety of

transfection conditions determined with the XTT proliferation assay

after 4 hours. No change in proliferation was present between any

of the groups suggesting YWHAB’s effects may be independent of

proliferation. XTT assay performed by Froese. Unpublished.

32

decreased in HepG2-GCGR cells overexpressing YWHAB compared to PcDNA3.1 control

(Figure. 14A). Densitometry showed an approximate 33% reduction of cell surface GCGR

expression with YWHAB overexpression (Figure. 14B). These results indicate that YWHAB’s

physical interaction with the GCGR may be involved in mediating its endocytosis into the cell

following ligand stimulation. If YWHAB enhances endocytosis of the GCGR, this may explain

the YWHAB-mediated suppression of cAMP and glucose production.

2.3.6 siRNA knockdown of YWHAB enhances cAMP production and decreases

GCGR gene expression

Thus far, overexpression of YWHAB has been the approach to study the function and

mechanism of YWHAB’s interaction with GCGR. While YWHAB has been shown to inhibit

glucose production in primary hepatocytes, it is possible that YWHAB is part of a larger process

and its individual contribution to this suppression is minimal. To further determine the impact of

YWHAB on glucose production, YWHAB siRNA knockdown was performed in primary mouse

hepatocytes. qPCR revealed high level of expression of YWHAB in primary mouse hepatocytes,

nearly 3-fold great than β-actin, making it a suitable candidate for siRNA knockdown (Figure. 15).

Figure 14. A. Cell surface expression of GCGR and GLUT2 (control) in transfected HepG2-GCGR cells

following stimulation with glucagon. B. Densitometry used to quantify expression of the GCGR

revealed a decreased of nearly one-third in cell surface expression of the GCGR in YWHAB over

expressing cells after normalizing bands to the loading control. Cell surface isolation performed by

Froese and Zhang, Western blot performed by Froese. * = p<0.05. Unpublished.

A B

33

Following siRNA knockdown, primary hepatocytes

were either stimulated with glucagon and subjected to

measurements of cAMP production, or lysed and

prepared for qPCR. Stimulation of primary

hepatocytes with YWHAB knockdown showed a

significantly higher glucagon-induced cAMP response

compared to the scramble control (Figure. 16). The

fact that knockdown of YWHAB was able to reverse

the effects on cAMP production seen with

overexpression indicates that YWHAB alone may

contribute to the suppression of gluconeogenesis upon

glucagon stimulation of the GCGR. Endogenous

levels of YWHAB may be significant contributors to

suppression of GCGR signalling.

Interestingly,

qPCR in primary

mouse hepatocytes

with knockdown of

YWHAB showed a

significant reduction

in mRNA expression

of the GCGR

(Figure. 17). When

overexpressed,

YWHAB caused a

significant reduction

in expression of

GCGR on the cell

Figure 15. qPCR measurements of

endogenous expression of YWHAB and

GCGR in primary hepatocytes revealed an

abundance of both genes, indicating that

YWHAB and GCGR are viable targets for

siRNA knockdown. qPCR performed by

Froese. ** = p<0.01. Unpublished.

Figure 16. cAMP production in primary hepatocytes following siRNA

knockdown of YWHAB lead to an increase in glucagon-induced cAMP

production compared to the scramble control at a number of different

doses of glucagon. siRNA knockdown and cAMP assay performed by

Froese. * = p<0.05 Unpublished.

34

surface (Figure. 14). Ordinarily, an increase in endocytosis

of GCGR mediated by YWHAB would lead to an increase

in the recycling and degradation of the receptor73,74,79,80,81.

Degraded GCGR must be then replaced via transcription of

mRNA and translocation to the cell membrane. Decreased

YWHAB expression would lead to decreased endocytosis

of the GCGR, and thus less GCGR would need to be

replaced via transcription, which may explain in part the

observation of decreased GCGR mRNA expression. This

decrease in GCGR mRNA expression following

knockdown of YWHAB may further explain the

mechanism through which YWHAB inhibits

gluconeogenesis.

Figure 17. mRNA expression of the

GCGR in primary hepatocytes

following siRNA knockdown of

YWHAB. Knockdown of YWHAB caused

a significant decrease in the expression

of GCGR compared to control,

indicating endogenous YWHAB may

lead to increases in GCGR mRNA

expression. siRNA knockdown and

qPCR performed by Froese. ** =

p<0.05 Unpublished.

35

Chapter 3

Discussion

3.1 Summary

Protein-protein interactions have been shown to be critical regulators of GPCR function,

and vast interacting protein (interactome) networks have been identified for a number of receptors,

including the GLP-1R by our lab119. These accessory proteins can have significant effects on

receptor function, activity and cell surface expression, but little is known about the glucagon

receptor’s interactome. Affinity purification and tandem mass spectrometry performed previously

by the Wheeler lab revealed 33 potential GCGR interactors, 8 of which were selected based on

their previously reported functional roles in signal transduction, as well as their cellular locations.

Following selection of these 8 interactors, identification of their functional and mechanistic

characteristics was determined.

3.1.1 Effects of select glucagon receptor interactors on receptor function

From 8 select GCGR interactors, 5 were successfully validated with co-

immunoprecipitation and were selected for further functional and mechanistic characterisation. In

the context of T2D, chronic activation of the GCGR by elevated glucagon levels leads to an

exaggerated increase in gluconeogenesis. Glucose production was therefore selected as the initial

functional output to determine the effects of these 5 validated interactors. Two interactors, CAV1

and GALK1 were found to enhance glucose production under basal glucagon conditions, while

the remaining three, LDLR, TMED2 and YWHAB, were found to significantly alter glucose

production following glucagon stimulation in primary hepatocytes, with LDLR and TMED2

enhancing glucagon-stimulated glucose production and YWHAB supressing it. LDLR, TMED2

and YWHAB were selected for further analysis based on their function under elevated glucagon

conditions.

Since glucagon-stimulated glucose production is mediated by the cAMP-PKA pathway,

the effects of overexpression of these three interactors on cAMP production in primary mouse

hepatocytes was measured. Glucagon-cAMP dose response curves were determined for transfected

CHO and HepG2 cells to ensure proper pathway function, and to determine the optimal dose of

36

glucagon that would allow both increases and decreases in cAMP production to be measureable.

Mirroring the trends seen in glucose production, LDLR and TMED2 significantly enhanced

glucagon-stimulated cAMP production, while YWHAB overexpression significantly impaired

cAMP production. To validate these results, cAMP production was measured in interactor

overexpressing CHO and HepG2-GCGR cells. In CHO cells, LDLR and TMED2 significantly

increased cAMP production, while YWHAB supressed it, consistent with the results seen in

primary hepatocytes. Interestingly, in HepG2-GCGR cells, only YWHAB altered cAMP

production, lowering cAMP production as seen in the primary hepatocytes and CHO cells.

Forskolin stimulated cAMP production was consistent between control and YWHAB

overexpressing CHO cells indicating that YWHAB’s effects on cAMP production are likely not

the result of an overall inhibition of cAMP production, and is rather the result of its interaction

with the GCGR itself, or Gas.

3.1.2 Effects of interactor overexpression on expression of key

gluconeogenic genes

In addition to phosphorylation of key gluconeogenic enzymes by cAMP-PKA activation,

changes to gluconeogenic gene expression provide a further increase in glucose production and

suppression of glucose storage and breakdown following glucagon stimulation. Thus, the

expression of key gluconeogenic genes was measured in primary hepatocytes overexpressing

LDLR, TMED2 or YWHAB. Two genes, PEPCK and G6Pase, regulate key steps in

gluconeogenesis and were selected for qPCR analysis. qPCR revealed that LDLR overexpressing

primary hepatocytes significantly increased PEPCK and G6Pase expression following glucagon

stimulation. TMED2 had no effect on PEPCK expression, but was shown to enhance expression

of G6Pase. On the other hand, YWHAB overexpressing primary hepatocytes has significantly

lower levels of both PEPCK and G6Pase. These results suggest that in addition to their effects

exerted through direct interaction with the GCGR, LDLR, TMED2 and YWHAB may have

additional effects on glucose production by altering expressing of key gluconeogenic genes. CREB

regulates transcription of key gluconeogenic genes, and these alterations to cAMP production with

GCGR receptor overexpression may in part explain these results. Each of these three interactors

are highly interesting GCGR interactors which profoundly alter glucose production in primary

hepatocytes and two cells lines, and represent potential targets for future therapeutic research.

37

3.1.3 Potential mechanism of YWHAB mediated reduction in cAMP

production

YWHAB was shown to inhibit glucose production in primary hepatocytes, and inhibited

cAMP production in both primary hepatocytes and two cells lines. In the context of T2D, the

ability to supress glucose production is of particular importance. Elevated glucagon levels in type

2 diabetics leads to chronic activation of the GCGR, leading to exaggerated glucose production.

Since YWHAB lowers the activity of the GCGR, it was selected for further mechanistic studies

Since YWHAB has been shown to play a role in cell cycle regulation and proliferative

pathways, it was first determined whether the inhibition of glucose production previously

identified was the result of changes to cell proliferation. Cell proliferation assays revealed no

difference in proliferation of CHO cells overexpressing YWHAB compared to control, further

suggesting YWHAB’s ability to supress glucose production is the result of its direct interaction

with the GCGR.

Next, the cell surface expression of the glucagon receptor in cells overexpressing YWHAB,

both in the presence and absence of glucagon stimulation was investigated. Decreased cell surface

expression of the GCGR could in part explain the effects of YWHAB. Indeed, cell surface

expression studies revealed a significant decrease in cell surface expression of the GCGR

following overexpression of YWHAB. Thus, YWHAB may mediate the internalization of GCGR,

decreasing its cell surface expression and lowering cAMP-PKA pathway activation in hepatocytes.

Overexpression of YWHAB was shown to decrease glucose and cAMP production in

primary hepatocytes, but the significance of YWHAB alone was yet to be determined. To this end,

siRNA knockdown of YWHAB was used to determine the impact of YWHAB alone on cAMP

production. YWHAB knockdown lead to a partial rescue of the attenuated glucagon stimulated

cAMP production, indicating that YWHAB has a direct and significant role in regulating glucose

production in primary hepatocytes. High levels of endogenous YWHAB mRNA expression in

primary mouse hepatocytes as determined through qPCR further supports this notion.

Last, to further explore the mechanism of YWHAB’s suppression of glucose production,

mRNA expression of the GCGR was measured to determine if the observed decreased cell surface

expression of the GCGR was the result of changes to receptor gene transcription. Endogenous

38

mRNA expression of the GCGR in primary hepatocytes following knockdown of YWHAB was

significantly decreased compared to scramble control which may be explained by my hypothesis

that YWHAB increases internalization of the GCGR. Increased internalization and degradation of

the GCGR would lead to an increase in GCGR transcription. A decrease in YWHAB, as in my

knockout model, would prevent increased internalization and reduce GCGR degradation,

eliminating the need to synthesize new GCGR, decreasing the level of GCGR mRNA. The current

working model of YWHAB’s effects on the GCGR are summarized schematically in Figure. 18.

Figure 18. Schematic of the proposed mechanism through which YWHAB decreases glucose production

and GCGR signalling. YWHAB mediates the interaction between the GCGR and endocytotic machinery.

Increased YWHAB leads to an increase in the endocytosis of the GCGR, leading to an eventual increase

in degradation and an increase in gene expression to restore normal levels of the receptor.

39

Chapter 4 Future Directions and Conclusions

4.1 Future Directions

The proposed mechanism through which YWHAB lowers glucose production in primary

hepatocytes involves the mediation of receptor endocytosis following ligand stimulation,

decreasing cell surface expression of the GCGR and lowering activity of the GCGR-cAMP-PKA

pathway. Future studies involving fluorescently labelled receptor, such as the fluorogen activating

peptide system, in the presence and absence of YWHAB would allow for the visualization of

GCGR expression and would provide compelling evidence to support or refute this proposed

mechanism.

While PEPCK and G6Pase were selected as key gluconeogenic genes, glycogen synthase

also plays an important role in glucose regulation, as glycogen synthase is responsible for storing

glucose as glycogen. Future studies should investigate gene expression of glycogen synthase in

the presence of these GCGR interactors, as well as expression of glycogen synthase kinase, the

enzyme responsible for the activation of glycogen synthase.

While the focus of this thesis was the mechanism through which YWHAB inhibits glucose

production in primary hepatocytes, exploration of the mechanism of LDLR and TMED2 mediated

increases in glucose production would also be highly beneficial to the study of type 2 diabetes.

Determining the mechanism through which GCGR interactors enhance glucose production could

lead to the revelation of potential targets for suppression that would in turn decrease glucose

production. In addition, two of the other early identified GCGR interactors, CAV1 and GALK1

should be characterized and explored further.

Furthermore, future studies could identify the GCGR interactome in primary human

hepatocytes. In this study, the interactome of the human GCGR was performed in CHO cells,

which may lack proteins found in primary human hepatocytes. Performing an AP-MS screen in

primary hepatocytes may reveal addition GCGR accessory proteins that have potent effects on

receptor function.

40

The use of YWHAB knockout mice may prove invaluable in determining the true clinical

impact this GCGR accessory protein has. It is possible that knockout of YWHAB may be fatal, as

YWHAB belongs to a family of highly conserved proteins that may be critical for survival.

However if YWHAB knockout mice survive, fasting glucose and oral glucose tolerance tests may

reveal interesting changes to blood glucose regulation in diabetic mice. The true impact that

YWHAB may have on GCGR activity can only be assessed in animal models, and is a logical next

step for future investigation.

4.2 Conclusions

GPCR accessory proteins have been shown to have profound effects on receptor activity

and expression, with the GCGR being no exception. Three were shown to significantly alter

glucose production in primary hepatocytes following glucagon stimulation, with one, YWHAB,

significantly inhibiting glucose production. These effects appear to be the result of YWHAB’s

mediation of endocytosis of stimulated GCGR, reducing its cAMP-PKA activation. My project

has identified novel regulators of GCGR function, and has identified potential novel targets for

future T2D treatments.

41

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