Comprehensive Summaries of Uppsala Dissertationsfrom the Faculty of Medicine 1105

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Alpha-2 Adrenergic Receptorsand Signal Transduction

Effector Output in Relation to G-Protein Couplingand Signalling Cross-Talk

BY

JOHNNY NÄSMAN

ACTA UNIVERSITATIS UPSALIENSISUPPSALA 2001

Dissertation for the Degree of Doctor of Philosophy (Faculty of Medicine) in Physiologypresented at Uppsala University in 2002

ABSTRACT

Näsman, J. 2001. Alpha-2 Adrenergic Receptors and Signal Transduction. Effector Output inRelation to G-Protein Coupling and Signalling Cross-Talk. Acta Universitatis Upsaliensis.Comprehensive Summaries of Uppsala Dissertations from the Faculty of Medicine 1105. 64pp. Uppsala. ISBN 91-554-5195-0.

The alpha-2 adrenergic receptor (α2-AR) subfamily includes three different subtypes, α2A-,α2B- and α2C-AR, all believed to exert their function through heterotrimeric Gi/o-proteins. Thepresent study was undertaken in order to investigate subtype differences in terms of cellularresponse and to explore other potential signalling pathways of α2-ARs.

Evidence is provided for a strong Gs-protein coupling capability of the α2B-AR,leading to stimulation of adenylyl cyclase (AC). The difference between the α2A- and α2B-ARsubtypes, in this respect, was shown to be due to differences in the second intracellular loopsof the receptor proteins. Substitution of the second loop in α2A-AR with the correspondingdomain of α2B-AR enrolled the chimeric α2A/α2B receptor with functional α2B-AR properties.Dual Gi and Gs coupling can have different consequences for AC output. Using coexpressionof receptors and G-proteins, it was shown that the ultimate cellular response of α2B-ARactivation is largely dependent on the ratio of Gi- to Gs-protein amounts in the cell. Also Gi-and Go-proteins appear to have different regulatory influences on AC. Heterologousexpression of AC2 together with Gi or Go and the α2A-AR resulted in receptor-mediatedinhibition of protein kinase C-stimulated AC2 activity through Go, whereas activation of Gipotentiated the activity.

α2-ARs mobilize Ca2+ in response to agonists in some cell types. This response wasshown to depend on tonic purinergic receptor activity in transfected CHO cells. Eliminationof the tonic receptor activity almost completely inhibited the Ca2+ response of α2-ARs.

In conclusion, α2-ARs can couple to multiple G-proteins, including Gi, Go and Gs. Thecellular response to α2-AR activation depends on which receptor subtype is expressed, whichcellular signalling constituents are engaged (G-proteins and effectors), and the signallingstatus of the effectors (dormant or primed).

Key words: Adrenergic, receptor, G-protein, adenylyl cyclase, cAMP, calcium, cross-talk.

Johnny Näsman, Department of Physiology, Uppsala University, Biomedical Center, P.O.Box 572, SE-751 23 Uppsala, Sweden

Johnny Näsman 2001

ISSN 0282-7476ISBN 91-554-5195-0

Printed in Sweden by Uppsala University, Tryck & Medier, Uppsala 2001

The thesis is based on the following papers, which are referred to in the text by their

Roman numerals:

I Dual signalling by different octopamine receptors converges on adenylate

cyclase in Sf9 cells. Näsman J., Kukkonen J.P., Åkerman K.E.O. Insect

Biochem. Mol. Biol., In press 2001.

II The second intracellular loop of the α2-adrenergic receptors determines

subtype-specific coupling to cAMP production. Näsman J., Jansson C.,

Åkerman K.E.O. J. Biol. Chem., 272, 9703-9708, 1997.

III Role of G-protein availability in differential signaling by alpha 2-

adrenoceptors. Näsman J., Kukkonen J.P., Ammoun S., Åkerman K.E.O.

Biochem. Pharmacol., 62, 913-922, 2001.

IV Endogenous extracellular purine nucleotides redirect α2-adrenoceptor

signaling. Åkerman K.E.O., Näsman J., Lund P.-E., Shariatmadari R.,

Kukkonen J.P. FEBS Lett., 430, 209-212, 1998.

V Modulation of adenylyl cyclase type 2 activity by Gi/o-protein coupled

receptors. Opposite regulation by Gi and Go. Näsman J., Kukkonen J.P.,

Holmqvist T., Åkerman K.E.O. Manuscript 2001.

Reprints were made with permission from the publishers.

CONTENTS

Abbreviations............................................................................................................ 3

1 Introduction ........................................................................................................... 4

1.1 General background............................................................................................ 4

1.2 G-protein coupled receptors and signal transduction......................................... 5

1.2.1 Receptors .............................................................................................. 6

1.2.2 Transducers .......................................................................................... 7

1.2.3 Effectors ............................................................................................. 10

1.2.3.1 Adenylyl cyclase, cAMP, and PKA..................................... 10

1.2.3.2 Phospholipase C, Ca2+, and PKC ......................................... 13

1.3 α2-adrenergic receptors .................................................................................... 15

1.3.1 A subfamily of adrenergic receptors .................................................. 15

1.3.2 Physiological functions ...................................................................... 17

1.3.3 Cellular signalling .............................................................................. 19

1.4 Other receptors utilized in the study................................................................. 21

1.4.1 Octopamine receptors......................................................................... 21

1.4.2 Purinergic receptors............................................................................ 22

1.4.3 Muscarinic receptors .......................................................................... 23

1.5 Why study heterologously expressed receptors? ............................................. 23

2 Aims of the study................................................................................................. 25

3 Materials and methods......................................................................................... 26

3.1 Cell cultures........................................................................................... 26

3.2 BEVS and infection procedures ............................................................ 26

3.3 Receptor quantification ......................................................................... 27

3.4 Receptor−G-protein interactions ........................................................... 27

3.5 Measurement of cAMP ......................................................................... 28

3.6 Measurement of Ca2+............................................................................. 29

3.7 Mathematical modelling........................................................................ 29

4 Results ................................................................................................................. 30

4.1 Receptor signalling in Sf9 cells............................................................. 30

4.2 Propensity of α2-ARs for Gs coupling................................................... 32

4.3 Signalling cross-talk .............................................................................. 34

5 Discussion............................................................................................................ 36

5.1 Suitability of Sf9 cells for receptor characterization............................. 36

5.2 Promiscuous G-protein coupling........................................................... 39

5.3 Adenylyl cyclase regulation by the Ca2+ signalling pathway ............... 42

5.4 The α2-AR−Ca2+ link ............................................................................ 43

6 Summary and conclusions................................................................................... 46

7 Acknowledgements ............................................................................................. 48

8 References ........................................................................................................... 50

3

ABBREVIATIONS

AC adenylyl cyclase

AR adrenergic receptor (as in α-AR and β-AR)

BEVS Baculovirus Expression Vector System

[Ca2+]i intracellular concentration of free Ca2+

CaM calmodulin

CaMK calmodulin kinase

CHO Chinese hamster ovary (a cell line)

Ctx cholera toxin

DAG diacylglycerol

GPCR G-protein coupled receptor

Gx-protein heterotrimeric GTP-binding protein with x identity of α subunit

Gαx α subunit of heterotrimeric G-protein with x identity

Gβγ βγ subunit complex of heterotrimeric G-protein

i2/i3-loop second/third intracellular loop of receptors

IBMX 3-isobutyl-1-methylxanthine

IP3 inositol 1,4,5-trisphosphate

PDE phosphodiesterase

PH pleckstrin homology

PI phosphtidylinositol

PIP2 phosphtidylinositol 4,5-bisphosphate

PK protein kinase (as in PKA and PKC)

PL phospholipase (as in PLC, PLA2, PLD)

Ptx pertussis toxin

Sf9 Spodoptera frugiperda (a cell line)

TM transmembrane domain of receptors

TPA 12-O-tetradecanoyl phorbol-13-acetate

4

1 INTRODUCTION

1.1 General background

All cells, whether single cell organisms or entities within multicellular organisms,

need to sense the exterior to be able to adapt to changes. In the course of evolution, a

multitude of such sensing molecules has evolved. A glimpse of this multitude comes

from the realization that cells of our own bodies are regulated by a diversity of signals

ranging from physical phenomena, such as light, through elementary ions, like

calcium, to numerous hormones and neurotransmitters. The action of hormones and

neurotransmitters is of prime importance and interest, which is reflected by the fact

that the majority of drugs used in medical care are targeted toward the sites of action

of these molecules. Some hormones are lipophilic and, thus, readily traverse the cell

membrane to exert their effects in the cell. Other hormones, and the neurotransmitters,

bind to and activate plasma membrane receptors to convey the signal to the interior of

the cell. In this way, a cell will be predisposed to the action of a particular hormone

only if the appropriate receptor is expressed, thereby ascertaining a level of selectivity

in hormone signalling. A majority of the cell surface receptors, including the α2-

adrenergic receptors (α2-ARs), belong to the superfamily of G-protein coupled

receptors (GPCRs).

Intracellular signalling by GPCRs is an area that has generated a large amount

of scientific knowledge during the last decades. A major breakthrough in elucidation

of hormone action via receptors came from work by Earl Sutherland in the 1950s and

1960s. Studying the effect of adrenaline and glucagon on glycogen metabolism in the

liver, he discovered that the hormone action on glycogen phosphorylase can be

reconstituted in a cell-free system, as long as the particulate fraction of the cell

material is included. This led to the discovery of adenosine cyclic 3’,5’-monophosphate

(cAMP), a messenger molecule between hormone receptor and target enzyme, and the

identification of the enzyme responsible for its synthesis, adenylyl cyclase (AC)

(reviewed in Sutherland, 1972). Since then, cAMP has been implicated, not only in

metabolic regulation, but in virtually all physiological processes studied. Most of the

5

actions of cAMP are mediated by protein kinase A (PKA). Another ubiquitous

enzyme, involved in signal interpretation, is protein kinase C (PKC). A role for PKC

in intracellular signalling was not recognized until it was demonstrated that the

enzyme was activated by diacylglycerol (DAG) (reviewed in Nishizuka, 1984), a

product of phosphatidylinositol (PI) breakdown resulting from receptor activation

(Berridge, 1987). Activation of PKC is often implicated in physiological processes

such as cellular proliferation and differentiation (see e.g. Watters & Parsons, 1999).

The PI breakdown also has consequences for the homeostasis of calcium ions (Ca2+)

in cells. A physiological role for Ca2+ has been known for a long time (Ringer, 1883),

but many aspects of Ca2+ signalling by receptors, and cellular consequences of Ca2+

transients, are still not clarified.

cAMP, DAG, and Ca2+ are usually classified as second messenger molecules.

The regulation of the cellular concentration of second messengers by GPCRs is

fundamental to a precise regulation of the cellular machinery. One way to achieve an

understanding of this precision is to elucidate the function of individual receptors in

relation to the different signalling components with which they interact. The goal of

the present study was to accomplish a step in this direction.

1.2 G-protein coupled receptors and signal transduction

Over one percent of the human genome encodes for more than 1000 putative GPCRs

(Flower, 1999). These have probably evolved as a consequence of the variety of

signals a highly developed organism needs to respond to. GPCRs are intimately

involved in most physiological processes that are under hormonal regulation. In

addition, synaptic transmission, vision, chemotaxis, as well as perception of taste and

smell, are also governed by GPCRs. It is therefore not surprising that the same

signalling theme has a long evolutionary history; the yeast pheromone response

pathway regulating mating behaviour also being relayed by GPCRs and G-proteins

(Blumer & Thorner, 1991).

GPCRs are, as the name implies, coupled to guanosine-5’-triphosphate (GTP)-

binding proteins (G-proteins) of heterotrimeric α, β, γ composition. The G-proteins are

6

known as ’transducers’, transducing the signals to effector proteins. The signal

transition from receptor to effector is thus dependent on protein−protein interactions

and is delimited to the plasma membrane. The signal is further conveyed from the

effector through diffusible second messengers, enabling a spreading of the

intracellular signal.

As indicated earlier, GPCR signalling is often a target for therapeutic

intervention. Perturbation of normal signalling can sometimes have severe

consequences. This is exemplified by the numerous activating mutations of GPCR

signalling components found in different kinds of tumours (Marinissen & Gutkind,

2001). Other examples are the bacterial exotoxins of Bordetella pertussis (pertussis

toxin, Ptx) and Vibrio cholerae (cholera toxin, Ctx), both of which perturb G-protein

function (Milligan, 1988).

1.2.1 Receptors

The term ’receptive substance’, or receptor as we now call it, was coined by J. N.

Langley in the early 20th century when studying the interaction of nicotine and curare

on neuromuscular transmission (Langley, 1905). A long time passed before the

’receptive substances’ took the form of defined structural proteins. The first GPCR to

be purified, sequenced (Ovchinnikov, 1982), and eventually cloned (Nathans &

Hogness, 1983) was bovine rhodopsin. Based on the predicted structural topography,

with seven hydrophobic amino acid stretches presumed to adopt membrane-spanning

α helices, the rhodopsin protein showed similarities to bacteriorhodopsin, a bacterial

proton pump for which the structure had been determined (Henderson & Unwin,

1975). However, it was not clear from the rhodopsin sequence that other receptors

would have a similar structural arrangement. The purification of the β-ARs was

accomplished in the early 1980s (Shorr et al., 1981, Shorr et al., 1982) and

reconstitution experiments with receptor, G-protein, and AC confirmed the hypothesis

of a three-component signalling system (May et al., 1985, Feder et al., 1986). In 1986,

two independent groups reported the cloning of β-ARs, the turkey β1-AR (Yarden et

al., 1986) and the hamster β2-AR (Dixon et al., 1986). The β-ARs show an overall

7

structural similarity to rhodopsin, with seven putative transmembrane helices (TMs)

connected by hydrophilic intra- and extracellular loops, although the primary amino

acid sequence homology to rhodopsin is weak.

After the successful cloning of the β-ARs, it was soon noted that GPCRs

constituted a large family of similarly structured proteins. The multitude of cloned

GPCRs reported during the following years set the starting point for heterologous

expression and thorough investigation of receptor function. All GPCRs are believed to

function through a common molecular mechanism. The binding of an agonist causes

conformational changes in the receptor protein. These changes then lead to activation

of G-proteins by promoting GTP for GDP exchange of the α subunit. The agonist

action on the receptor protein is a poorly understood process, although direct evidence

has been obtained for conformational changes (Gether et al., 1995). How the activated

receptor triggers G-protein activation is also rather poorly understood (Wess, 1998).

On the other hand, molecular cloning of GPCRs provided the means for advanced

structure−function studies and the mapping of receptor interaction sites. The first study

on chimeric α2/β2-receptors, i.e. receptor hybrids with domains from both parental

receptors, was reported in 1988 (Kobilka et al., 1988). It was concluded from the

experiments that a particular section of the β-AR, including transmembrane helices

(TM) five and six and the third intracellular loop (i3-loop) connecting the helices, is

involved in the G-protein interaction. The chimeric receptor approach was soon

realized to be a very efficient mean to map receptor−G-protein interactions, and a

wealth of information has been gathered on this subject (see e.g. Wess, 1997).

1.2.2 Transducers

G-proteins belong to a superfamily of GTPases. A protein that transduces the signal

from receptor to effector was first anticipated by Dr. Rodbell and coworkers in 1971 in

light of the guanine nucleotide requirement for glucagon action on AC (Rodbell et al.,

1971). The AC stimulatory G-protein, Gs, was later purified by Dr. Gilman’s group

and found to be heterotrimeric, composed of an α, a β, and a γ subunit (Northup et al.,

8

1980). Subsequently, the AC inhibitory G-protein, Gi, was identified (Katada & Ui,

1982, Codina et al., 1983, Bokoch et al., 1984). A few years earlier, it had been shown

that catecholamine action on erythrocyte membranes stimulates GTPase activity

(Cassel & Selinger, 1976). Analysis of the purified components revealed that the α

subunits possess the GTPase activity. In the ground state a GDP molecule is bound

and the α subunit is associated with the βγ complex. Receptor activation of the

heterotrimer lowers the affinity for GDP, allowing GTP to bind. Based on in vitro

studies, the effect can be explained by an increase in the affinity of G-proteins for

Mg2+, which, in turn, promotes the nucleotide exchange (Birnbaumer & Birnbaumer,

1995). The α-GTP and βγ complex then dissociates from each other to associate with,

and affect, effector proteins. The dissociation theory is widely used to explain G-

protein action, although not universally accepted (see e.g. Rebois et al., 1997, Chidiac,

1998). The intrinsic GTPase activity of the α subunit then returns the G-protein to the

ground state, accompanied by subunit association.

As for all other components in signal transduction, molecular cloning has revealed a

diversity of G-proteins (reviewed in Gilman, 1987, Hepler & Gilman, 1992,

Hildebrandt, 1997). Four major subfamilies can be outlined based on α subunit

structure: Gs, Gi, Gq, and G12.

The Gs subfamily contains the short and long isoforms of αs, two splice variants

of a single gene, and. a closely related αs isoform, αolf. Golf is primarily found in

olfactory neurons and mediates the effects of odorants to a specific isoform of AC

(Menco et al., 1992). Gs-proteins are ADP-ribosylated by the A (active) protomer of

Ctx, rendering the αs subunit constitutively active by stabilizing the GTP-bound form

of the α subunit (Cassel & Selinger, 1977, Kahn & Gilman, 1984).

The Gi subfamily is the largest, containing three αi subunits, two αo subunits,

αgust, αz, and the transducins αtr and αtc. All Gi subfamily proteins, except αz, are

substrates for Ptx-catalyzed ADP ribosylation, which uncouples the Gi subfamily

proteins from receptors (Ui, 1984, Milligan, 1988). It was by the use of Ptx that Gi was

9

identified as the inhibitory G-protein of AC (Katada & Ui, 1982). Gi subfamily

proteins are also involved in intracellular Ca2+ homeostasis through inhibitory actions

on voltage-gated Ca2+-channels and stimulation of PLCβ isoforms. These functions

are presumably mediated by the Gβγ subunits and can thus also be ascribed to other

G-proteins. A clear-cut effector for Go-proteins has not been identified, despite its

great abundance in the brain (Neer et al., 1984, Sternweis & Robishaw, 1984). Little is

known about Gz, but it may be similar in action to Gi (Kozasa & Gilman, 1995).

The Gq subfamily of proteins include αq, α11, α14, α15, and α16, all of which

activate PLC-β enzymes (reviewed in Rhee, 2001). Expression of G16, and the mouse

orthologue G15, is restricted to cells of hematopoietic origin, whereas the expression

patterns of Gq and G11 are wide.

The function of G12 and G13 is largely unknown, but they appear to possess high

transforming capacity when constitutively activated (for review, see Gudermann et al.,

2000).

The Gβγ subunit family is made up of five β subunit genes and eleven γ subunit genes

(Hildebrandt, 1997). The different possible combinations of these, and their potential

selectivity for specific Gα subunits, may contribute to specificity in receptor

signalling. However, there does not seem to be much specificty in the association of

either Gβγ dimers or Gαβγ trimers, except in a few cases (Fawzi et al., 1991, Schmidt

et al., 1992, Yan et al., 1996). Initially, Gβγ subunits were considered inhibitory

modules of Gα subunits by stabilizing the inactive GDP-bound form of Gα (Cerione

et al., 1986b). Receptor activation of G-proteins reguires the heterotrimeric composi-

tion (Fung, 1983), indicating a role in receptor recognition as well. In addition, Gβγ

subunits are hydrophobic, partially due to a γ-associated prenyl group, having an

important role in anchoring the Gα subunits to the membrane (Sternweis, 1986). The

first demonstrated direct effector action of Gβγ was the activation of K+-channels in

atrial myocytes (Logothetis et al., 1987). Subsequently, Gβγ subunits were shown to

affect several AC isoforms, PLCs of the β family, PLA2, and voltage-gated Ca2+-

10

channels (reviewed in Sternweis, 1994). An interesting aspect of Gβγ signalling is that

the β subunits contain WD40 repeats, about 40 amino acid long stretches with

Trp(W)-Asp(D) dipeptide sequences (Wang et al., 1994). These structures are

believed to specify protein−protein interactions by binding to pleckstrin homology

(PH) domains found in a multitude of signalling molecules, including PLCs and β-AR

kinases (Shaw, 1995).

1.2.3 Effectors

Classically, GPCRs are linked to effectors by G-proteins. This is not true in all cases

(Hall et al., 1999). Nevertheless, a multitude of G-protein−effector interactions have

been demonstrated. The widely accepted effectors, directly affected by G-proteins,

include adenylyl cyclase (AC), cGMP phosphodiesterase (cGMP PDE), phospholipase

C (PLC), and Ca2+- and K+-channels. Here the focus will be on AC and PLC.

1.2.3.1 Adenylyl cyclase, cAMP, and PKA

AC is a ubiquitous enzyme synthesizing cAMP from Mg2+-ATP. Before the molecular

cloning era, the mammalian membrane-bound ACs were divided into a common Ca2+-

insensitive isoform and a neuronal Ca2+/CaM-activated isoform. Pfeuffer and

colleagues managed to purify these rare membrane protein species by the use of

forskolin affinity matrix (Pfeuffer et al., 1985a, Pfeuffer et al., 1985b). In 1989, the

cDNA for the type 1 neuronal isoform (AC1) was reported (Krupinski et al., 1989),

and subsequently a whole family a related isoforms were identified (reviewed in

Sunahara et al., 1996). The topographical structure of the ACs shows similarity to

membrane transporters such as P glycoprotein and the cyctic fibrosis transmembrane

conductance regulator (CFTR). The protein traverses the membrane 12 times in two

cassetts, adopting a pseudo-dimeric structure. The cytoplasmic regions between the

cassettes and distal to the second cassett make up the catalytic center, and are also the

sites of regulation by G-proteins and protein kinases (Tang & Hurley, 1998). ACs are

subject to many regulatory inputs, indicating that the principal control of cellular

11

cAMP concentration lies at the level of its synthesis, and that fine-tuning of the

enzymes is central to the control.

Molecular cloning of the bovine AC1 and the successful expression in Sf9

insect cells (Tang et al., 1991) were important pioneering work for establishing

regulatory properties of ACs. Heterologous expression of different isoforms of ACs

has since then greatly improved our understanding of AC regulation. The mammalian

ACs can be largely grouped into three subfamilies based on regulatory properties

(Table 1): the Ca2+/CaM-stimulated isoforms, the Gβγ-stimulated isoforms, and the

Ca2+-inhibited isoforms. Several AC isoforms, homologous to mammalian ACs, are

also found in invertebrates (Levin et al., 1992, Iourgenko et al., 1997, Korswagen et

al., 1998).

As can be seen from table 1, there is a large potential for cross-talk between

traditional Ca2+ mobilizing receptors and cAMP signalling already at the stage of

cAMP synthesis. Overall, the number of positive and negative regulators of ACs are

well in balance to tightly regulate the cAMP synthesis through a variety of receptors.

An enigma, in this regard, is the Gβγ-stimulated subgroup, and in particular the AC2

isoform that has been demonstrated to have high basal activity (Pieroni et al., 1995),

but no inhibitory input from receptors.

12

Table 1. Regulators of mammalian AC isoforms.

Physiological regulators

Subfamily Isoform Stimulators Inhibitors

Ca2+/CaM-

stimulated

AC1

AC3b

AC8

Gsa, Ca2+/CaM

Gs, Ca2+/CaM

Gs, Ca2+/CaM

Gi, Go, Gβγ, CaMKIV

Gi, CaMKII

Gi

Gβγ-stimulated AC2

AC4

AC7

Gs, Gβγ, PKC

Gs, Gβγ

Gs, Gβγ, PKC

none?

PKCc

?

Ca2+-inhibited AC5d

AC6

AC9f

Gs, PKC

Gs

Gs

Ca2+, Gi, PKA, Gβγe

Ca2+, Gi, PKA, Gβγe

Ca2+/calcineurin, Gi?a AC1 may not be stimulated by Gs activation in vivo due to concomitant inhibition by Gβγ

(Wayman et al., 1994), but it is stimulated in vitro by Gαs (Tang et al., 1991).b Stimulation of this isoform by Ca2+/CaM is dubious, since stimulation in vitro reguires

supraphysiological concentrations of Ca2+ (Choi et al., 1992). c activated PKC reduces Gs- but not forskolin-stimulated activity (Zimmermann & Taussig,

1996).d Inhibition of this isoform by Ca2+ is questionable.e Gβγ effect determined by cotransfection of ACs and Gβγ in COS-7 cells (Bayewitch et al.,

1998).f AC9 is usually not included in this subfamily, but it is inhibited by Ca2+ via calcineurin

(Antoni et al., 1998).

13

cAMP is chemically stable, but it is degraded in cells by PDEs to the inactive

metabolite 5’-AMP. There are at least 15 gene products for cyclic nucleotide PDEs,

grouped into seven subfamilies (Spina et al., 1998). Some of these are specific for

cAMP, some for cGMP, and some are unselective. Since the activity of most of the

PDEs is regulated mainly by substrate availability, the inhibition by different natural or

synthetic agents is in the focus of PDE research. Theophylline (1,3-dimethylxanthine)

is the archetypal PDE inhibitor. IBMX (3-isobutyl-1-methylxanthine) is closely related

to theophylline, show little subtype selectivity (Ukena et al., 1993), and often used in

cell culture experiments to address AC or cAMP functions.

PKA is a multifunctional cAMP-dependent serine-threonine kinase that mediates most

of the effects of cAMP (Walsh & van Patten, 1994). The PKA family of enzymes are

assembled from the products of four Regulatory and, at least, two Catalytic subunit

genes. The R subunits homodimerize, and each R binds one C subunit forming a

tetrameric holoenzyme. Activation is triggered by cAMP binding to the R subunits

and subsequent dissociation of the C subunits. Besides the tight control of the cellular

cAMP concentration, which is crucial for PKA activation, the increased awareness of

compartmentalization in signalling has also led to identification of multiple A Kinase

Anchoring Proteins (AKAPs), providing a degree of subcellular specificity of PKA

action (Dell’Acqua & Scott, 1997).

1.2.3.2 Phospholipase C, Ca2+, and PKC

Phospholipase Cs are a group of enzymes catalyzing the hydrolysis of phosphatidyl-

inositol 4,5-bisphosphate (PIP2) to inositol 1,4,5-trisphosphate (IP3) and diacylglycerol

(DAG). IP3 mobilizes Ca2+ from intracellular depots and DAG is an activator of PKC.

In the 1970s, Ca2+ was being recognized as a messenger molecule of hormone

action (reviewed in Rasmussen, 1970, Exton, 1981). At the same time, the involve-

ment of phosphatidylinositol (PI) in signalling was subjected to investigation (Michell,

1975). Sir Michael Berridge pulled these findings together and showed, with the help

of colleagues, that IP3 was the missing link between receptor activation and Ca2+

14

mobilization (Streb et al., 1983, reviewed in Berridge & Irvine, 1984). This discovery

set the starting point for a whole new research branch trying to characterize PLCs, IP3

action, and Ca2+ signalling. Today at least 11 different isoforms of PLCs, grouped into

four subfamilies, β, γ, δ, and ε, have been identified (Rhee, 2001). The PLC-β

isoforms are subject to GPCR signalling through Ptx-insensitive Gq subfamily of

proteins as well as through Ptx-sensitive G-protein βγ subunits. Four different

isoforms of PLC-βs, β1-4, are all activated by Gαq/11 proteins. PLC-β2 appears to be

restricted to hematopoietic cells, and is likely to be the target of Gα16, which also

activates PLC-β1 and -β3 isoforms (Kozasa et al., 1993). Gβγ has been shown to

interact with PLC-β1, -β2, and -β3, with the highest affinity for PLC-β2 (Runnels &

Scarlata, 1999), by binding to the PH domain of the PLC-βs (Wang et al., 1999).

The IP3 product of PLC action mobilizes Ca2+ from intracellular stores, mainly the

endoplasmatic reticulum. This, in turn, evokes a capacitative inflow of Ca2+ from

extracellular space. Ca2+ acts as a universal messenger involved in crucial events of

life cycle, such as fertilization, muscle contraction, secretion, proliferation, metabo-

lism, and gene expression (Berridge, 1997). It is therefore not surprising that a

multitude of molecules are involved in regulating intracellular Ca2+ homeostasis and

Ca2+ actions. The reviewing of these is beyond the scope of this thesis.

Dr. Nishizuka and colleagues discovered PKC in 1977 (Inoue et al., 1977). This

enzyme was initially identified as a Ca2+-activated, phospholipid-dependent serine-

threonine kinase, with no obvious relation to signal transduction. Around 1980, it was

shown that DAG greatly increases the affinity of PKC for Ca2+ and thereby, in a

sense, activates it (Takai et al., 1979, reviewed in Nishizuka, 1984). As DAG is one of

the products of PIP2 hydrolysis, it is obvious that PKC is a target for PLC-activating

receptors. PKC has long been implicated in cellular transformation due to the tumour

promoting activity of phorbol esters, which bind to and activate PKC (Castagna et al.,

1982).

15

As can be anticipated, the PKC enzymes constitute a family of related proteins that

differ in their tissue distribution and regulatory properties (reviewed in Hug & Sarre,

1993). They can be grouped into three subfamilies based on activation requirements:

the classical PKC isoforms are activated by DAG and phorbol esters in a Ca2+-

dependent way, the novel PKC isoforms are activated by DAG and phorbol esters in a

Ca2+-independent way, and the atypical PKC isoforms are not activated by

conventional GPCR signalling.

1.3 α2-adrenergic receptors

1.3.1 A subfamily of adrenergic receptors

Noradrenaline and adrenaline exert their effects through a large family of GPCRs

encoded by nine different genes in humans, three of each of β-ARs, α1-ARs and α2-

ARs (Pepperl & Regan, 1994). The α-ARs were first divided from β-AR by Dr.

Ahlqvist in the 1940s based on agonist potency differences of evoked physiological

responses (Ahlquist, 1948). Later, the α-ARs were further subdivided into α1- and α2-

ARs based on micro-anatomical location as pre- or postsynaptic receptors, respectively

(Langer, 1974). Isotope labelling of ligands finally enabled a pharmacological

distinction between these subtypes using α1-selective [3H]prazosin and α2-selective

[3H]yohimbine (Starke, 1981). The original classification by Dr. Langer did not hold

for very long as α2-ARs were found both on postsynaptic sites and in nonsynaptic

locations, such as platelets (Berthelsen & Pettinger, 1977).

The human platelet α2A-AR was cloned in 1987 (Kobilka et al., 1987). The

predicted protein is highly homologous to the β-ARs in TM regions. The intracellular

domains differ from the β-ARs, the α2A-AR having a relatively long i3-loop and a

short carboxyl terminus whereas the opposite is true for β-ARs. The expressed

receptor shows similar ligand binding properties as the pharmacologically defined

α2A-AR (Bylund, 1985), with high affinity for yohimbine and low affinity for

16

prazosin. Soon after cloning of the first α2-AR, the α2C-AR (Regan et al., 1988) and

the α2B-AR (Lomasney et al., 1990) subtypes were cloned. All three subtype

homologues in rat and mouse have also been cloned and sequenced (see e.g. Pepperl

& Regan, 1994). The rodent α2A- and α2B-ARs are of some interest to the present

study. The mouse α2B-AR subtype (Chruscinski et al., 1992) is virtually identical to

the human orthologue in the TM regions, but only 64% homologous in the i3-loop,

suggesting that this region need not be highly conserved for preserved functions. The

i2-loops are identical between mouse and human α2B-AR.

The rodent α2A-AR has different antagonist binding properties than the human

α2A-AR. This finding initially classified the rodent receptor as a new pharmacological

subtype, α2D-AR (Lanier et al., 1991). The α2D-AR is now accepted as the orthologue

of human α2A-AR and will be referred to as α2A-AR throughout this study. The basis

for the different affinities of human and mouse α2A-ARs for yohimbine has been

shown to be due, at least partially, to a single amino acid difference in TM5 (Link et

al., 1992).

In the original paper by Dr. Kobilka and collegues on chimeric α2/β2-receptors,

the TM7 of both parent receptors was strongly implicated in agonist and antagonist

binding specificity (Kobilka et al., 1988). However, TM7 has an important role in

intramolecular interactions, stabilizing the three-dimensional conformation of the

receptor (Suryanarayana et al., 1992), and may not be directly involved in ligand

interactions of the α2-ARs. It has been shown that a conserved aspartic acid residue in

TM3 (Asp113 in α2A-AR) provides the counterion to the positively charged amino

group of catecholamine ligands (Wang et al., 1991, for review, see also Savarese &

Fraser, 1992). A serine residue in TM5 (Ser204 in human α2A-AR) is believed to

interact with the para-hydroxyl group of the catechol moiety of the natural ligands

(Wang et al., 1991). Another serine residue (Ser200 in human α2A-AR) has also been

implicated in ligand binding affinity and signalling potency (Wang et al., 1991,

Rudling et al., 1999).

17

1.3.2 Physiological functions

Central effects of α2-AR agonists include hypotension, bradycardia, sedation, sleep

induction, analgesia, mydriasis, and modulation of growth hormone release from the

pituitary (reviewed in Ruffolo et al., 1993, Aantaa et al., 1995). Clonidine has long

been used as an antihypertensive drug in clinical practice. Clonidine stimulation of

central α2-ARs reduces the sympathetic outflow to the periphery, leading to a

reduction in arterial blood pressure and bradycardia. The exact site of action is a

matter of debate, but it appears to include several centers in the medulla including

nucleus tractus solitarius, a center that receives baroreceptor input (Ruffolo et al.,

1993, Aantaa et al., 1995, Nicholas et al., 1996, Singewald & Philippu, 1996).

Sedation is a common side effect of clonidine, and the α2-AR agonists xylasine

and medetomidine are frequently used in veterinary medicine for sedation and also for

analgesia. Locus coeruleus, the predominant noradrenergic nucleus in the mammalian

brain, is rich in α2-ARs and is probably the site of action for the sedative/hypnotic

effect of α2-AR agonists (Correa-Sales et al., 1992).

In the periphery, noradrenaline released from sympathetic nerve endings and

adrenaline released from the adrenal medulla exert the effects of sympathetic

stimulation by activation of adrenergic receptors located presynaptically on nerve

endings, or post- and/or extrasynaptically on target tissues. As Langer pointed out in

his subdivision of adrenergic receptors, α2-ARs are predominantly located on

presynaptic nerve terminals, and when activated inhibit neurotransmitter release

(Langer, 1974). Presynaptic α2-ARs are found on all sympathetically innervated

tissues examined (Ruffolo et al., 1993). Post- and/or extrasynaptic α2-ARs are found

in vascular beds of both arterial and venous origin. Activation of these receptors leads

to smooth muscle contraction and vasoconstriction (reviewed in Langer & Hicks,

1984). It may also lead to vasodilation through increased production of nitric oxide in

the endothelia (Bockman et al., 1993). The arterial α2-ARs are believed to be mainly

extrasynaptic, responding to circulating adrenaline, whereas the postsynaptic receptors

18

would be of α1-AR origin (Ruffolo et al., 1993). Another smooth muscle tissue with

α2-ARs is the uterus (Hoffman et al., 1981). An intersting α2−β2-AR cross-talk in

relation to pregnancy appears to occur in this tissue (Mhaouty et al., 1995). At mid-

pregnancy the myometrial α2A-AR predominates and has the ability to potentiate β-

AR-stimulated cAMP synthesis. This could be a mechanism of keeping the muscles in

a relaxed state. At term, the α2B-AR is upregulated and attenuates the β-AR response

to provide for a forced contraction at delivery.

In several target tissues, α2-AR activation is associated with inhibitory

functions (Ruffolo et al., 1993). In the gastrointestinal tract, secretion and motility is

decreased, and in pancreas, insulin release is inhibited by beta cell α2-ARs. In adipose

tissue, α2-ARs inhibit lipolysis, and in the kidney, α2-ARs counteract vasopressin

action.

A long known target for circulating catecholamines with undisputable

extrajunctional α2-ARs are the platelets. Adrenaline induces aggregation of platelets

and potentiates aggregation induced by other agents such as ADP and thrombin

(Thomas, 1967). However, the physiological benefit of α2-AR-mediated platelet

aggregation is not clear, neither is the intracellular signalling cascade regulating it

(Ruffolo et al., 1993, Keularts et al., 2000).

Most classical functions of α2-ARs are elucidated using clonidine, p-

aminoclonidine, and UK14,304 as selective α2-AR agonists (Berlan et al., 1992,

Hieble et al., 1995). These ligands are unselective for the different subtypes, and most

functions have been related to the α2A-AR subtype based on prazosin insensitivity

(Hieble et al., 1995). Lately, the techniques of gene knock-out or knock-in in mice

have been introduced in an effort to elucidate the in vivo function of different α2-AR

subtypes (reviewed in Kable et al., 2000). Using knock-out mice, it has been shown

that the long-lasting antihypertensive effect of agonists is mediated by the α2A subtype.

Upon administration of the drugs to animals, there is an initial hypertensive pressor

response. This pressor response is absent in α2B-AR knock-out mice, suggesting that

19

the peripheral pressor response is mediated by α2B-AR action on the vasculature.

Other subtype-related effects demonstrated in knock-out mice are the salt-induced

hypertension, which is abolished in α2B-AR knock-outs (heterozygotes), and the

presynaptic inhibition of noradrenaline release, which appears to lack an α2B-AR

component, at least in sympathetic nerves innervating the heart.

1.3.3 Cellular signalling

A diminution of cAMP synthesis through Gi-proteins is the most frequently observed

cellular signalling function of α2-AR activation (Fain & Garcia-Sainz, 1980). The

white adipocytes provides a good example of functional consequences of traditional

inhibition of AC activity. A hormone-sensitive lipase (HSL) in these cells is

responsible for triglyceride breakdown and lipolysis. This HSL is stimulated by PKA-

mediated phosphorylation, following that α2-AR activation decreases the activity of

the AC−PKA−HSL pathway and counteracts β-AR stimulation of the same (Lafontan

& Berlan, 1993).

In general, though, a noticeable direct physiological consequence of α2-ARs

mediated via inhibition of cAMP synthesis seems unusual. Events like inhibition of

insulin release (Ullrich & Wollheim, 1984), inhibition of neurotransmitter release

(Lipscombe et al., 1989), platelet aggregation (Haslam et al., 1978), and mitogenic

signalling through MAPK (Kribben et al., 1997) all appear independent of the cAMP

pathway. Cellular activation of PLC (Dorn et al., 1997), PLA2 (Sweatt et al., 1986),

and PLD (Jinsi et al., 1996) by α2-ARs is even more distant to a cAMP decrease.

Nevertheless, cAMP is easily measured and AC regulation serves as the most reliable

functional indicator for α2-AR activation.

The inhibition of cAMP synthesis by α2-ARs has been studied in numerous

clonal cell lines, expressing endogenous or ectopic α2-ARs, as well as in primary cell

cultures. In some cell types, the cAMP synthesis is increased upon α2-AR activation

or exhibit U-shaped inhibitory and stimulatory regulation depending on agonist conc-

20

entration. Table 2 lists a number of such "anomalous" behaviours of defined receptor

subtypes expressed in different cell types.

Table 2. Stimulatory cAMP responses via α2-ARs.

Receptor Cell type Effect on cAMPa References

α2A CHO

CHW

COS-7

PC-12

JEG-3

HEK 293

Rat-1

U

↑(Ptx)

↑(Ptx)

↑

U

Ub

↑c

U,↑(Ptx)

(Fraser et al., 1989, Eason et al., 1992)

(Jones et al., 1991, Wang et al., 1991, Eason et

al., 1992, Airriess et al., 1997, Rudling et al.,

1999)

(Cotecchia et al., 1990)

(Federman et al., 1992, Eason & Liggett, 1995)

(Duzic & Lanier, 1992)

(Pepperl & Regan, 1993)

(Chabre et al., 1994)

(Sautel & Milligan, 1998)

α2B CHO

PC-12

JEG-3

S115

Sf9

↑(Ptx)

↑

↑

↑b

↑(Ptx)

↑

(Eason et al., 1992)

(Pohjanoksa et al., 1997, Rudling et al., 2000)

(Duzic & Lanier, 1992)

(Pepperl & Regan, 1993)

(Jansson et al., 1994)

(Jansson et al., 1995)

α2C CHO

JEG-3

↑(Ptx)

↑(Ptx)b (Eason et al., 1992, Rudling et al., 2000)

(Pepperl & Regan, 1993) a ↑ means stimulation, ↑(Ptx) means stimulation revealed after Ptx treatment, and U means

U-shaped responses. b indirect assay via CREB-mediated transcription.c cells overexpressing Gs-proteins.

21

In many of the studies referred to in table 2, a comparison of signalling between

receptor subtypes has been made in the same cell type. With this background

information, it is evident that the α2C-AR is very resticted in stimulatory cAMP

effects. Comparison between α2A-AR and α2B-AR suggests that the α2B-AR is more

disposed for stimulation, although comparable results for α2A-AR are obtained in some

cell types.

Whereas a stimulation of cAMP synthesis is usually not seen with endogenous

α2-ARs, an increase in [Ca2+]i in response to agonists is observed in tissue

preparations of smooth muscle (Aburto et al., 1993), in primary cultures of astrocytes

(Salm & McCarthy, 1990), and in clonal cell lines expressing endogenous receptors,

e.g. HEL human erythroleukemia cells (Michel et al., 1989) and NG108-15 rat

neuroblastoma×glioma cells (Holmberg et al., 1998). Ca2+ signalling is also evident in

several transfected cell lines (Åkerman et al., 1996, Dorn et al., 1997, Kukkonen et al.,

1998).

Modulation of ion channel activity is crucial for α2-AR-mediated inhibition of

neurotransmitter release. Both K+- and Ca2+-channels have been implicated in this

function (Isom & Limbird, 1988). K+-channel activation and Ca2+-channel inhibition

are presumed to be dependent on protein−protein interaction and, thus, independent of

cAMP (Lipscombe et al., 1989, Surprenant et al., 1992).

1.4 Other receptors utilized in the study

1.4.1 Octopamine receptors

Octopus salivary glands contain enormous amounts of octopamine (Erspamer &

Boretti, 1951, Saavedra, 1974). Octopamine is also found in neuronal as well as non-

neuronal tissues of most other invertebrates (reviewed in Roeder, 1999). The

invertebrate octopaminergic system is thought to be homologous to the vertebrate

adrenergic system. This is not only due to the structural analogy of octopamine and

22

noradrenaline, but also for the restricted localization of octopaminergic neurons,

similar to adrenergic neurons, in CNS, release of octopamine in stress situations, and

the functional consequences of the release (Roeder, 1999).

The first receptor to be characterized for octopamine was found in the CNS of

the cockroach Periplaneta americana (Nathanson & Greengard, 1973). A

classification of octopamine receptors was introduced by Dr. Evans in the early 1980s.

Using the locust extensor tibiae muscle as a model, three different subtypes could be

recognized on functional and pharmacological criteria, class 1, 2A and 2B (Evans,

1981). Later a pharmacologically distinct receptor subtype, class 3, was characterized

in neuronal tissue of the locust (Roeder, 1992). Many of the characterized octopamine

receptors seem to be coupled to AC activation (class 2 and 3 receptors), but receptors

coupled to Ca2+ mobilization (class 1 receptors) are also found (Roeder, 1999). The

Sf9 cell line is one of the few clonal cell lines expressing endogenous octopamine

receptors, making it useful for studies of octopamine receptor signalling (Orr et al.,

1992).

1.4.2 Purinergic receptors

Adenine nucleotides are released from cells either from vesicles or in response to

stress or hypoxia. Cellular effects of extracellular adenine nucleotides are mediated by

purinergic receptors, which are expressed, in some form, by most cell types (Ralevic &

Burnstock, 1998).

A physiological effect of administered adenosine was shown more than 70

years ago (Drury & Szent-Györgyi, 1929). Much later, adenosine was demonstrated to

either stimulate (Sattin & Rall, 1970) or inhibit (van Calker et al., 1978) cAMP

production in brain tissue, thereby setting a need for classification into adenosine A1

and A2 receptors (van Calker et al., 1979). In the 1970s, evidence were accumulating

for specific ATP receptors as well, and the purinergic receptors were classified into P1

(adenosine) and P2 (ATP) receptors (Burnstock, 1980). Now, it is generally accepted

that the P1 receptors encompass four subtypes of adenosine receptors linked to

stimulation (A2A, A2B) or inhibition (A1, A3) of AC (Fredholm et al., 1994). The P2

23

receptor family contains numerous ionotropic (P2X) and metabotropic (P2Y) receptors

(Fredholm et al., 1994). The P2Y receptors are mostly linked to PLC activation through

Gq subfamily proteins (O’Connor et al., 1991).

1.4.3 Muscarinic receptors

The sympathetic action of noradrenaline is in most tissues counteracted by the

parasympathetic action of acetylcholine on muscarinic receptors. The name for this

subfamily of acetylcholine receptors comes from the plant alkaloid muscarine, a potent

agonist at these receptors noticed by Sir Henry Dale some 90 years ago. Muscarinic

receptors, in contrast to adrenergic receptors, are also found in invertebrates (Shapiro

et al., 1989). Molecular cloning of the muscarinic receptors, in humans as well as in

rodents, has revealed a family of five distinct subtypes, M1-M5 (Ashkenazi & Peralta,

1994). Expression studies of these receptors have coined a consensus view of

functional coupling to the Gq subfamily (M1, M3, M5) or the Gi subfamily (M2, M4) of

proteins.

1.5 Why study heterologously expressed receptors?

Examination of distinct receptor subtypes in vivo is extremely difficult for several

reasons. First, in the case of adrenergic receptors, the sympathetic innervation of

tissues makes it difficult to distinguish responses of pre- and postsynaptic receptors

(Berlan et al., 1992). Second, the available quantities of tissues are restricted, and the

ethical aspects are of high weight in experimental research involving animals. Third,

single cells may contain multiple receptor subtypes with similar ligand binding

properties, e.g. adipocytes may contain up to five different adrenergic receptors

(Lafontan & Berlan, 1993). Fourth, ligands with sufficient subtype selectivity are not

available for distinction between α2-AR subtypes. Fifth, the amount of receptors in

native tissues is usually very low, reflected by the fact that an overall 80,000 -

24

100,000-fold purification is necessary to obtain a homogenous α2-AR population from

human platelets (Regan et al., 1982).

Another way to study receptors is by the use of immortalized cell lines.

However, the establishment of cell lines with endogenous distinct receptor subtypes is

not an easy task. For α2-ARs, this has been a secondary profit of already established

cell lines in some fortunate cases. These include human HT-29 cells, expressing α2A-

AR (Bylund et al., 1988), rat NG108-15 cells, expressing α2B-AR (Bylund et al.,

1988), opossum OK cells and human HepG2 cells, expressing α2C-AR (Murphy &

Bylund, 1988, Schaak et al., 1997). A human cell line expressing only the α2B-AR

subtype has not been found.

Heterologous expression of receptors is a way of getting unlimited amounts of

single receptor subtypes in a defined setting. Most receptors appear to behave naturally

also in an unnatural environment, i.e. human receptors can be expressed in non-human

cells and vice versa, and retain their capacity to bind ligands and relay signals.

Heterologous expression systems also have their limitations. One such important

limitation is that the signalling machinery is defined by the cells and made up of fixed

tranducers and effectors. Endogenous effector proteins, such as ACs, are often

heterogenous in composition (Marjamaki et al., 1997, Varga et al., 1998), and lack of

selective blockers for these makes it impossible to hint particular isoforms as targets of

receptor action. A way to overcome this obstacle is by genetic engineering of cells to

either impair endogenous expression of isoforms or overexpress exogenous isoforms.

Heterologous expression of receptors and signalling molecules has led to the

discovery of a vast diversity of receptor signalling pathways. Some of these may be

physiologically redundant, or even non-existing, in vivo. Nevertheless, given the

difficulties of examining receptors in native tissues, our current understanding of

receptor structure and function would not have been possible without heterologous

expression.

25

2 AIMS OF THE STUDY

The aims of this study were:

- to characterize receptor signalling cascades in Sf9 insect cells and to optimize the

Baculovirus Expression Vector System (BEVS) for functional studies of GPCRs.

- to determine what factor(s) is responsible for the opposite coupling of α2A- and α2B-

ARs to cAMP production in Sf9 cells.

- to characterize α2-AR signalling to specific AC isoforms.

- to characterize the Ca2+ signalling mechanism of α2-ARs.

- to highlight the signalling differences between α2-AR subtypes.

26

3 MATERIALS AND METHODS

This section is a brief summary of the material and methods used in the different

studies and mostly a complement to these. For more details the reader is referred to

individual papers.

3.1 Cell cultures

The Sf9 cell line is a clonal cell line isolated from Sf21 cell culture, which originates

from Spodoptera frugiperda pupal ovarian tissue (Vaughn et al., 1977). The Sf9 cell

line was maintained as suspension culture in glass spinner bottles with constant

stirring at 25-27°C. The cells also grow as adherent cultures and were usually seeded

on tissue culture dishes for the purpose of infection with recombinant baculovirus (I,

II, III, V). The buffer used during experimental conditions was Na+-based. As K+ is

the most abundant cation in the culture medium, and in the hemolymph of lepidopteran

insects, a K+-based medium (Vachon et al., 1995) was tested in functional assays with

α2-ARs. No significant differences in the outcome compared to the Na+-based buffer

were detected (data not shown).

Stable α2-AR transfectants of Chinese hamster ovary (CHO) cells (Pohjanoksa

et al., 1997) were obtained through Dr. Mika Scheinin (University of Turku, Turku,

Finland), and grown in MEM alpha supplemented with antibiotics and 5% foetal calf

serum at 37°C (IV). The clones expressed comparable levels of the different human

α2-AR subtypes (Pohjanoksa et al., 1997, Kukkonen et al., 1998).

3.2 BEVS and infection procedures

The Baculovirus Expression Vector System (BEVS) uses the Autographa californica

Nuclear Polyhedrosis Virus (AcNPV, family Baculoviridae) to deliver genes into

insect cells for efficient eukaryotic expression. There are several commercially

available systems for generation of recombinant baculovirus. The basic for all of them

is to replace a non-essential gene of the baculovirus genome with the gene of interest.

All recombinant virus constructs used in this study are replacing the polyhedrin gene,

27

and use the polyhedrin gene promoter to drive the expression of the recombinant

genes.

Infection of cells can be done simply by adding a small amount of virus to cell

cultures and allow spreading of infection by diffusion. This method is inefficient for

coexpression studies involving multiple viruses. To obtain efficient coexpression, and

a synchronized infection of all cells, the infection was done by sequential overlay of

cells with high-titer virus stocks (III, V). The efficiency of coexpression was tested by

sequential infection with wild-type virus, producing occlusion bodies visible under

light microscope, and a recombinant virus expressing Green Fluorescent Protein

(GFP), which can be detected with fluorescense microscopy. Examination of cells 48 h

post infection indicated coexpression in virtually all cells (> 90%).

3.3 Receptor quantification

α2-AR expression was monitored by specific binding of [3H]RX821002 ([3H]2-

methoxy-idazoxan) to cell homogenates (II) or membrane fractions of homogenates

(III). [3H]RX821002 is a selective α2-AR antagonist, that exhibits low non-specific

binding, and binds with high affinity also to the rodent α2A orthologue, in contrast to

[3H]rauwolscine and [3H]yohimbine (Erdbrugger et al., 1995, Deupree et al., 1996).

Saturation binding studies of the human α2-AR subtypes expressed in Sf9 cells give

affinity konstant (Kd) values slightly under 1 nM for the α2A-AR and around 3.5 nM

for the α2B-AR (Uhlen et al., 1998). These values are similar to the values obtained for

the human subtypes in CHO cells (Pohjanoksa et al., 1997), corresponding rat

subtypes in COS-1 cells, and corresponding porcine subtypes in tissue preparations

(Uhlen et al., 1998).

3.4 Receptor−G-protein interactions

Pertussis toxin (Ptx) was used to ablate receptor−Gi/o-protein interactions (II, V).

There has been a debate regarding Ptx sensitivity of Sf9 cell G-proteins. Some

investigators have failed to abolish responses with Ptx (Vasudevan et al., 1992,

28

Mulheron et al., 1994), while others have not encountered problems (Richardson &

Hosey, 1992, Ng et al., 1993). In the paper first describing the expression of α2A-AR

in Sf9 cells (Jansson et al., 1995), Ptx significantly inhibited the receptor response, but

did not abolish it. We have also noticed that the inhibition of AC2 (V) is not

completely abolished by Ptx treatment when using high concentrations of UK14,304

(> 3 µM) at the human α2A-AR. Such problems have not been met with the M4

receptor.

The nonhydrolyzable GTP analogue GTPγS can be used to activate G-proteins

independently of receptors in membranes or permeabilized cells. When applied at low

concentrations, it can also be used to monitor receptor-catalyzed G-protein activation

(III). An increase in GTPγS binding to Gi/o-proteins is readily observed upon receptor

stimulation. Receptor-catalyzed binding to Gs-proteins is more difficult to obtain, and

usually has a low signal-to-noise ratio (Wieland & Jakobs, 1994). A similar problem

is encountered in GTPase assay, as a result of the extremely low rate of GTP hydrolysis

by Gs-proteins (Milligan, 1988).

3.5 Measurement of cAMP

Gs-protein coupled receptors increase AC activity, which is easily measured as an

increase in the cAMP content of cells. To measure Gi-protein coupled receptor action,

a stimulant needs to be included. Forskolin, a diterpene isolated from the plant Coleus

forskohlii, is known to activate AC independently of receptor activation (Seamon et

al., 1981). Agonist-dependent inhibition of forskolin-stimulated cAMP production is

therefore often used as a measure of Gi-protein activation by receptors.

To measure cAMP accumulation (I, II, III, IV, V), cells were incubated with

[3H]adenine to label the ATP pool, from which cAMP is formed. Cells were then

stimulated, whereafter the cAMP was extracted, separated from other labelled

contaminants by ion exchange chromatography and adsorption to insoluble aluminium

oxide salt, and quantified by liquid scintillation counting. The method lacks spatial

resolution of cAMP concentrations, but gives highly reproducible results of total

cellular cAMP accumulation. The absolute concentration of cAMP cannot be obtained

29

by this method, since the radiolabelled cAMP (and ATP) makes up only part of the

total cellular cAMP content. The cAMP level is instead related to the labelled ATP

and ADP pools, eluted from the ion exchange step (Krishna et al., 1968), and gives a

relative measure of the amount of conversion of ATP to cAMP.

3.6 Measurement of Ca2+

Fura-2, a fluorescent probe highly selective for Ca2+, is delivered into cells as an

acetoxymethyl ester conjugate (Fura-2 AM) (Grynkiewicz et al., 1985). The fura-2

molecule is trapped inside the cells after the removal of the AM group. Fura-2 shows

a spectrum shift upon binding Ca2+ ions, 380 nm being the wavelength for maximal

excitation in the absence and 340 nm in the presence of Ca2+, providing the basis for

measurements. The affinity constant for the fura-2−Ca2+ complex at 37°C, and

physiological salt concentrations, is ∼220 nM (∼280 nM at 22°C), setting the

sensitivity and the limitations of the probe for Ca2+ changes. Most cell types can be

assayed for Ca2+ signalling in suspension using fura-2 (I, IV). However, suspension

measurements do not provide information about cell heterogeneity nor spatial

resolution of signalling. In addition, perfusion of cells is not technically possible. For

the above purposes, the fura-2-loaded cells, on cover slips, were excited under

microscopic view (IV).

3.7 Mathematical modelling

A mathematical model of receptor-regulated AC activity was developed to account for

promiscuous Gi/Gs-protein coupling (III). The model includes three different steps.

The first step describes the agonist activation of the receptor, in which agonist

concentration and efficacy can be varied to give different concentrations of activated

receptor. The second step describes the activation of two different G-proteins, Gi and

Gs, by the activated receptor from the first step. In the second step, the outcome, i.e.

relative concentrations of activated Gi and Gs, can be modified by varying the

receptor’s affinity for the different G-proteins as well as by varying mutual

competitiveness between the G-proteins for activation by the receptor. The third step

30

describes how Gi and Gs allosterically modulate AC to either decrease (Gi) or increase

(Gs) the catalytic activity.

Although sufficiently sophisticated to enable the modelling work performed in

paper III, the model suffers from some limitations and a refined model has been

described after the publication of paper III (Kukkonen et al., 2001).

4 RESULTS

4.1 Receptor signalling in Sf9 cells

Stimulation of Sf9 cells with octopamine led to an elevation of [Ca2+]i and cAMP

production (I). The potency of octopamine was similar for both events. The [Ca2+]i

increase originated from intracellular stores as well as from the extracellular space.

Activation of the expressed human adenosine A2A receptor, linked to Gs-proteins,

resulted in cAMP elevation with no concomitant increase in Ca2+, excluding the role

of cAMP in Ca2+ mobilization. When Ca2+ was chelated with EGTA, there was an

attenuation of the cAMP increase in response to octopamine, suggesting a role for

Ca2+ in AC activation. However, activation of the muscarinic M3 receptor, linked to

Gq-proteins, increased [Ca2+]i, but had no effect on basal cAMP production. Using a

panel of octopamine receptor/α2-AR antagonists, we found that the dual octopamine

responses are mediated by different receptor populations in Sf9 cells.

It was not tested whether octopamine increases the IP3 production, indicative of

PLC activation. The muscarinic Gq-protein coupled receptors do increase the IP3

concentration in Sf9 cells (Kukkonen et al., 1996). It is also interesting in this context

to note that the heterologously expressed human α2B-AR is coupled to IP3 production

and [Ca2+]i increase in Sf9 cells (Holmberg et al., 1998), and apparently uses the same

dual coupling mechanism as the octopamine receptors to increase cAMP production

(Figure 1).

31

cAM

P ac

cum

ulat

ion

0

1

2

3

4

5

EGTA

Ca2+

Iono

NA_ NA NA

Figure 1. Effects of Ca2+ chelation on α2B-AR-mediated cAMP production.Sf9 cells expressing recombinant human α2B-AR were assayed for noradrenaline (NA)-stimulated cAMP production in the presence of 1 mM Ca2+ or in the presence of 1 mMEGTA. Some cells were pretreated with ionomycin (Iono) in the presence of EGTA to depletecells of Ca2+. Experiments were performed in the presence of 30 µM forskolin. Theconcentration of NA was 100 µM. Data are means ± SEM from three different experimentsperformed in triplicate.

The endogenous AC(s) in Sf9 cells integrated two signals, Gs and Ca2+, to increase the

total cAMP synthesis. How this is mechanistically regulated is at present unknown.

Comparison to the mammalian AC1, which is directly activated by the Ca2+/CaM

complex (Tang et al., 1991), indicated that the Sf9-AC is regulated in a different

manner, requiring a primary stimulant for Ca2+ effects.

In paper V we investigated receptor regulation of the mammalian AC2,

heterologously expressed in Sf9 cells. The PLC-linked M3 receptor was able to

stimulate this AC isoform via PKC. The PKC isozyme(s) involved probably belong to

32

the classical subfamily based on phorbol ester (TPA) sensitivity (V) and a Ca2+-

dependence (data not shown).

All α2-AR subtypes can inhibit forskolin-stimulated Sf9-AC activity (Oker-

Blom et al., 1993, Jansson et al., 1995, II, III, V). In the case of human α2B-AR, the

inhibition was increased when coexpressed with mammalian Gi-proteins (III). The

complex regulation of AC2 by Go-proteins, with both inhibitory and stimulatory input

depending on primary stimulus, was also obtained with the endogenous G-proteins

(V).

4.2 Propensity of α2-ARs for Gs coupling

In paper II we investigated if the opposite coupling of the heterologously expressed

α2A- and α2B-ARs to cAMP production (Jansson et al., 1995) resided in the receptor

proteins as sequence differences. The mouse orthologues of these subtypes were

expressed in Sf9 cells at similar densities. The α2B-AR action on cAMP production

was U-shaped, with inhibition at low and potentiation at higher noradrenaline

concentrations. The α2A-AR inhibited the AC activity in the equivalent concentration

range. The potentiating effect of the α2B-AR was hypothesized to result from

promiscuous coupling to Gs-proteins and, if so, ought to involve cytoplasmic domains

of the receptor. An exchange of the i3-loops between the subtypes did not alter the

responses of the parent receptors. The carboxyl terminal domain neither seemed to be

involved. Of the remaining cytoplasmic domains, the i2-loop from α2B-AR conferred

the stimulatory effect to α2A-AR. Only a few amino acid residues of the i2-loops differ

between the subtypes. Site-directed mutagenesis of single amino acid residues in the

i2-loop of α2A-AR was not enough to convincingly demonstrate an α2B-AR

functionality, but a double mutant (S134→A, L143→S) showed an enhanced stimulatory

capacity, suggesting that several amino acid residues are involved in the coupling

specificity.

In paper III we wanted to provide evidence for true Gs-protein coupling of α2B-

AR, and get insight into the molecular mechanisms that define the U-shaped

33

regulation of cAMP production. For these purposes, we coexpressed Gi- or Gs-proteins

together with the human α2B-AR in Sf9 cells. An interaction between Gs and α2B-AR

was demonstrated by enhanced GTPγS binding to Gs-proteins upon receptor

activation. An increase in the potency of noradrenaline to stimulate cAMP production,

and an increased maximal response, were also evident with Gs coexpression, indicative

of an interaction in the cellular milieu as well.

The α2-ARs are coupled to an inhibition of cAMP synthesis in many cell types.

An increase in the Gi-protein level should thus, in the case of α2B-AR, if not displace,

then at least counteract the effect of Gs on AC. Gi coexpression did attenuate the

forskolin stimulation via α2B-AR, but part of the stimulatory component remained.

However, the potentiating effect with noradrenaline could be overcome with longer

infection times, when the concentration of Gi-proteins was prominently increased

(Figure 2).

There was an obvious difference in the activation mode of α2B-AR by different

agonists. Noradrenaline produced U-shaped dose-response curves, while UK14,304

elicited the stimulatory phase only weakly in the same concentration range (II, III).

The phenomenon, whereby different agonists can induce different responses through

the same receptor, has been termed agonist-specific coupling (Robb et al., 1994). As

can be seen from figure 2, this can have a major impact on AC regulation via

promiscuous Gs/Gi-protein coupling. In this context, it is worth pointing out that the

partial agonist clonidine, with apparent low intrinsic efficacy for Gs coupling, induces

inhibitory responses that are "fuller" than those of certain "full agonists".

34

cAM

P (p

erce

nt c

hang

e of

fors

kolin

)

-50

0

50

100

150

200α2B (25 h p.i.)α2B + Gi (25 h p.i.)α2B + Gi (38 h p.i.)

NA Oxy UK Clon

Figure 2. Ligand activity at α2B-AR coexpressed with Gi-proteins.Sf9 cells expressing recombinant human α2B-AR alone or together with Gi-proteins wereassayed for agonist-mediated changes in forskolin-stimulated cAMP production at differenttimes post infection (p.i.). The agonists were noradrenaline (NA), oxymetazoline (Oxy),UK14,304 (UK) and clonidine (Clon). The concentrations used were 100 µM for all agonists.Part of these data are presented in paper III, table 1. Data are means ± SEM (means ± SD,38 h p.i.) from three different experiments (one experiment, 38 h p.i.) performed in triplicate.

4.3 Signalling cross-talk

The AC activity in Sf9 cells gave an example of cross-talk between different receptors

at the effector level. Two distinct signal pathways, elicited by different octopamine

receptor populations, activating different G-proteins, converge on the AC activity to

amplify the cAMP increase (I). In this system, Gs-protein activation, by one type of

receptor, has an obligatory role for the potentiating effect of Ca2+ on the AC activity.

In paper IV it was shown that activation of purinergic Gq-coupled receptors was, in a

similar way, obligatory for α2-AR-mediated increase in [Ca2+]i in CHO cells.

Stimulation of α2-ARs resulted in an increase in [Ca2+]i of about 200 nM. Treatment

of cells with apyrase to hydrolyze extracellular purine nucleotides significantly

reduced the α2-AR-mediated [Ca2+]i increase, as did the block of P2Y receptors with

suramin. Neither apyrase nor suramin unspecifically influenced the G-protein coupling

35

of α2-ARs, as judged from the AC inhibition. With continuous perfusion of cells, to

remove released purine nucleotides, the Ca2+ response to α2-AR stimulation was

virtually absent, indicating that activation of Gq subfamily proteins is a prerequisite for

PLC activation through α2-ARs.

Among the α2-ARs, only the α2B-AR subtype mediates a Ca2+ response in Sf9

cells (Holmberg et al., 1998). Continuous perfusion of these cells did not attenuate the

α2B-AR-mediated Ca2+ response (Holmberg et al., 1998). In an effort to elucidate the

mechanism for the Ca2+ response in this cell type, we coexpressed the α2B-AR with Gi-

or Gs-proteins. The Ca2+ response was attenuated about 70% with Gi coexpression,

whereas Gs had no major influence on the response (Figure 3).

∆ [C

a2+] i

0

100

200

300

400

500

600

700

α2B-AR + Gi + Gs

Figure 3. Effect of G-protein expression on α2B-AR-mediated Ca2+ signalling.Sf9 cells expressing recombinant human α2B-AR alone or together with Gi- or Gs-proteinswere assayed for changes in [Ca2+]i in response to noradrenaline (1 µM). Data are means ±SD from two experiments.

36

In paper V another example of cross-talk between receptors was demonstrated. PKC

activation, by TPA or M3 receptor activation, elevated the cAMP content of Sf9 cells

expressing AC2, but not of wild-type or AC1 expressing cells. The stimulation was

almost totally inhibited by activation of the α2A-AR or the M4 receptor expressed in

the same cells. Since AC2 has been considered refractory to inhibitory G-proteins, this

led us to further investigate this apparent discrepancy. We could show that

concomitant activation of Gs-proteins switches the response of α2A-AR to stimulatory,

presumably an effect via Gβγ, as it was sensitive to Gβγ scavenging by Gα11. To

identify the G-protein species mediating the inhibition, we developed a method that

take advantage of the viral turn-off of host protein synthesis. This enabled us to

eliminate most of the receptor coupling to endogenous G-proteins by what we call

"time-restricted pertussis toxin pretreatment", i.e. Ptx-sensitive endogenous G-proteins

were uncoupled by Ptx pretreatment prior to infection with viruses coding for signal-

ling components, including G-proteins. Since Ptx was removed soon after infection,

the newly synthesized G-proteins could escape ADP ribosylation. Using this approach,

it was shown that Go-proteins could inhibit TPA-stimulated activity and potentiate Gs-

stimulated activity of AC2. Expression of Gi, on the other hand, potentiated both of

these primary stimulus events. The identity of Gβγ subunits were the same for both G-

protein species, indicating that the identity of Gα subunit determines the outcome.

5 DISCUSSION

5.1 Suitability of Sf9 cells for receptor characterization

Early reports on GPCRs in Sf9 cells described the successful expression of different

receptors, and purification of functional receptors from these cells (George et al.,

1989, Parker et al., 1991, Reilander et al., 1991, Vasudevan et al., 1991). Expression

of GPCRs in Sf9 cells ususally results in high receptor densities, well suited for

studying pharmacological binding characteristics (Parker et al., 1991, Mouillac et al.,

1992, Quehenberger et al., 1992, Mills et al., 1993, Oker-Blom et al., 1993, Hartman

37

IV & Northup, 1996). The use of Sf9 cells for functional studies became somewhat

dubious by a report stating an absence of Gi-proteins in these cells (Quehenberger et

al., 1992). Nevertheless, soon afterwards, inhibition of Sf9-AC activity via the 5-HT1B

receptor (Ng et al., 1993) and via the α2C-AR (Oker-Blom et al., 1993) was reported.

We have later shown that all three α2-AR subtypes can inhibit forskolin-stimulated AC

activity in Sf9 cells (Jansson et al., 1995, II, III). In the case of the human α2B-AR, the

inhibition was increased when coexpressed with mammalian Gi-proteins, suggesting

Sf9-AC sensitivity toward this G-protein as well (III).

The inhibitory G-proteins in Sf9 cells have not been characterized in detail, but

at least a Go-like G-protein appears to be expressed (Ng et al., 1993, Mulheron et al.,

1994). Go-proteins are expressed predominantly in neural tissues, but are also found in

other places. The Gαo subunit identified in Drosophila melanogaster is also expressed

in oocytes and ovarian nurse cells, in addition to its neuronal localization (Schmidt et

al., 1989). Having in mind that Sf9 cells originate from ovarian tissue, it is not unlikely

that a Spodoptera Go is expressed in these cells. The endogenous inhibitory G-protein

can obviously substitute for the mammalian Go-protein for the inhibition of AC2 (V),

supporting this hypothesis.

Sf9 cells have often been used to address G-protein selectivity of various

receptors by expression of selected receptor−G-protein pairs (Butkerait et al., 1995,

Grunewald et al., 1996, Hartman IV & Northup, 1996, Barr et al., 1997, III). This has,

on some occasions, led to confirmation of presumptive G-protein coupling of receptors

(Butkerait et al., 1995, Barr et al., 1997, III). One reason for utilizing Sf9 cells for

establishing receptor−G-protein coupling preferences is that relatively high levels of

expression of desired proteins are obtained, improving the signal-to-noise ratio. An-

other feature of Sf9 cells is the low level of endogenous GPCRs interfering with the

ectopic receptors (Hu et al., 1994, Butkerait et al., 1995, Barr et al., 1997), as well as

low levels of endogenous G-proteins functionally coupled to expressed receptors, as

determined by lack of GTP-sensitive high-affinity agonist binding sites (Quehenberger

et al., 1992, Butkerait et al., 1995, Grunewald et al., 1996).

38

Assessment of signalling pathways in whole cells could have some advantages of the

above notions. For example, purinergic receptor signalling is obviously "disturbing"

the cellular signalling status when working with isolated cell cultures (IV, Florio et

al., 1999, Ostrom et al., 2000). We have not detected changes in cAMP production, or

Ca2+ homeostasis, with the purinergic receptor agonists ATP and adenosine in

uninfected Sf9 cells, or in cells infected with wild-type virus. This suggests that Sf9

cells could be a cell line of choice when such tonic signalling is not to interfere with

the experimental outcome. Potentially interfering receptors expressed by the Sf9 cells

are the octopamine receptors (Orr et al., 1992, Hu et al., 1994, I). This is especially

relevant in adrenergic receptor research due to the structural similarity of ligands

acting on receptors from these families. It has also been shown that octopamine

activates α2-ARs (Airriess et al., 1997, Rudling et al., 2000). When tested on

uninfected Sf9 cells, noradrenaline had an effect on the [Ca2+]i and cAMP production

at high concentrations (> 3 uM) (Figure 4). The effect of clonidine was prominent on

the [Ca2+]i level, but modest on cAMP accumulation. UK14,304 did not seem to

activate the endogenous octopamine receptors (data not shown), and could be the

agonist of choice for studying α2-ARs. Unfortunately, UK14,304 appears not to

activate the α2-ARs in the same manner as noradrenaline (see Figure 2).

39

NA Oxy Clon Oct

∆ cA

MP

accu

mul

atio

n

0.0

0.2

0.4

0.6

0.8

1.0

NA Oxy Clon Oct

∆ [C

a2+] i

0

50

100

150

200

250

300

Figure 4. Ligand activity at octopamine receptors in Sf9 cells.Uninfected Sf9 cells were assayed for α2-AR agonist-mediated changes of cAMPaccumulation or [Ca2+]i. The agonists were: noradrenaline (NA), oxymetazoline (Oxy),clonidine (Clon) and p-octopamine (Oct). The concentrations used were 100 µM for allagonists. The data for cAMP accumulation are given as changes from basal level and aremeans ± SD, N=3. Data for [Ca2+]i are given as changes from basal level and are means ±SD, N=2.

A drawback of the BEVS is that the viral infection is lethal to cells. This renders the

Sf9 cells unable to maintain ionic gradients and metabolic equilibrium status late in

infection. A primary reason for this deterioration is the virally mediated turn-off of

host protein synthesis. However, this can be turned to an advantage in receptor

research by Ptx pretreatment prior to infection to abolish receptor coupling to endo-

genous G-proteins, and subsequent recovery of signal pathways by heterologous

expression of desired G-proteins (V).

5.2 Promiscuous G-protein coupling

A receptor is primarily coupled to a specific subset of G-proteins and activation of

these by the receptor is efficacious and occurs with high agonist potency. For example,

pioneering work with purified platelet α2-AR and G-proteins, reconstituted in

phospholipid vesicles, revealed that this receptor activates Gi and Go very efficiently,

whereas activation of Gt and Gs is negligible (Cerione et al., 1986a). It has later been

shown that many receptors are not that strict in selecting G-protein partners, but

40

instead they exhibit additional coupling to G-proteins from distantly related

subfamilies (Gudermann et al., 1996). It is this phenomenon that is usually referred to

as promiscuous G-protein coupling.

Activation of α2-ARs results in a seemingly controversial stimulation of cAMP

synthesis in some cell types. This phenomenon was initially shown to be mediated by

PLA2 activation (Jones et al., 1991), but subsequent studies also revealed a probable

Gs-protein interaction of the α2A-AR in transfected CHO cells (Eason et al., 1992). In

Sf9 cells, the α2B-AR is linked to an increase in cAMP production, whereas the α2A-

AR subtype decrease forskolin-stimulated AC activity (Jansson et al., 1995). A

productive Gs-protein coupling of the α2B-AR in these cells was demonstrated in paper

III. Work from Dr. Liggett’s lab has suggested that there are subtle differences in the

propensities of the different α2-AR subtypes for Gs activation with the endogenous

catecholamine ligands (Eason et al., 1994). Our initial studies pointed to an all-or-none

difference between α2B-AR, on the one hand, and α2A-AR and α2C-AR on the other

(Oker-Blom et al., 1993, Jansson et al., 1995). A true difference between the α2A- and

α2B-AR subtypes, although not as dramatic, was demonstrated in paper II, in which

the coupling differences cannot be ascribed to differences in receptor densities. It now

seems clear that the α2B-AR subtype is more propensive for Gs coupling than the other

subtypes (Eason & Liggett, 1993, Pepperl & Regan, 1993, II, Rudling et al., 2000,

III). The weak capability of the α2A-AR to stimulate cAMP production in the presence

of surplus Gs-proteins also supports this notion (III).

The structure of the i2-loop appears to be a determinant for this subdivision

(II). The whole selection structure for Gs coupling is probably not confined to this

area, since potent Gs activation is also obtained with α2A in different cell types (see

Table 2). The first study on this issue showed that deletion of the amino terminus of

the i3-loop of α2A-AR eliminates stimulation of AC activity, indicating a Gs coupling

domain in this area (Eason & Liggett, 1995). However, substitution of this domain

with corresponding domain from β2-AR, the prototype Gs coupled receptor, attenuated

the Gs coupling when compared with the intact α2A-AR (Eason & Liggett, 1995). On

41

the other hand, substitution of the i2-loop with β2-AR sequence significantly

improved the Gs coupling (Jewell-Motz et al., 1997), supporting our hypothesis about

the importance of i2-loop structure for Gs coupling (II).

The dose-response curve forms for α2-AR action on AC activity varies a lot

from cell type to cell type and from receptor subtype to receptor subtype, including U-

shaped relations in addition to monophasic stimulatory and inhibitory curves. Even

more intriguing, all these different forms of dose-response curves can be obtained in a

single cell type with a single receptor subtype (III). To be able to explain these

behaviours as a simple consequence of dual Gi and Gs coupling, we developed a

mathematical model that monitor AC activity in relation to Gi and Gs activation (III).

Using this model, it was possible to reproduce the different curves obtained in

experiments, suggesting that relative affinities of the receptor for Gi and Gs activation

can alone explain the different behaviours. The results from the modelling also

suggested that different agonists, like noradrenaline and UK14,304, differentially

activate the receptor and thereby dictate relative affinity differences of the receptor for

the two G-protein species.

It is difficult to correlate the promiscuous G-protein coupling of α2-ARs to a

specific physiological effect. It is clear though, that a decrease in cAMP synthesis

cannot explain many effects of α2-AR activation observed in vivo (Isom & Limbird,

1988). A comment on recent progress in β-AR research on this topic may be valid

here. Prior to cloning of the β-AR, purification enabled reconstitution of this receptor

with G-proteins. From these experiments it was concluded that the β-AR cross-reacted

with Gi (Cerione et al., 1985). A couple of years ago, it was shown that β-ARs evoke

a Ca2+ signal in submandibular gland cells through a mechanism involving activation

of Gi-proteins (Luo et al., 1999). Very recently, Dr. Kobilka’s group provided

evidence for promiscuous coupling of β2-AR to Ptx-sensitive G-proteins in myocyte

cultures from β1-AR knock-out mice, the result of which was recorded as a decrease

in contraction rate (Devic et al., 2001). These studies implicates that promiscuous

coupling may be a general mode of diversifying receptor signalling and not simply a

42

laboratory phenomenon due to high expression levels of receptors, or abnormal

signalling environments of used cell types.

5.3 Adenylyl cyclase regulation by the Ca2+ signalling pathway

AC regulation by Ca2+/CaM is perhaps the most well conserved regulatory theme of

cAMP synthesis. Pathogenicity of Bacillus anthracis and Bordetella pertussis is

partially due to secreted, soluble Ca2+/CaM-stimulated AC enzymes (Danchin, 1993).

In higher organisms, Ca2+-regulated AC activity is particularly associated with neural

functions (Dudai & Zvi, 1984, Xia & Storm, 1997, Chern, 2000). In Sf9 cells, the AC

activity was not stimulated by an increase in [Ca2+]i, but the primary, Ca2+-

independent, cAMP elevation with octopamine, and with α2B-AR activation, was

highly potentiated by a concomitant increase in [Ca2+]i. The potency of forskolin for

AC stimulation was similar in the absence and presence of elevated [Ca2+]i, suggesting

that Ca2+ does not alter the affinity for the primary stimulant. A likely explanation is

that Ca2+ increases the activity of the enzyme, integrating the Ca2+ signal with the

direct activator. Signalling molecules with the property of integrating multiple signals

are called coincidence detectors (Iyengar, 1996). Coincidence detection of ACs is

thought to play important roles in transcription, synaptic plasticity and endocrine

function.

AC2 is a coincidence detector for Gαs and PKC as well as Gβγ subunits

(reviewed in Sunahara et al., 1996). A cross-talk between PKC and AC was suggested

in 1987 in a paper showing phorbol ester-mediated phosphorylation of AC in

erythrocytes (Yoshimasa et al., 1987). However, it is still unclear what physiological

role PKC play in cAMP signalling. AC2 has been suggested to play a role in synaptic

plasticity (Defer et al., 2000), although this has never been demonstrated. The AC2

isoform has in several instances been implicated in growth and development. In NIH-

3T3 cells, ectopic expression of AC2 is sufficient to inhibit proliferation in response

to serum (Smit et al., 1998). Neuronal, as well as mesodermal, differentiation of the

teratocarcinoma P19 cells is accompanied by an upregulation of AC2 mRNA

(Lipskaia et al., 1997, Lipskaia et al., 1998). A related isoform of the mammalian AC2

43

is expressed in Caenorhabditis elegans. Loss of function of this isoform results in

early larval lethality, indicating an important role in development (Korswagen et al.,

1998).

In mammals, AC2 is widely expressed in the brain with no specific area

preference (see e.g. Mons & Cooper, 1995). This suggests a role in common neuronal

action or development. In light of the results presented in paper V, it is tempting to

speculate, as follows, about such a common action. Neurotransmitter release is a

complicated process triggered by [Ca2+]i increases. The amount of Ca2+ entering the

nerve terminal is strictly regulated by voltage-gated Ca2+-channels. Some of these

channels are targets for PKC-mediated upregulation of activity (Zamponi et al., 1997),

whereas attenuation is transmitted from GPCRs, such as α2-ARs, via activation of Go-

or Gi-proteins (for review, see e.g. Dolphin, 1998). Recent studies have implicated a

role also for cAMP in the neurotransmitter release process (Trudeau et al., 1996,

Chavis et al., 1998, Chang et al., 2000). Thus, if AC2 and Ca2+-channels were co-

localized in the nerve terminals, both a coordinated stimulation of AC2 and Ca2+-

channels via PKC, or a coordinated inhibition of the same molecules via Go-proteins,

could account for synergistic effects on transmitter release.

5.4 The α2-AR−Ca2+ link

There are now compelling evidence that α2-ARs can alter the [Ca2+]i by other

mechanisms than through ion channel modulation. Activation of α2-ARs in a variety

of cell types mobilizes Ca2+ from intracellular stores (Michel et al., 1989, Salm &

McCarthy, 1990, Åkerman et al., 1996, Dorn et al., 1997). In light of the multitude of

Gi/o coupled receptors that have been shown to mobilize Ca2+, it is no wonder that the

α2-ARs behave similarly. It was early reported that, for some receptors, the link to

Ca2+ mobilization and PLC activation is sensitive to Ptx (see e.g. Ui et al., 1988). The

finding that PLC-βs are stimulated by Gβγ subunits gave a satisfactory explanation to

this phenomenon. For the α2-ARs, a Gβγ-dependent Ca2+ mobilization has been de-

monstrated in COS-7 cells (Dorn et al., 1997).

44

Characterization of PLC-β isoforms in vitro suggested a direct activation by

Gβγ, without conditioning by Gq subfamily proteins (Camps et al., 1992, Katz et al.,

1992). In CHO cells, expressing either subtype of α2-ARs, the α2-AR-mediated Ca2+

mobilization was highly dependent on tonic stimulation of Gq coupled receptors (IV).

This led us to hypothesize that Gβγ-mediated PLC signalling requires concomitant

αq/11/14/16 activation. The mechanistic explanation has been elaborated in a study by

Dr. Wong and colleagues, showing that Gi-protein coupled opioid receptors could not

activate PLC, unless coexpressed with Gα16, in COS-7 cells (Chan et al., 2000). On

the same theme, they showed that the opioid receptors acquire the ability to stimulate

PLC in cells cotransfected with constitutively activated Gαq or Gα14. Further support

for the relevance of cross-talk or promiscuous G-protein coupling in Ca2+ signalling

has appeared from two papers describing signalling in knock-out mice deficient in Gq

family proteins. First, PLC activation by thrombin, a Gq and Gi coupled receptor, is

abolished in platelets from Gαq-deficient mice, indicating that Gi activation alone is

insufficient for PLC activation (Offermanns et al., 1997). Second, the C5a receptor,

which is strongly coupled to Ca2+ mobilization through Ptx-sensitive G-proteins,

looses the Ca2+-mobilizing ability in macrophages from Gα15 knock-out mice, while

MAPK activation is normal (Davignon et al., 2000).

Most Gi-protein coupled receptors in hematopoietic cells, such as the C5a

receptor described above, usually respond to agonists with robust [Ca2+]i increases.

Hematopoietic cells express the Gα15/16 subtype of G-proteins (Amatruda et al., 1991).

The G15/16-protein has been found rather unselective for receptor interactions

(reviewed in Milligan et al., 1996). Thus, if there is promiscuous receptor coupling to

G15/16, the Ca2+ response should largely benefit from this. In HEL cells, α2-ARs give

prominent Ca2+ responses (Michel et al., 1989, Kukkonen et al., 1997, Jansson et al.,

1998), compared to for example primary astrocytes (Enkvist et al., 1996) and NG108-

15 cells (Holmberg et al., 1998). A reason for this may be promiscuous receptor−G16-

protein coupling in HEL cells, which has been shown for purinergic receptors in this

cell line (Baltensperger & Porzig, 1997).

45

Apyrase treatment of CHO cells did not totally abolish, especially in the case of the

α2B-AR, the Ca2+ response of α2-ARs (IV). In some instances, the Ca2+ signalling

mediated by α2-ARs is not abolished by Ptx treatment. In Ptx-treated NG108-15 cells,

the Ca2+ response of the endogenous α2B-AR is inhibited by about 65% (Holmberg et

al., 1998). In CHO cells, the α2B-AR-mediated [Ca2+]i increase is attenuated by about

80% in Ptx-treated cells, whereas α2A- and α2C-AR responses are completely blocked

(Kukkonen et al., 1998). Of the α2-ARs, only the α2B-AR subtype mediates a Ca2+

response in Sf9 cells, and the response in this cell type is insensitive to Ptx (Holmberg

et al., 1998). Contrary to expectations, the Ca2+ response of α2B-AR was attenuated by

Gi coexpression in these cells (Figure 3), suggesting a Gβγ-independent pathway.

Although the α2B-AR is promiscuously coupled to Gs-proteins, the Ca2+ response is

probably not associated with this G-protein species (I). The hypothesis is that another

G-protein than Gi/o or Gs is involved. A Gq subfamily protein is a good candidate.

However, we have not detected any change in the Ca2+ signalling mode of α2B-AR in

Sf9 cells with coexpression of the mammalian G11-protein, which, if coupled to the

receptor, would be expected to work in an analogous fashion to Gs-protein expression,

increasing agonist potency and/or maximum response (III). This does not exclude the

possibility of the insect Gq as the linker, but additional work will be needed to either

exclude or include this, as well as other, potential pathways in α2-AR-mediated Ca2+

signalling.

46

6 SUMMARY AND CONCLUSIONS

The use of BEVS appears well suited for functional characterization of receptor

signalling. The signalling components of Sf9 cells are well recognized by mammalian

receptors, and heterologous expression of complementary signalling molecules can be

used to assess these in signal transduction of particular GPCRs. The lack of

endogenous receptors for most mammalian hormones and neurotransmitters in Sf9

cells provides a good cellular background for heterologous receptor characterization.

In addition, the predetermined fate of baculovirus-infected cells, with the concomitant

inhibited host protein synthesis, can be used advantageously for examination of Ptx-

sensitive G-protein function.

The α2B-AR was demonstrated to couple promiscuously to Gs-proteins both in

membrane preparations and live cells. The ability of the mouse and the human α2B-AR

subtype to induce stimulatory cAMP responses in Sf9 cells through endogenous G-

proteins suggests a similarly productive Gs coupling, and an interspecies similarity in

this regard. Using a chimeric receptor approach, it was demonstrated that the structure

of the i2-loop of α2B-AR is a determinant for Gs coupling, and that it is this loop that

differentiates the G-protein coupling selectivities of the α2A- and α2B-ARs. The

influence of receptor density on the cellular response is also obvious from the studies.

The human α2B-AR, expressed at relatively high levels, induces mainly stimulatory

responses. The chimeric mouse α2A/α2B-i2 loop receptor is also primarily coupled to

stimulation at high receptor densities, but exhibits U-shaped inhibitory and stimulatory

responses at lower densities. The mouse α2B-AR, expressed at relatively low density,

exhibits a U-shaped cAMP response.

The AC stimulatory capacity of α2-ARs may not always be associated with a Gs-

protein coupling, as shown in paper V. The results of paper V demonstrate that a

selective activation of Gi or Go has distinct consequences for AC2 regulation.

47

Coupling of the α2A-AR to Go resulted in inhibition of the PKC-stimulated AC2

activity. In contrast, Gi activation, by the same receptor, resulted in potentiation of the

PKC-stimulated AC2 activity. A Gβγ-mediated potentiation of Gs-stimulated AC2

activity was evident for both types of G-proteins, indicating that the α subunits of Go

and Gi have different roles for this AC isoform.

Ca2+ mobilization by α2-ARs is sensitive to Ptx in some cell types and is likely to be a

result of Gβγ-mediated stimulation of PLC. In CHO cells, the Ca2+ response of α2-

ARs was attenuated, almost to zero response in some cases, if extracellular purine

nucleotides were removed. This implicated purinergic receptors in the signalling

cascade, which was confirmed by attenuation of α2-AR response in the presence of

suramin, a purinergic receptor antagonist. The results suggest a receptor cross-talk at

the level of PLC enzymes. The tonic purinergic receptor activation will provide PLCs

with GTP-loaded α subunits of the Gq subfamily proteins and thereby prime the PLC

for Gβγ signalling. Thus, Gα subunit activation seems to be obligatory for Gβγ

signalling in this model system.

A trend of the α2A-AR subtype being promiscuously coupled to Gs, whereas the α2B-

AR is nearly unproductive in Gs coupling, was inferred from the first paper on

promiscuous G-protein coupling of α2-ARs (Eason et al., 1992). In our hands, the α2B-

AR is far more propensive for Gs coupling than the α2A-AR. Regarding the Ca2+

signalling, all α2-AR subtypes have the ability to regulate PLC activity in CHO cells.

In Sf9 cells, only the α2B-AR is coupled to a [Ca2+]i increase, suggesting again a

fundamental difference between α2-AR subtypes on functional criteria.

48

7 ACKNOWLEDGEMENTS

This study was conducted at the Department of Physiology, Uppsala University under

supervision of professor Karl Åkerman. The study was funded by The European

Commission, The Medical Research Council of Sweden, The Cancer Research Fund

of Sweden, The Clas Groschinsky Foundation, The Åke Wiberg Foundation and The

Lars Hiertas Minnesfond

I would like to thank:

Professor Karl Åkerman for providing an opportunity for a young biochemistry

student to take part in up to date scientific research, for sharing his knowledge in

cellular signalling, and for being a friend, and a solver of all kinds of problems.

Professor Gunnar Flemström, my co-supervisor, for his advice and guidance on a

variety of matters.

Dr Jyrki Kukkonen, co-author and friend, for invaluable help with pharmacological

issues and computer managing. Dr Kukkonen has always been open for discussions of

all kinds of matters and is much appreciated for this. Dr Kukkonen also read this thesis

and supplied valid criticism.

Dr Per-Eric Lund, co-author and friend, for numerous discussions of ion channels, cars

and other every day matter.

Dr Staffan Uhlén for sharing his excellent knowledge of adrenoceptor pharmacology

and for our discussions about child behaviour.

Dr Mikael Jolkkonen for fruitful cooperation in my second project on muscarinic

toxins.

Dr Viktoria Göransson for helpful guidance of thesis writing.

49

All former and present PhD student friends at the department, and especially those of

the Åkerman group: Ramin Shariatmadari, co-author, Tomas Holmqvist, co-author,

Sylwia Ammoun, co-author, and Elena Krioukova, for all their help and encourage-

ment. Tomas is also acknowledged for his appreciated humorous inputs in everyday

misery and for critical reading of the thesis before printing.

Technical assistant Karin Nygren for taking care of all kinds of different matters

required for on-going research and for the friendly welcome of the "Finns" to the

department.

All other persons at the department and especially the technical personnel for help

with all matters associated with being a PhD student.

My former colleagues at the Åbo Akademi University, Turku, Finland. Some of the

persons deserve a mentioning here: Dr Christian Jansson, co-author, for teaching me

the basic of cAMP and how to measure it, Dr Christian Oker-Blom for introducing the

BEVS, Pekka Ojala and Heli Hämäläinen for their excellent supervision of molecular

biology techniques.

My parents for their love and support.

My life partner, Pi, and our children, Max and My, for sharing their lives with me.

50

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