g-protein coupled receptor oligomerization in neuroendocrine pathways

Frontiers inNeuroendocrinology

www.elsevier.com/locate/yfrne

Frontiers in Neuroendocrinology 24 (2004) 254–278

G-protein coupled receptor oligomerizationin neuroendocrine pathwaysq

Karen M. Kroeger, Kevin D.G. Pfleger, and Karin A. Eidne*

Molecular Endocrinology Research Group/7TM Receptor Laboratory, Western Australian Institute for Medical Research,

Centre for Medical Research, University of Western Australia, Sir Charles Gairdner Hospital, Hospital Avenue, Nedlands, Perth, WA 6009, Australia

Abstract

Protein–protein interactions are fundamental processes for many biological systems including those involving the superfamily of

G-protein coupled receptors (GPCRs). A growing body of biochemical and functional evidence supports the existence of GPCR–

GPCR homo- and hetero-oligomers. In particular, hetero-oligomers can display pharmacological and functional properties distinct

from those of the homodimer or oligomer thus adding another level of complexity to how GPCRs are activated, signal and traffick

in the cell. Dimerization may also play a role in influencing the activity of agonists and antagonists. We are only beginning to

unravel how and why such complexes are formed, the functional implications of which will have an enormous impact on GPCR

biology. Future research that studies GPCRs as dimeric or oligomeric complexes will enhance not only our understanding of

GPCRs in cellular function but will also be critical for novel drug design and improved treatment of the vast array of GPCR-related

conditions.

� 2003 Elsevier Inc. All rights reserved.

Keywords: GPCR; Homo-dimerization; Hetero-dimerization; Oligomerization; Neuroendocrine; Cross-talk

1. Introduction

The realization in the early 1980s that a complex in-

terplay of neurotransmitters is responsible for modu-

qAbbreviations used: ATII, angiotensin II; AT1R, type 1 angio-

tensin receptor; BRET, bioluminescence resonance energy transfer;

CCR5, chemokine receptor 5; CFP, cyan fluorescent protein; ECL,

extracellular loop; ER, endoplasmic reticulum; EYFP, enhanced

yellow fluorescent protein; FITC, fluorescein isothiocyanate; FRET,

fluorescence resonance energy transfer; FSH, follicle stimulating

hormone; GFP, green fluorescent protein; GH, growth hormone;

GnRH, gonadotropin releasing hormone; GPCR, G-protein coupled

receptor; GRK, G-protein coupled receptor kinase; ICL, intracellular

loop; LH, luteinizing hormone; a-MSH, a-melanocyte stimulating

hormone; MT1R, melatonin receptor 1; MT2R, melatonin receptor 2;

mGluR1, metabotropic glutamate receptor 1; PKC, protein kinase C;

PRL, prolactin; RAMP, receptor activity modifying protein; RFP, red

fluorescent protein; Rluc, Renilla luciferase; SST, somatostatin; SSTR,

somatostatin receptor; TM, transmembrane; TR-FRET, time-resolved

FRET; TRH, thyrotropin releasing hormone; TSH, thyroid stimulat-

ing hormone; trunc, truncated; P2Y1 receptor, P2 ATP purinoceptor;

VFTM, venus fly-trap motif; VIP, vasoactive intestinal polypeptide;

YFP, yellow fluorescent protein.* Corresponding author. Fax: +61-8-9346-3838.

E-mail address: [email protected] (K.A. Eidne).

0091-3022/$ - see front matter � 2003 Elsevier Inc. All rights reserved.

doi:10.1016/j.yfrne.2003.10.002

lating hypophysiotropic hormone function has greatly

contributed towards the expansion of the field of neu-

roendocrinology. The neurotransmitters, norepineph-

rine, dopamine, acetylcholine, serotonin, excitatory

amino acids (EAAs), and c-aminobutyric acid (GABA)

have all been shown to modulate pituitary hormone

secretion, either at the hypothalamic level or by directaction on the pituitary [131]. A fundamental challenge

was to understand how these classical neurotransmitters

in the brain could integrate their signals with those of

the neuropeptides and it was postulated that intra-

membrane receptor/receptor interactions could occur

[6]. The discovery that G-protein coupled receptors

(GPCRs) could directly interact to form dimers and

higher-order complexes (oligomers) has been a majordevelopment in the last decade and their ability to

link together to form these complexes has shed some

light on how different receptor pathways intersect and

cross-react.

Whilst GPCR oligomerization is a not a new concept

[61,101,136], it is only recently that this has begun to

remodel the thinking of researchers in the GPCR field.

The currently held view is that oligomerization is a

mail to: [email protected]

K.M. Kroeger et al. / Frontiers in Neuroendocrinology 24 (2004) 254–278 255

fundamental component of GPCR regulation andfunction, providing a mechanism by which distinct sig-

nalling pathways can be directly linked and receptor

functions integrated, which obviously has vast thera-

peutic implications in human disease. This review will

summarize the progress that has been made in our un-

derstanding of GPCR oligomerization, the mechanisms

involved and the role it plays in receptor function. The

contribution to the neuroendocrine system and its reg-ulation through direct linkage of different receptors will

be discussed, including how this information may then

be translated into novel treatment strategies.

2. GPCR homo- and hetero-dimerization

Evidence for the existence of GPCR homodimers oroligomers is now substantial, with a large number of re-

ports suggesting that this is a general phenomenon for

this receptor superfamily (recently reviewed in

[8,16,50,109] and summarized in Table 1). Soon after the

first reports describing homo-dimerization emerged, it

became apparent that GPCRs could also interact with

other members of the receptor superfamily (Table 1).

Interactions have been reported between different re-ceptor subtypes (Table 1) and splice variants, truncated

and mutant receptors have also been shown to dimerize

with wild-type receptors. Often this can affect wild-type

receptor function by acting in a dominant-negative or

positive manner [14,28,34,54,170,189]. Depending on

structural features, GPCRs are classified into different

classes (Classes A, B, and C) (reviewed in [32]). Dimer-

ization between receptors within the same GPCR classand also between different GPCR classes has now been

widely demonstrated with many heterodimers displaying

novel pharmacological and/or functional properties

(Tables 1 and 2). The fact that this process has been re-

ported for such a variety of receptors across different

classes is suggestive that dimer and oligomer formation is

a universal process amongst this superfamily of recep-

tors. If so, we are only at the initial stages of decipheringhow and why this occurs for different receptor systems.

2.1. Early studies

Traditionally GPCRs were thought to exist as mo-

nomeric structures coupling to G-proteins with a 1:1

stoichiometry. However, much earlier under-appreci-

ated studies provided indirect evidence for the existenceof higher-order GPCR structures. Initial pharmacolog-

ical studies involving complex radioligand binding ex-

periments utilizing both agonists and antagonists or

dimeric ligands were interpreted as evidence of positive

or negative cooperativity between ligands explained by

physical interactions between individual receptor

monomers [60,92,101]. Antibody-induced receptor acti-

vation also provided additional indirect evidence thatGPCRs could function as dimers. A gonadotropin re-

leasing hormone (GnRH) antagonist was shown to ac-

tivate receptor signalling in the presence of a bivalent

antibody directed towards the antagonist, suggesting

that receptor aggregation could induce signalling [21].

Additional functional evidence supporting the existence

of GPCR dimers was also gained from radiation inac-

tivation studies which indicated that GPCRs couldfunction as complexes that were larger than predicted

by their monomeric structure [23,91,168]. However,

despite these observations dimerization until recently

was not generally accepted as an alternative to the single

receptor model.

2.2. Trans-complementation studies

More compelling evidence for GPCR dimers came

from studies involving functional reconstitution fol-

lowing co-expression of two non-functional mutant or

chimeric receptors. Maggio et al. [96] used two a2-ad-renergic/M3 muscarinic chimeric receptors each com-

posed of transmembrane domain (TM) 1–5 of one

receptor and TM 6–7 of the other. These chimeric re-

ceptors were non-functional when expressed alone,however, binding of both muscarinic and adrenergic li-

gands was restored upon co-expression. More recently,

co-expression of two truncated dopamine receptors that

when individually expressed are non-functional, either

TM 1–5 of the dopamine D2 receptor (D2trunc) with

TM 6–7 of the dopamine D3 receptor (D3tail), or the

converse D3trunc with D2tail, both resulted in the res-

cue of the full pharmacological and functional proper-ties of each receptor. This provides strong evidence that

D2 and D3 receptors interact [150]. A similar trans-

complementation event was also observed between two

angiotensin AT1 receptor mutants [111]. Both Lys102

(TM3) and Lys99 (TM5) mutant AT1 receptors do not

bind angiotensin II, however, upon co-expression the

normal binding site is restored. The dominant-negative

and -positive effects observed for certain mutants onwild-type receptor function also provides evidence for

direct interactions between receptors [54,189]. The nat-

urally occurring chemokine receptor 5 (CCR5) mutant,

ccr5D32, was found to interfere with the surface ex-

pression of the wild-type receptor, providing a molecu-

lar explanation for the mechanism of delayed onset of

AIDS in CCR5/ccr5D32 individuals [14]. In contrast, the

co-expression of wild-type b2-adrenergic receptor witha constitutively desensitized palmitoylation deficient

mutant was able to rescue normal receptor function [64].

2.3. Co-immunoprecipitation (biochemical techniques)

Many reports describe the use of co-immunoprecipi-

tation to demonstrate the existence of GPCR dimers

Table 1

Summary of studies on homo- and hetero-dimerization in different GPCR classes and the techniques employed

GPCR Technique Refs

Homo-oligomerization

Class A

a1a-adrenergic receptor Immunoprecipitation, FRET [161]

a1b-adrenergic receptor Immunoprecipitation, FRET [161]

b1-adrenergic receptor BRET [88,105]

b2-adrenergic receptor Immunoprecipitation [7,63]

BRET [7,88,102,105]

TRH receptor 1 BRET [57,84]

Immunoprecipitation [188]

TRH receptor 2 BRET [57]

GnRH receptor Photobleaching FRET [24,67]

BRET [84]

D2 dopamine receptor Immunoprecipitation [89]

FRET [175]

D1 dopamine receptor Immunoprecipitation [45]

d opioid Immunoprecipitation [26,46]

BRET, TR-FRET [51,102]

j opioid Immunoprecipitation [74]

l opioid Immunoprecipitation [46]

BRET [51]

Somatostatin receptor 5 Immunoprecipitation, FRET [144]

Cholecystokinin receptor BRET [19]

LH receptor Photobleaching FRET [146]

a-Factor receptor FRET [124]

CXCR2 Immunoprecipitation [165]

CXCR4 BRET [10,68]

Chemokine receptor CCR5 Immunoprecipitation [169]

BRET [68]

Chemokine receptor CCR2 Immunoprecipitation [145]

Complement C5a receptor FRET [40]

Melatonin M1 receptor Immunoprecipitation, BRET [9]

Melatonin M2 receptor Immunoprecipitation, BRET [9]

Thyrotropin (TSH) receptor FRET [87]

Neuropeptide Y FRET [29]

Adenosine A1 BRET [182]

Adenosine A2 BRET [77]

Oxytocin BRET [164]

Vasopressin V1a BRET [164]

Vasopressin V2 BRET [164]

Class C

Calcium-sensing receptor Functional complementation of inactive

mutant receptors, immunoprecipitation

[11,12,71]

BRET

Metabotropic glutamate receptor 5 Immunoprecipitation [147]

Metabotropic glutamate receptor 1 Immunoprecipitation [140]

Crystal structure of N-terminus [86]

Hetero-oligomerization

Class A and Class A

SSTR1 and SSTR5 Photobleaching FRET [144]

SSTR2A and SSTR3 Immunoprecipitation [134]

SSTR2A and l opioid receptor Immunoprecipitation [135]

D2 dopamine receptor and SSTR5 Photobleaching FRET [143]

Adenosine A1 and dopamine D1 receptor Immunoprecipitation [48]

256 K.M. Kroeger et al. / Frontiers in Neuroendocrinology 24 (2004) 254–278

Table 1 (continued)

GPCR Technique Refs

Adenosine A2A and dopamine D2 receptor Immunoprecipitation [65]

BRET [77]

AT1A and AT2 receptor Immunoprecipitation [3]

AT1A and bradykinin B2 receptor Immunoprecipitation [2]

j and d opioid receptor Immunoprecipitation [74]

BRET [51]

l and d opioid receptor Immunoprecipitation [46,49]

CCR2 and CCR5 Immunoprecipitation [103]

FRET [103]

D2 and D3 dopamine receptor Functional reconstitution [150]

TRHR1 and TRHR2 BRET [57]

5-HT1B and 5-HT1D Immunoprecipitation [176]

Melatonin M1 and M2 Immunoprecipitation, BRET [9]

b1 and b2-adrenergic Immunoprecipitation, BRET [88,105]

Adenosine A1 and P2Y1 BRET [182]


b2-adrenergic and d opioid BRET, TR-FRET [102]


b2-adrenergic and j opioid Immunoprecipitation [76]

a2a-adrenergic and b1-adrenergic Immunoprecipitation [177]

a1a-adrenergic and a1b-adrenergic FRET [161]

Oxytocin and Vasopressin V1a BRET [164]

Oxytocin and Vasopressin V2 BRET [164]

Vasopressin V1a and V2 BRET [164]

Class C and Class C

GABABR1 and GABABR2 Immunoprecipitation [73,80,173]

Functional reconstitution

Muscarinic M2 and M3 Receptor (chimeric) reconstitution studies [99]

Calcium sensing receptor and metabotropic glutamate 1 receptor Immunoprecipitation [43]

Calcium sensing receptor and metabotropic glutamate 5 receptor Immunoprecipitation [43]

Class A and Class C

Muscarinic M3 and a2-adrenergic Chimeric receptor reconstitution studies [96]

5HT1A and GABABR2 Immunoprecipitation [148]

Adenosine A1 and Metabotropic glutamate receptor 1 Immunoprecipitation [20]

Adenosine A2A and metabotropic glutamate receptor 5 Immunoprecipitation [39]


(Table 1) and most of these studies involve co-expres-

sion of differentially epitope-tagged receptors. However,

due to the highly hydrophobic nature of these seven

transmembrane receptors and the extreme levels of

protein expression often used, there is a concern that

artefactual aggregation may occur following cell lysis

and solubilization [137,148]. Attempts have been made

to address these concerns by using various detergentcombinations and controls. For example, dimers have

only been observed upon co-expression rather than

following mere mixing of lysed cells expressing individ-

ual receptors [74]. Therefore, when appropriate controls

are used, co-immunoprecipitation can represent a valid

technique for demonstrating GPCR dimerization.

Ideally studies should involve the investigation of

the presence and function of receptor heterodimers incell lines or tissues in which they are endogenously

expressed. This is often problematic due to low receptor

expression levels in these cell types and also the diffi-

culties of obtaining specific receptor antibodies. The

existence of heterodimer formation between endoge-

nously expressed receptors has been demonstrated for

the angiotensin AT1 and bradykinin B2 receptors in rat

smooth muscle cells [2], as well as human platelets and

omental vessels [4]. GABABR1 and GABABR2 ex-pressed in rat cortex could also be co-immunoprecipi-

tated using specific antibodies [80]. A direct association

of the adenosine A1 and the P2 ATP purinoceptor

(P2Y1) receptors was shown in co-immunoprecipitation

studies using membrane extracts from different regions

of rat brain [182]. Interactions between the calcium

sensing receptor and the metabotropic glutamate 1 and

5 receptors were detected by co-immunoprecipitationfrom rat brain [43].

Table 2

Summary of studies on hetero-dimerization in different GPCR classes and the possible functional roles of GPCR dimerization

GPCR Functional Roles References

Hetero-oligomerization

Class A and Class A

SSTR1 and SSTR5 receptor Allows internalization of SSTR1 [144]

SSTR2A and SSTR3 receptor Results in inactivation of SSTR3 function [134]

SSTR2A and l opioid receptor Cross-modulation of receptor phosphorylation, internalization and desensitization [135]

D2 dopamine and SSTR5 receptor Increased ligand binding affinity and enhanced signalling [143]

Adenosine A1 and dopamine D1 receptor Altered D1 receptor signalling [48]

Adenosine A2A and dopamine D2

receptor

Co-desensitization and co-internalization [65]

AT1A and AT2 receptor AT2 receptor antagonizes function of the AT1A receptor [3]

AT1A and bradykinin B2 receptor Increased signalling activity [2]

j and d opioid receptor Altered ligand binding, potentiated signalling and altered internalization [74]

l and d opioid receptor Change in binding properties of opiate ligands and altered G-protein coupling [46,49]

CCR2 and CCR5 Greatly increases the sensitivity and range of signalling pathways activated.

Cell adhesion rather than chemotaxis.

[103]

D2 and D3 dopamine receptor Increased affinity and potency for agonists and antagonists [150]

TRHR1 and TRHR2 Altered trafficking and b-arrestin interactions [57]

b1- and b2-adrenergic receptor Inhibits internalization and ERK activation by b2AR [88]

Adenosine A1 and P2Y1 receptor Altered ligand binding [180]

b2-adrenergic and d opioid receptor Altered internalization and MAPK signalling [76]

b2-adrenergic and j opioid receptor Altered internalization and MAPK signalling [76]

a2a- and b1-adrenergic receptor Altered ligand binding and internalization [177]

a1a- and a1b-adrenergic receptor Altered internalization [161]

Class C and Class C

GABABR1 and GABABR2 GABABR2 serves as a molecular chaperone for GABABR1 and is required for full

receptor function

[73,80,173]

Muscarinic M2 and M3 Altered internalization [99]

Class A and Class C

Adenosine A1 and metabotropic

glutamate receptor 1

Alteration of signalling [20]

Adenosine A2A and metabotropic

glutamate receptor 5

Enhanced ERK activity [39]


2.4. Evidence from the resolution of three-dimensional

structures

Three-dimensional crystal structures can provide

invaluable information about the inactive and active

conformations of GPCRs and currently the only crys-

tal structures available for full-length GPCRs are for

the light receptor, rhodopsin [123,127,163]. Crystallo-graphic data obtained for rhodopsin confirms that this

model family A GPCR exists as a structural dimer

[123,127,163]. With regards to receptor dimerization,

the exact nature of the dimer and the residues com-

prising the dimerization interface, can be determined

from crystallographic data. Crystal structures of the

extracellular ligand-binding domain of the metabo-

tropic glutamate receptor (mGluR1) reveal the exis-tence of disulfide linked dimers [86]. The two ligand

binding domains were found to be linked by a disulfide

bridge between Cys 140, confirming earlier reports us-

ing site-directed mutagenesis [166]. Furthermore, it was

demonstrated that glutamate binding resulted in

movement of the two lobes in each ligand binding

domain, stabilizing the active dimer configuration and

potentially transducing a conformational change to the

transmembrane and intracellular regions to activate

the receptor. Recently, it was demonstrated that the

residues involved in the mGluR1 dimer interface are

crucial in this domain rearrangement that leads to ac-

tivation [149], thus tightly linking dimerization with

receptor activation.

2.5. Biophysical techniques

Biophysical techniques represent a powerful tool as

they enable the detection and monitoring of GPCR di-

mers in living cells. Fluorescence (FRET) and biolumi-

nescence (BRET) resonance energy transfer have both

been used to demonstrate receptor dimers in living cells[33]. FRET and BRET technologies both rely on the

transfer of energy between an energy donor and accep-

tor which is strictly proximity-dependent, with donor

and acceptor being <100�AA apart, making these tech-

niques ideal for the study of dynamic protein–protein

interactions (Fig. 1).

Fig. 1. Schematic representation of the application of BRET to detect GPCR oligomerization. (A, C) Receptors fused with either the energy donor,

Renilla luciferase (Rluc) or the energy acceptor, green fluorescent protein (GFP) or enhanced yellow fluorescent protein (EYFP) are co-expressed. In

the absence of dimerization, no energy transfer is observed following addition of the Rluc cell permeable substrate, coelenterazine. Light is emitted

from Rluc at its peak wavelength of 470 nm. (B, D) If a constitutive or ligand-induced receptor–receptor interaction occurs bringing the donor and

acceptor tags within 100�AA, energy resulting from the degradation of coelenterazine by Rluc will be transferred to EYFP resulting in an emission of

additional light at 530 nm characteristic of EYFP. The principle of FRET is similar, with the energy donor being a fluorescent protein (e.g., cyan

fluorescent protein), instead of a bioluminescent molecule.


2.5.1. Fluorescence resonance energy transfer

In the case of FRET, both the donor and acceptor

molecules are fluorescent proteins or dyes, with an

overlap occurring between the emission spectrum of the

donor and the excitation spectrum of the acceptor

molecule. Through the use of FRET in conjunction

with imaging techniques, receptor dimerization has

been studied both spatially and temporally, in live cells[33] (Table 1). Commonly, cyan fluorescent protein

(CFP) and yellow fluorescent protein (YFP), are em-

ployed as donor and acceptor molecules, respectively.

However, other molecules have been used. The agonist-

mediated dimerization of the GnRH receptor

(GnRHR) was demonstrated using FRET with GFP as

the donor and red fluorescent protein (RFP) as the

acceptor [24]. Fluorescent dyes, fluorescein isothiocya-

nate (FITC) and rhodamine, conjugated to antibodies

directed towards HA-tagged somatostatin receptor 5

(SSTR5) and dopamine D2 receptor, were used to

detect the SSTR5/D2 hetero-dimerization using FRET

[143]. Time-resolved FRET (TR-FRET) has been used

to demonstrate the hetero-dimerization of the b2-ad-renergic and d-opioid receptors [102]. TR-FRET takes

advantage of the long-lived fluorescence of fluoro-phores such as europium3þ (donor) and allophycocy-

anin (acceptor), thus reducing the background due to

autofluorescence.

2.5.2. Bioluminescence resonance energy transfer

BRET is a naturally occurring phenomenon observed

in many marine animals including the jellyfish Aequorea

victoria and the sea pansy Renilla reniformis. In these


organisms bioluminescence resulting from the degrada-tion of the substrate coelenterazine by the luciferase

enzyme is transferred to green fluorescent protein

(GFP), which re-emits light at its characteristic wave-

length when the two proteins interact. This phenomenon

has been exploited in the BRET technique where it is

used to detect and monitor protein–protein interactions.

The two proteins of interest are fused with either Renilla

luciferase (Rluc) (energy donor) and GFP or a red-shifted variant of GFP, enhanced yellow fluorescent

protein (EYFP) (energy acceptor). The degradation of

the cell-permeable substrate coelenterazine by Rluc re-

sults in the emission of light that is transferred to the

EYFP when in close enough proximity (<100�AA). It is

then re-emitted at a wavelength characteristic of EYFP

[178].

BRET methodology was first developed to study thedimerization of the light-sensitive circadian clock pro-

tein, KaiB in Escherichia coli [178]. Since then it has been

used to demonstrate the homo-dimerization of several

GPCRs including the b2-adrenergic receptor [7], thy-

rotropin releasing hormone (TRH) and GnRH receptors

[84] and oxytocin and vasopressin V2 receptors [164]

(Table 1). BRET has also been used to demonstrate the

existence of heterodimers, including interactions be-tween the adenosine A1 and P2Y1 receptors [182] and

between the b1- and b2-adrenergic receptors [88]. The

majority of homo- and hetero-dimers detected using

BRET are pre-formed in the absence of ligand, with

several reports describing either an increase or a decrease

in the BRET signal following receptor activation

[7,19,84,182]. Ligand modulation of GPCR dimerization

will be discussed in more detail later.

2.5.3. Advantages and disadvantages of BRET and FRET

Biophysical techniques have generated important

new evidence that strongly supports the existence of

GPCR dimers in living cells, providing advantages over

other more traditional methodologies. However, both

BRET and FRET suffer from limitations. It should be

noted that both FRET and BRET are extremely de-pendent on the distance between donor and acceptor

molecules and orientation, such that it is possible that

an interaction could be occurring which puts the donor

and acceptor molecules in spatial arrangements that are

unfavourable for energy transfer. These biophysical

approaches overcome some of the limitations of more

conventional techniques, primarily because they allow

protein interactions to be monitored in live cells in real-time. Biophysical techniques allow receptor interactions

to be studied in live cells with the proteins expressed in

the correct location in the cell, thus overcoming the

limitations of biochemical methods such as co-immu-

noprecipitation which could potentially lead to artefac-

tual aggregation of receptors. BRET provides an

advantage over FRET in that it avoids the need for

excitation and with this the associated problems ofautofluorescence, photobleaching, cell damage, and

signal loss. Furthermore, the reduced background fluo-

rescence associated with BRET makes it an extremely

sensitive technique allowing the detection of weak

interactions or low-level protein interactions [184].

A potential limitation of BRET, however, is the in-

ability to determine where in the cell the interaction is

occurring. The development of single-cell BRET imagingwould overcome this problem and represent a significant

advancement in the BRET technology, including its ap-

plication to the study of GPCR interactions. Imaging of

BRET has been performed on E. coli colonies [178] and

on CHO cell extracts [172]. One study reports measuring

single cell BRET in HEK293 cells, however, subcellular

resolution was not obtained [9].

Biophysical techniques predominantly involve heter-ologous expression systems. To date all studies designed

to investigate GPCR dimerization by utilizing biophys-

ical techniques have been performed in cell lines trans-

fected with the receptors (often epitope tagged) of

interest and often expressed at non-physiological levels.

As such, it has been suggested that BRET characterizes

artefactual interactions occurring merely due to protein

over-expression [137]. However, by monitoring interac-tions using high-sensitivity BRET instrumentation ca-

pable of detecting BRET signals between proteins

expressed at levels near or below physiological levels,

BRET can indeed provide evidence that a protein in-

teraction is occurring at endogenous expression levels.

Reports are now emerging where BRET has been used

to monitor receptor dimerization occurring between

receptors expressed at levels similar to those observed innative tissues [9,57,68,164] giving strength to the argu-

ment that homo- and hetero-dimerization detected by

BRET is not merely a result of over-expression and

could reflect the in vivo situation. Currently it is not

possible to measure BRET between endogenously ex-

pressed receptors due to a lack of appropriate donor and

acceptor molecules that could be conjugated to receptor

specific antibodies. The sensitivity of certain FRETapproaches allows measurements of receptor interac-

tions in single cells, and the availability of receptor-

specific antibodies for GPCRs would make it more

feasible to analyze interactions in live cells endogenously

expressing the receptors.

3. Mechanism of oligomerization

Unravelling the structural mechanisms for GPCR

dimerization will involve the identification of the di-

mer interface, which is largely unknown at present.

Several models have been proposed with respect to

how dimer or oligomer formation occurs. These are (i)

covalent bonds (i.e., disulfide bonds) formed between


extracellular domains, (ii) interaction of intracellulardomains particularly the C-terminal tail (C-tail), and

(iii) interactions between transmembrane domains.

However, as more data emerges it seems probable that

GPCRs use a combination of these mechanisms.

3.1. Extracellular domains

Oligomerization of Class C receptors such as the cal-cium-sensing receptor and the metabotropic glutamate

receptor-1 (mGluR1), both characterized by their extra

large N-terminal domains, is via disulfide bond forma-

tion between N-terminal cysteine residues. Mutation of a

single cysteine residue (Cys 140) in mGluR1 disrupts di-

mer formation [138]. The role of cysteine residues was

clearly demonstrated when the crystal structure of the N-

terminus of the mGluR1 was obtained (see Section 2.4)[86]. In the case of the calcium sensing receptor, whilst

mutation of cysteine residues disrupts disulfide bonding

and increases the proportion of monomer, significant

levels of dimer and oligomer remain, suggesting disulfide

bonding is not the only mechanism involved in dimer-

ization of this receptor class [187]. Other classes of re-

ceptors might also dimerize through disulfide bond

formation, for example, dimer/oligomer formation waslost between the 5HT1A and 5HT1D receptors when

SDS–PAGE gels were run under disulfide bond-reducing

conditions [148]. Intermolecular disulfide bonds in the

second extracellular loop of the M3 muscarinic receptor

were also found to be involved in dimerization, however,

they were found not to be essential with non-covalent

interactions being sufficient for dimerization [186].

3.2. C-terminal tails

Other GPCRs, namely the metabotropic GABAB

receptors, form heterodimers (GABABR1/GABABR2)

via coiled-coil domains within their intracellular car-

boxy terminal tails (C-tails) [78]. However, more recent

data has demonstrated that the C-tail of the GABABR2

masks an endoplasmic reticulum (ER) retention signalin the C-tail of the GABABR1. Thus the interaction

between the C-tails may be necessary for export from

the ER and cell surface expression [100,126]. Further-

more, C-terminally truncated GABAB receptors lacking

this motif still interact [17,126]. The C-tail may also be

involved in homo-dimerization of the d opioid receptor,

a Class A GPCR. Whilst this receptor does not contain

a coiled-coil domain, a mutant receptor where a portionof the C-tail is deleted does not exhibit a significant level

of dimer formation [26,139].

3.3. Domain swapping versus lateral packing

There are currently two theories as to how the trans-

membrane helices of rhodopsin-like GPCRs interact to

form dimers. �Contact dimerization� (lateral packing)would enable the heptahelical integrity of the monomer

to be maintained, but would require the presence of

additional interacting sites on the exterior of the trans-

membrane bundle (Fig. 2A). In contrast, �domain-swap-

ping� would require the separation of two independent

folding units within the receptor. The same interaction

sites as used in forming the monomer could then be uti-

lized to form the dimer (Fig. 2B). There is increasingevidence that rhodopsin-like GPCRs are made up of two

distinct folding units, one consisting of the N-terminus to

intracellular loop 3 (ICL3) including TM1–TM5 (Do-

main A) and the other from ICL3 to the C-terminus

including TM6 and TM7 (Domain B) [53]. For the

b2-adrenergic receptor [83], M2 and M3 muscarinic re-

ceptors [97], and the dopamine D2 receptor [150], these

domains are not functional when expressed alone, how-ever, co-expression of both receptor fragments results in

the restoration of binding and signalling. A consequence

of having two distinct units is the possibility of greater

flexibility within the heptahelical GPCR structure. ICL3

could act as a hinge, thereby allowing the units to sepa-

rate. This could facilitate ligand binding and/or receptor

activation, and would be a pre-requisite for dimerization

by domain-swapping as discussed below [53].Contact dimerization would theoretically enable

high-order oligomers to be formed, however, domain-

swapping can only produce dimers (Fig. 2C). An alter-

native possibility is that higher-order structures such as

tetramers consist of two domain-swapped dimers that

interact by lateral contact.

The strongest evidence for domain-swapping in dimer

formation arises from the work of Maggio et al.[96,98,99] and Monnot et al. [111]. Chimeric receptors

were created, comprising the a2-adrenergic receptor

domain A and M3 muscarinic receptor domain B (and

vice versa). These chimeras did not bind adrenergic or

muscarinic receptor ligands when expressed alone,

however, co-expression restored the binding of both.

The work of Schoneberg et al. [153] using the M3

muscarinic receptor again demonstrated that co-ex-pression of domains A and B could restore wild-type

binding affinity for a range of ligands. Additionally, they

showed that receptors split in ICL2 or extracellular loop

2 (ECL2) could also bind agonists and antagonists, al-

though with affinities 2.5- to 20-fold lower than ob-

served at the wild-type receptor depending on the

particular ligand [153]. This indicates the possibility of

different folding units that can be swapped to form di-mers. Monnot et al. [52,111] demonstrated receptor

rescue by complementation of two mutated type 1 an-

giotensin receptors (AT1R), one mutated in TM3 and

the other in TM5, postulating that swapping of TM5 to

TM7 was occurring using ECL2 as a hinge.

Studies using the V2 vasopressin receptor have pro-

vided evidence for both models. The domain-swapping

Fig. 2. Schematic diagram depicting the potential mechanisms involved in formation of GPCR dimers and oligomers. (A) In the contact dimer or

lateral packing model, GPCRs directly contact one another using residues on the exterior of the transmembrane domains, additional to those used in

intra-molecular interactions. This model does not require any change in monomeric integrity. (B) In contrast, the domain swapping model requires

the swapping of independently folded transmembrane domains between monomeric units, with the same interaction sites used in forming the

monomer and the dimer. (C) Contact or lateral packing dimerization would theoretically enable higher-order oligomers to form with a second set

of interaction points being used to form the oligomer. Alternatively, two domain-swapped dimers could interact by lateral packing to form an

oligomeric structure.


mechanism was supported by the functional rescue of

mutant V2 receptors by the co-expression of a V2 re-

ceptor C-terminal fragment (ICL3 to C-terminus in-

cluding TM6 and TM7) [154,155]. In contrast, similar

studies using an N-terminal receptor fragment (N-ter-

minus to ICL3 including TM1-TM5) did not restore thefunction of receptors containing mutations in the N-

terminal region, despite evidence of their interaction

[156]. Furthermore, co-expression of full-length recep-

tors, one with an N-terminal mutation and one with a C-

terminal mutation, again did not result in restoration of

function. This suggests dimerization involves lateral in-

teraction (contact dimerization) as opposed to domain-

swapping. Furthermore, dopamine D2 receptor function

could not be restored by co-expression of mutant and

truncated receptors, implying that dimerization does not

occur by domain-swapping [89]. This again indicatesthat different receptors are likely to utilize different

dimerization mechanisms. Indeed, the evidence for

receptors dimerizing in a manner not involving

domain-swapping, presumably by lateral interaction, is

increasing.


3.4. Transmembrane domain motifs/residues involved in

dimerization

For many GPCRs it is still not clear what residues

will be involved in the dimerization interface. For those

receptors that do form complexes via their transmem-

brane domains, the question arises as to which residues

are involved and whether a universal sequence motif is

implicated. Herbert et al. [63] have demonstrated theexistence of a ‘‘dimer motif’’ (LXXXGXXXGXXXL

where X is any amino acid) within the cytoplasmic side

of TM6 of the b2-adrenergic receptor. However, this

motif is not highly conserved in other class A GPCRs

and the peptide used representing TM6 of the b2-ad-renergic receptor did not disrupt dimer formation of

other GPCRs tested suggesting that each receptor may

have unique residues that are involved in oligomeriza-tion. Similarly, peptides representing TM6 and TM7 of

the dopamine D2 receptor also blocked dimerization

[117]. In contrast, similar experiments on the dopamine

D1 receptor were not able to inhibit dimerization, al-

though signalling was compromised [45]. This adds

further evidence to the view that oligomerization for

different receptors is likely to occur via different mech-

anisms. It should be noted that with this approach tomapping dimer interfaces, the results do not necessarily

implicate TM6 and TM7 as dimerization sites, as the

binding of the peptide to a specific site may interfere

with the receptors ability to form a dimer through an-

other region of the receptor. Recently, using cysteine

cross-linking, TM4 was identified as a symmetrical in-

terface in the dopamine D2 dimer, rather than TM6 or

TM7 as previously described [55].Computer simulations and data mining approaches,

such as evolutionary trace and correlated mutational

analysis, have also highlighted the importance of amino

acid residues within TM5 and TM6 for GPCR dimer-

ization, although these techniques cannot distinguish

between contact and domain-swapped dimers [27,52,53].

These theoretical approaches involve multiple sequence

alignments of both conserved amino acids and knownreceptor mutations, based on the tendency for such

mutations to accumulate at protein interfaces. In addi-

tion to identifying functional sites within TM5 and

TM6, these studies have also discovered a secondary

interface between TM2 and TM3. Possible functional

roles that have been suggested for these domains are:

formation of higher-order complexes (Fig. 2), hetero-

dimerization, or interaction with non-GPCR proteins[27]. If GPCRs do exist as oligomers rather than dimers,

more than one dimer interface must exist. Recent data

from native membranes of mouse retina showed that

rhodopsin was arranged in an oligomeric array of

structural dimers, with potential contact points between

monomers within the dimer in TM4 and 5, whereas

contacts to form oligomers or rows of dimers exist in

TM1 and 2 and ICL3 [90]. These findings have beensupported by atomic force microscopy which provides

images of such clarity that vertical resolution can be

exhibited in the region of 2�AA [41].

TM1 was found to be the primary dimerization in-

teraction site for yeast a-factor (STE2 gene product)

receptor when analyzed using FRET with various re-

ceptor deletion mutants. TM2 and the N-terminal ex-

tracellular domain were found to further stabilize thedimer [125]. In this case, dimerization is thought to oc-

cur by the contact-dimer (lateral packing) model rather

than by domain-swapping as receptor fragments con-

taining only the N-terminal extracellular domain and

TM1 can self-associate. In another study, this time ex-

amining the bradykinin B2 receptor, the N-terminal

extracellular domain was also found to be involved in

dimerization, which was induced following treatmentwith agonist [1]. A recent study on the a1b-adrenergicreceptor revealed using receptor fragments and chime-

ras, that TM1, and to a lesser extent TM7, were involved

in its dimerization [161]. The GXXXG motif present in

certain GPCRs and reported to be important in dimer-

ization of the b2-adrenergic receptor [63], is located in

TM2 and 7, however, it was found not to be involved in

a1b-adrenergic receptor homo-dimerization. A similarapproach was used to demonstrate that the chemokine

receptor, CXCR2 homo-dimerizes through a region

comprising ECL1, TM3, and ICL2 [165]. Interestingly,

CXCR2 was also shown to heterodimerize with the

AMPA-type glutamate receptor 1 (GluR1), resulting in

drastically compromised CXCR2 function. The inter-

action was mediated through a region partially over-

lapping with that involved in homo-dimerization,comprising TM1, ICL1, TM2, ECL1, and TM3 [165].

From the findings to date, it is probably more likely

that different residues will be involved for different re-

ceptors, rather than a general ‘‘dimerization consensus

sequence’’ being present. Furthermore, for a given re-

ceptor the regions involved may be different for the

homodimer compared to those involved in the forma-

tion of heterodimers. Defining the interfaces and resi-dues involved in GPCR dimerization may help to

disrupt dimer formation, potentially providing clues to

the role of dimerization in receptor function. There is

also the possibility that specific GPCR dimers could be

targeted directly, providing a means to alter or modulate

receptor homo- and/or hetero-dimerization and receptor

function.

3.5. Receptor dimer or oligomer

The proportion of monomeric and oligomeric recep-

tors, and the question of whether GPCRs can function

as monomers, still requires resolution. Even the nature

of the oligomer (dimer or higher-order oligomer) re-

mains unclear. Unfortunately, many current techniques,

Fig. 3. Visualization of rhodopsin arranged in an oligomeric array of receptor dimers. (A) Deflection image of rod outer-segment disc membranes

from mouse retinae, visualized by atomic force microscopy showing the textured topography consisting of densely packed lines. Numbers 1, 2, and 3

represent the three types of surfaces present—cytoplasmic side of the disc membrane, lipid and mica support, respectively. Scale bar represents

200 nm. (B, C) Higher magnification images of the organization and topography of cytoplasmic surface of rhodopsin obtained using atomic force

microscopy. (B) Shows the regular arrangement of rhodopsin in the membrane. (C) Shows the oligomeric arrangement of rhodopsin, with rows of

dimers. Occasional individual dimers (dashed circle) and monomers (arrow) are also seen. Scale bars : (B) 50 nm; inset 5 nm; (C) 15 nm. This figure is

reproduced, in part, with permission from Nature (Fotiadis et al., Nature, 2003, 421: 127) and the authors.


such as biophysical techniques, cannot easily distinguishbetween these two possibilities and the term dimer is

often used interchangeably with oligomer or multimer.

A recent study addressed this issue by describing the use

of a BRET competition assay. The BRET signal be-

tween either melatonin receptor MT1 or MT2 homodi-

mer BRET pairs was reduced linearly with increasing

amounts of untagged MT1 or MT2 receptor respec-

tively. This showed that the MT1 and MT2 homodimersexisted predominantly as dimers and not as either

monomers or higher-order structures [9]. The finding

supports the hypothesis that the receptor dimer repre-

sents the functional signalling unit. Another study used

quantitative BRET to determine the level of b2-adren-ergic receptor constitutive dimers at above 80% of the

total receptor pool, indicating that in live cells the vast

majority of the receptor exists pre-formed as a dimerrather than a monomer [105]. Rhodopsin has recently

been visualized as an oligomeric array of structural di-

mers in native membranes, with only a few individual

dimers and monomers observed (Fig. 3) [41,90].

4. Regulation of GPCR dimerization

In a single cell several GPCRs could be co-expressed,

potentially leading to a complex combination of homo-

and heterodimers being formed. However, little is

known about how dimerization is regulated. It is pos-

sible that homo- or heterodimer formation is influenced

by receptor expression levels and/or relative affinities of

particular receptor combinations [105,164]. Alterna-

tively, GPCR dimerization may require and/or be

modulated by additional proteins. These two mecha-nisms may be particularly relevant with regard to the

modulation of constitutive dimerization.

4.1. Ligand modulation of GPCR dimerization

There has been much controversy surrounding the

issue of whether ligands can modulate receptor oligo-

mers either by (i) promoting the association or dissoci-ation of dimers or (ii) binding to pre-formed oligomers

to alter the conformation of the oligomer, with evidence

existing that supports both constitutive and agonist-

promoted dimerization. Several studies have examined

the effect of ligands on dimerization using co-immuno-

precipitation and have observed an increase [63,144,188]

or decrease [26] in the amount of receptor dimer present

following agonist stimulation. Others saw no change[74,186] or only observed dimers after ligand binding

[1,145]. Furthermore, different agonists have been ob-

served to have different effects on the same heterodimer.

In the case of the dopamine D1 and adenosine A1

heterodimer, a dopamine receptor agonist decreased the

amount of dimers, whilst the adenosine A1 receptor

agonist had no effect [48]. From crystallization studies

the mGluR1 was shown to form dimers in the presenceor absence of ligand [86].

Biophysical techniques have also been used to eval-

uate the ability of ligands to modulate GPCR dimer-

ization. Agonist-induced increases in the BRET signal

have been reported for the b2-adrenergic receptor ho-

modimer [7], TRH and GnRH receptor homodimers

[84] and the adenosine A1/P2Y1 heterodimer [182].

Whilst for other GPCRs no change [10,68,71,102,164] or


even a decrease in the BRET signal was observed [19].Similarly, the use of FRET has demonstrated variations

in the effect of ligands on dimerization. FRET studies on

the yeast a-factor receptor indicate that dimerization of

this receptor is constitutive and unaffected by ligand

[124]. In contrast, the homo- and hetero-dimerization

of the SSTR5 [128,143,144] represents one of only a

few resonance energy transfer studies on GPCR dimer-

ization, along with those on the dimerization ofthe GnRHR [67,84], in which significant constitutive

dimerization was not detected [143,144].

The observed diversity of ligand-induced effects on

GPCR dimerization may reflect true differences that

exist between receptors. However, differences in the in-

terpretation of results obtained due to limitations and

differences in methodologies may also explain this di-

versity. Changes in the amount of dimer observed maynot necessarily reflect an increase or decrease in the

amount of dimer formed but rather an agonist-induced

conformational change in the dimer. In co-immuno-

precipitation studies, an observed increase in the quan-

tity of dimer could be explained by a ligand-induced

conformational change increasing accessibility to anti-

bodies, resulting in an increased detection of dimers.

Likewise, changes in FRET and BRET signals could beinterpreted as changes in the actual amount of dimer

present, or conformational changes induced in the dimer

that result in more or less favourable orientations of the

donor and acceptor tags. Evidence from studies on the

dimerization of the melatonin receptors 1 (MT1R) and 2

(MT2R) showed that ligand promoted BRET between

MT1R/Rluc and MT2R/EYFP, but not between

MT2R/Rluc and MT1R/EYFP [9]. Moreover, the factthat antagonists as well as agonists led to an increase in

BRET suggests that changes in the BRET signal are

related to conformational changes in the receptor, rather

than dimerization being linked to receptor activation.

To add to the controversy, studies utilizing different

techniques to investigate the dimerization of the same

receptor have reported contrasting observations. The

chemokine CCR5 receptor was shown to undergo li-gand-dependent dimerization using immunoprecipita-

tion [103], whilst using BRET another group were

unable to detect any changes in BRET signals following

agonist activation [68]. The lack of detection of ligand-

induced BRET seen for dimerization of several GPCRs,

may be explained by insufficient sensitivity in the de-

tection of what may be relatively small conformational

changes, especially if the pre-formed dimer is already ina conformation which brings donor and acceptor tags

into an optimum orientation.

Due to discrepancies in the role of ligand binding in

dimerization, largely contributed by differences in the

methodologies used to detect dimerization, it is often

difficult to understand the functional relevance of

GPCR homodimerization. The question of whether

GPCRs exist as stable pre-formed dimers or can bemodulated by ligand has received much attention over

recent years. Clarifying the role of ligand binding in

modulating/regulating receptor dimerization will help to

understand the role dimerization may play in receptor

function.

4.2. Site of synthesis of dimers

Many studies report the existence of pre-formed di-

mers and furthermore, there is evidence to suggest that

dimers are formed in the endoplasmic reticulum (ER).

An example mentioned previously in Section 3.2 is the

GABAB receptor, where hetero-dimerization between

GABABR1 and GABABR2 occurs in the ER [17,126].

The oligomerization of the yeast a-factor receptor

(STE2 gene product) was found to occur in the ER[125]. Using CFP and YFP tagged a-factor receptors,

FRET was monitored in a series of subcellular fractions.

The efficiency of energy transfer was found to be similar

in purified plasma membrane and ER fractions. Simi-

larly, constitutive BRET signals were obtained from

both plasma membrane and ER fractions prepared from

cells co-expressing Rluc and EYFP tagged CCR5 re-

ceptors [68] and also in a study investigating oxytocinand vasopressin V2 receptor homo- and hetero-dimer-

ization [164]. Investigations into CXCR2 homo-dimer-

ization revealed that the receptor still dimerizes in the

absence of glycosylated monomeric receptors, indicating

that the receptors dimerize during the biosynthetic

pathway before they reach the plasma membrane and

suggesting that perhaps dimerization is an essential step

for proper expression of receptors in the plasma mem-brane [165]. This suggests that perhaps GPCRs gener-

ally traffick to the cell surface as dimers, where upon

ligand binding the dimer may (a) dissociate, (b) increase

in number, (c) become oligomeric or (d) change its

conformation.

4.3. Role of additional proteins in modulating dimeriza-

tion

Dimer or oligomer formation may require the pres-

ence of a third party, in that receptor complex formation

may be modulated/regulated by additional proteins. It

would be interesting to determine if the dimerization

process reputedly occurring in the ER involves addi-

tional proteins or occurs spontaneously. Candidate

oligomerization regulatory proteins are GPCR chaper-ones such as the receptor-activity modifying proteins

(RAMPs) and others like calnexin, or the Homer pro-

teins [16,142]. The PDZ domain-containing proteins

may also potentially be involved in GPCR dimerization,

as they are known to interact with certain GPCRs.

These proteins are involved in the scaffolding and tar-

geting of proteins to specific subcellular domains, which


in turn may even cause or facilitate GPCR oligomeri-zation. For example, the PDZ domain protein PICK-1

has been shown to cause clustering of the prolactin-re-

leasing peptide receptor [93]. Although this study did

not give direct evidence that the receptor underwent

oligomerization, it is possible that such PDZ domain

proteins may have a role in this process.

5. Functional significance of GPCR homo-dimerization

It is often difficult to ascertain the functional rele-

vance of GPCR homodimerization, as there are many

conflicting reports regarding whether dimers are pre-

formed or regulated by ligand binding. In those recep-

tors that display constitutive oligomerization, oligomer

formation may occur in the ER and be a requirementfor trafficking to the cell surface (possibly by chaper-

one proteins) [73,80,173]. It is also possible that oligo-

merization may be required for receptor activation

and constitutive dimerization may be related to the

constitutive signalling of a receptor [7,84].

It has also been suggested that the general function of

GPCR homo-oligomerization is to form the interface

required for scaffolding of a multiprotein complex,consisting of receptor, G-protein, effector molecules and

adaptor proteins involved in internalization, thus cre-

ating a functional microdomain within the cell [31].

GPCR oligomerization may also allow multiple ligand

binding sites to be present, potentially creating an in-

creased gradient of receptor-mediated signalling that is

dependent on ligand concentration and receptor occu-

pancy. There is also the potential for co-operativitybetween different ligands, as well as between agonists

and antagonists. Furthermore, oligomerization could

provide a means of signal amplification, through the

activation of more than one receptor with only a single

ligand. The observation that functionality can be re-

stored upon co-expression of two non-functional mu-

tant receptors, one that can bind ligand and one lacking

the ability to couple to G-proteins, demonstrates howligand binding to one receptor might activate a neigh-

bouring receptor within the oligomeric complex. Indeed

this is the case with the GABAB receptor, whereby li-

gand binds to the GABABR1, inducing G-protein cou-

pling to GABABR2 [30,42,82,141]. It is thought that in

the GABAB heterodimer (and potentially in dimers of

other class C/family 3 GPCRs), a conformational

change in the large extracellular domain [venus fly trapmotif (VFTM)] following ligand binding is transduced

to the transmembrane domains. The resultant confor-

mational change in this region is thereby believed to

stabilize G-protein coupling.

As previously discussed, elegant studies using

atomic-force microscopy on rod outer-segment disc

membranes showed rhodopsin receptors arranged in

an oligomeric array consisting of closely packed re-ceptor dimers [41,90] (Fig. 3). This visualization of the

rhodopsin receptor organization is the first report to

clearly demonstrate that GPCRs exist as dimers or

oligomers in native tissues. Although this may be

peculiar to the rhodopsin system (the high density

packing of the receptor in the disk membranes could

be important for efficient photon absorption), this

oligomeric organization may have multiple implica-tions for other GPCR-mediated signal transduction

systems.

6. Functional significance of splice variants

Dimerization of receptors may provide a mechanism

by which splice variants and mutant receptors couldmodulate wild-type receptor function and have physio-

logical effects. Alternatively, it may provide a means

to rescue the function of mutant receptors in heterozy-

gous individuals, through dominant-positive receptor

interactions.

6.1. Truncated splice variants can inhibit the function of

full-length receptors

Several studies have shown that receptor splice vari-

ants can alter wild-type receptor function, with dimer-

ization being the inferred mechanism. In addition to the

full length GnRHR, a truncated splice variant lacking

128 bp has been isolated from human pituitaries [54].

This protein is non-functional when expressed alone,

however, upon co-expression in COS-7 cells with full-length GnRH receptor it inhibits trafficking of the re-

ceptor to the plasma membrane [54]. The physiological

role of a dominant negative GnRHR splice variant in

humans is presently unclear. Certain splice variants of

the bullfrog type 3 GnRHR have also been shown to

inhibit full-length receptor function and seasonal chan-

ges in full-length to splice variant ratio implies that there

may be a physiological regulatory role in amphibians[170]. Much debate has surrounded the possibility of a

second GnRH receptor in the human [108], particularly

following the cloning of type II GnRH receptors from

the marmoset [107], Green Monkey, and Rhesus Mon-

key [113]. Although the expression of a second full-

length human receptor appears increasingly unlikely,

the possibility of truncated splice variants remains [112].

It has been suggested that such receptor fragmentsmay have a role in the regulation of the type I GnRH

receptor [108].

There are examples of truncated splice variants of

other GPCRs potentially playing a role in human

disorders. It has been suggested that the dopamine D3

receptor and its splice variants play a role in schizo-

phrenia [79], the key to which may be splice variant


hetero-oligomerization [34,79,119]. The best character-ized of the dopamine D3 splice variants is D3nf, which

does not exhibit D3 receptor-like pharmacology

[34,79,94,119, 151]. This protein is a truncated form of

the D3 receptor that diverges from the full-length se-

quence in the region coding for ICL3 [94,151]. Follow-

ing the splice junction, the remaining sequence may code

for an alternative TM6 and extracellular C-terminal tail

[34].There is increasing evidence for the formation of D3/

D3 homo-oligomers and D3/D3nf hetero-oligomers as

demonstrated by immunoprecipitation and immunocy-

tochemistry in vitro and in vivo [34,79,119]. The mech-

anism by which D3/D3nf hetero-oligomerization

influences wild-type D3 receptor function is debatable.

D3nf may target a proportion of D3 receptors to a par-

ticular region of the dendritic tree [119]. Alternatively,hetero-oligomerization may have a dominant-negative

effect on D3 receptor function, either by preventing

trafficking to the plasma membrane [79], or by modu-

lating the D3 receptor in the membrane such that its

ability to bind ligand is compromised [34]. Two D3

receptor fragments, D3trunk (N-terminus to ICL3) and

D3tail (ICL3 to C-terminus), have been engineered and

used to demonstrate an inhibition of wild-type D3 re-ceptor expression [150]. Furthermore, greatly reduced

binding to D3 receptors was exhibited in mice hetero-

zygous for a D3 receptor mutation that produces a

truncated receptor [5]. This is an important consider-

ation for conditions resulting from heterozygous recep-

tor expression. Previously it was assumed that the

attenuated cellular function resulted from reduced ca-

pacity to produce fully functional receptor protein.However, it is becoming increasingly likely that expres-

sion of the mutated protein exacerbates the problem as a

result of this dominant-negative activity.

Another example of the dominant-negative activity

of a splice variant playing a role in human disease

appears to be in ductal pancreatic adenocarcinoma.

These tumors have been shown to be unresponsive to

nanomolar concentrations of secretin [72] despite ex-pression of the full-length secretin receptor [28]. Splice

variant expression exceeded that of full-length receptor

in pancreatic cell lines and tumors, but was not found

in normal pancreatic tissue. In addition, the splice

variant possessed dominant-negative activity in vitro.

BRET was used to demonstrate heterodimerization

with the full-length receptor, providing a mechanism

for the inhibitory effect [28]. The secretin receptor hasbeen shown to mediate growth inhibition and may

well have a physiological role in balancing growth

stimulation mediated by the receptor for vasoactive

intestinal polypeptide (VIP) [72]. Therefore, by ablat-

ing the growth inhibitory effect of secretin, the ex-

pression of the splice variant may contribute to tumor

progression [28].

6.2. Therapeutic potential of receptor fragments for the

restoration of receptor function

The ability to restore the function of a specific re-

ceptor by co-expression of a receptor fragment has great

therapeutic potential. Gene-delivery systems, such as the

use of adenoviruses, provide the possibility of inserting

genes into human cells in vivo [59]. However, a major

problem is the targeting to specific cells. A gene codingfor a functional full-length receptor delivered via an

adenoviral vector injected into the blood could poten-

tially result in the expression of that receptor in cells

throughout the body, resulting in aberrant cellular

responses. However, a receptor fragment that is non-

functional when expressed alone, but restores the func-

tion of an endogenously expressed mutant receptor,

could be delivered throughout the body and only influ-ence those cells in which it is required. X-linked neph-

rogenic diabetes insipidus is an example of a disease

state where such treatment could be applicable. In vitro

studies have already demonstrated that adenovirus-

mediated gene transfer can restore V2 receptor function

[155]. The next stage is to investigate the suitability of

such an approach in an animal model.

7. Functional significance of hetero-dimerization

The observation that hetero-dimerization can result

in the formation of receptor units with altered phar-

macological and functional properties compared to the

individual receptor monomers or homodimers has

enormous consequences for patient therapy and theunderstanding of biological systems as a whole. For

certain receptors, hetero-dimerization is essential for

receptor function. In the case of the GABAB receptor,

hetero-dimerization between the GABABR1 and

GABABR2 receptors is required for trafficking to the

cell surface and receptor function [73,80,173]. Hetero-

dimerization is also a required event for functioning of

the taste receptors. Taste receptor T1R3 complexes withT1R1 to form functional receptor units for recognizing

amino acids [115] and with T1R2 to sense sweet tastes

[114]. Indeed, hetero-dimerization may represent a

mechanism for generating diversity of function amongst

GPCRs and may explain some pharmacological activi-

ties not accounted for by individual receptors. Altera-

tions in ligand binding, signalling (both in the strength

of the signal generated and the type of signalling path-ways activated) and receptor trafficking, have all been

observed as a result of GPCR hetero-dimerization (Ta-

ble 2). Although many studies illustrate how hetero-di-

merization can diversify receptor-mediated responses,

hetero-dimerization has also been seen to inhibit or in-

activate receptor function. The hetero-dimerization of

the somatostatin type 2A (SSTR2A) and type 3 (SSTR3)


receptors was shown to result in inactivation of SSTR3function, with the heterodimer displaying pharmaco-

logical and functional characteristics resembling

SSTR2A [134]. Similarly, the angiotensin AT2 receptor

was found to interact with AT1R and antagonize

its function, without the requirement of AT2 receptor

activation [3].

Many of these novel hetero-dimeric or hetero-oligo-

meric receptor complexes involve neuroendocrine re-ceptors and hetero-dimerization represents a mechanism

by which signal cross-talk can occur between distinct

receptors. Such direct receptor–receptor interactions not

only add to the complexity of regulation of hormone

secretion by occurring between endocrine receptors,

they also play an important role in modulating neuro-

transmitter function and the interplay between neuro-

transmitters and the hypothalamic–pituitary axis. Asubset of these interactions and their possible physio-

logical roles are discussed in more detail in this section,

with a more exhaustive list summarized in Tables 1 and

2. The receptor interactions discussed below represent

just some of the hetero-dimerization events involving

neuroendocrine receptors. They are chosen to demon-

strate how such novel interactions can alter receptor

function and provide potential explanations for thephysiological cross-talk observed in the neuroendocrine

system.

7.1. Thyrotropin releasing hormone receptor

The hypothalamic neuropeptide, thyrotropin releas-

ing hormone (TRH) has well characterized roles in

controlling synthesis and release of prolactin and thyroidstimulating hormone (TSH) from the anterior pituitary

through its action at the TRH receptor (TRHR1).

However, TRH is also known to display extrapituitary

actions [15,66], including neurotransmitter actions. The

recent cloning of a second TRH receptor (TRHR2) from

the brain and spinal cord [18,69,122] provides a possible

explanation for these actions. A type 2 TRHR has

subsequently been cloned from mouse [58]. However,TRHR2 has not yet been described in humans. The

TRHR subtypes show a 50% overall identity, have sim-

ilar binding affinities for TRH and activate the same

signalling pathways [58,122,171]. Basal signalling has

been observed to be higher for TRHR2 than TRHR1

[162,171] and contradicting reports describe differences

in the internalization properties of the two receptors

[57,122,162].We have previously shown using BRET, that the

TRHR1 exists as pre-formed homo-dimers or oligomers

in live cells [84]. Agonist stimulation led to a time- and

dose-dependent increase in the amount of energy

transfer, however, whether this increase in BRET signal

was indicative of an increase in the amount of dimers

present or a conformational change in pre-existing

dimers, was not determined. Co-immunoprecipitationhas also been used to demonstrate TRHR dimerization

[188]. Using this approach receptors were isolated as

dimers under basal conditions, with the degree of di-

merization increasing upon agonist-stimulation due to

either a stabilization of the pre-formed dimer or an in-

crease in the amount of dimer formation. By employing

a C-terminally truncated TRHR it was also demon-

strated that this region is not required for dimerization[188], as previously suggested [57,84].

Hetero-dimerization between TRHR subtypes was

also observed, with the interaction shown to be specific

through the use of other tagged GPCRs and by per-

forming BRET competition assays using untagged

TRHRs. The TRHR1/2 heterodimer displayed altered

internalization properties compared to the individual

receptors, although ligand binding and signalling prop-erties were similar to the receptors expressed alone [57].

The TRHR2 was seen to internalize to a much lesser

extent and at a slower rate compared to the TRHR1,

whereas co-expression of both receptors in vitro resulted

in intermediate internalization kinetics. Interestingly,

using BRET it was demonstrated that the receptor in-

teractions with b-arrestin were altered in the heterodi-

mer, with b-arrestin 1 now binding to the TRHR2(normally a class A GPCR that preferentially interacts

with b-arrestin 2). It is thought that the heterodimer-

ization event leads to a receptor conformational change

more favourable for the binding of b-arrestin 1 and that

this may explain the change in internalization kinetics of

the heterodimer. As such, this represents the first ex-

ample of a GPCR heterodimer having altered associa-

tions, compared with homodimers, with intracellularregulatory proteins such as b-arrestins.

7.2. Gonadotropin releasing hormone receptor

Gonadotropin releasing hormone (GnRH) acts on

the anterior pituitary where it triggers the release of

luteinizing hormone (LH) and follicle-stimulating hor-

mone (FSH) from gonadotrope cells [158]. GnRH an-tagonists have been found to act as agonists if their

cross-linkage results in receptor microaggregation

[21,22,159] and more recent studies have demonstrated

agonist-induced GnRHR homo-oligomerization [24,67,

70,84]. Therefore, homo-dimerization may well be an

important component of GnRHR function. To date no

hetero-dimeric interactions have been described for the

GnRHR.

7.3. Dopamine D2 receptor

The five dopamine receptor subtypes, D1–D5, are

divided into two classes. D1 and D5 belong to the D1-

like class and are coupled to G-proteins that stimulate

adenylate cyclase. D2, D3, and D4 belong to the D2-like


class and are coupled to G-proteins that inhibit aden-ylate cyclase [110]. D2 dopamine receptors are present in

the anterior and intermediate lobes of the pituitary

gland. Hypothalamic dopamine acts via these receptors

to inhibit secretion of prolactin (PRL) [13,35,104] and

a-melanocyte-stimulating hormone ða-MSH) [25,160].

Immunoprecipitation experiments identified two im-

munoreactive species, of approximately 44 and 93 kDa,

believed to represent the D2 receptor monomer anddimer [116–118]. Further evidence for receptor oligo-

merization was provided by the inhibition of wild-type

D2 receptor expression by mutant or truncated D2 re-

ceptors [89,150]. Recent FRET studies have also pro-

vided additional evidence that dopamine D2 receptors

can exist as dimers in live cells [175]. Interestingly,

[125I]azidophenethylspiperone was found only to label

the D2 monomer whereas [125I]4-azido-5-iodonemona-pride labelled both monomers and dimers [117,185].

This phenomenon is likely to be clinically relevant as it

provides an explanation for the substantially different

D2 receptor densities observed in humans using positron

tomography with ligands such as [11C]methylspiperone

and [11C]raclopride [36,37,121,132,167,174]. The recep-

tor configuration appears to be altered by dimerization

such that certain ligands are not able to bind the dimericcomplex. These findings have important implications for

drug discovery. If oligomerization results in different

pharmacology compared to the monomer, the ability of

drugs to select between dimers and monomers has great

potential for increasing drug specificity.

Hetero-dimerization has been described for the do-

pamine D2 receptor with the SSTR5 [143], the adenosine

A2A receptor [65] and the dopamine D3 receptor [150],with these events providing possible explanations for the

complex functional interactions observed clinically and

experimentally, between the dopaminergic and other

receptor systems. By immunocytochemistry, dopamine

D2 receptors and SSTR5 were shown to co-localize in

specific subsets of rat neurons and FRET indicated that

heterodimerization was occurring between these recep-

tors when co-expressed in CHO-K1 cells [143]. Theheterodimer showed a greatly increased affinity for the

SSTR5 ligand upon co-treatment with a D2 agonist, but

a reduced affinity in the presence of a D2 antagonist. The

affinity of the D2 ligand for its receptor was also in-

creased in the presence of SSTR5 agonist. This phar-

macological synergy consequently results in potentiated

signalling from the heterodimer. Behavioural and clinical

evidence indicates interactions are occurring between thedopaminergic and somatostatinergic systems, with D2/

SSTR5 hetero-dimerization providing a possible expla-

nation for such observed cross-talk. Enhancement of

dopaminergic and somatostatinergic transmission has

been observed in vivo upon administration of somato-

statin (SST) or dopamine agonists. This hetero-dimer-

ization event may therefore have important clinical

implications. For example, treatment could be alteredsuch that co-administration of a SST agonist does

not produce the unwanted side-effects associated with

activation of the dopaminergic system.

Adenosine A2A/dopamine D2 receptor hetero-dimer-

ization has been demonstrated by co-immunoprecipita-

tion and BRET [77] with the receptor complex showing

co-desensitization and co-internalization upon treatment

with one or both agonists [65]. This A2A receptor-med-iated antagonism of the D2 receptor via hetero-dimer-

ization may provide a possible explanation for the

reduced effect of the D2-acting drug LL-DOPA used to

treat Parkinson�s disease. Adenosine is thought to in-

crease in Parkinson�s patients chronically treated with

LL-DOPA [38,120]. Therefore, chronic activation of both

the A2A receptor and D2 receptors may occur in these

individuals and, due to the hetero-dimerization, causean even greater desensitization of both receptors re-

sulting in increased LL-DOPA tolerance. Co-treatment of

A2A receptor antagonists with LL-DOPA may therefore

represent an improvement in the therapeutic approach

to Parkinson�s disease.Using the approach of re-constitution of function by

co-expression of split receptors, the D2 receptor was

shown to heterodimerize with the related D3 receptor[150]. Cells co-expressing both receptors displayed en-

hanced inhibition of adenylate cyclase when stimulated

with both receptor ligands. It is thought that this ob-

servation may explain why hypothermia induced in rats

by a D3-selective ligand can be inhibited by a D2-se-

lective antagonist [56,106]. Consequently this interaction

may be functionally significant in vivo in humans.

7.4. Somatostatin receptors

Somatostatin receptors consist of five subtypes

(SSTR1–SSTR5), which all bind the naturally occurring

somatostatin-14 (SST-14) and somatostatin-28 (SST-28)

with high affinity. All five subtypes couple to G-proteins

that inhibit adenylate cyclase, with selective subtypes

also coupling to various ion channels and phospholip-ases [130]. Somatostatin is secreted from the hypothal-

amus and acts on receptors in the anterior pituitary to

inhibit secretion of growth hormone (GH) and PRL, as

well as regulating the release of TSH. GH regulation

appears to occur via SSTR2A and SSTR5 on somato-

tropes, however, the receptor subtypes mediating se-

cretion of the other pituitary hormones have yet to be

established [152]. Immunohistochemistry has been usedto identify all five receptor isoforms in the normal rat

pituitary [85]. SSTR5 was the predominant subtype

identified in somatotropes, followed by SSTR2. SSTR3

and 4 were present in about a fifth of somatotropes, with

SSTR1 in a small percentage.

Using FRET, SSTR5 has been shown to homo-di-

merize in an agonist-dependent manner at physiological


expression levels [128,129,144], whereas SSTR1 appearsto function as a monomer [128]. Furthermore, SSTR5

forms heterodimers with SSTR1, but not SSTR4, im-

plying that interactions between subtypes are specific

and may be functionally relevant [128,144]. SSTR1 is

unable to internalize when expressed alone. However,

following binding of the SSTR1 selective ligand to the

SSTR1/5 heterodimer, SSTR1 was seen to internalize.

The observation that SSTR1 only internalizes whenpresent as part of a heterodimer has important

implications for the regulation of this receptor by

SSTR5 ligands.

Investigation of SSTR2A and SSTR3 using co-im-

munoprecipitation has revealed constitutive homo-dimer-

ization of these subtypes, as well as hetero-dimerization

when co-expressed [134]. The heterodimer exhibits

similar pharmacology to the SSTR2A homodimer, withSSTR2A acting as an antagonist to inactivate SSTR3

function. This interaction may provide an explanation

for why SSTR3 specific binding and signalling is diffi-

cult to detect in mammalian tissues [56]. The hetero-

dimerization of SSTR2A/3 may have implications in the

treatment of neuroendocrine tumours which often over-

express several subtypes of somatostatin receptor. A

tumour co-expressing SSTR2A and SSTR3 may bemore responsive to treatment with SSTR2A-specific

ligands than SSTR3-specific ligands, whilst a tumour

expressing only SSTR3 could be treated with SSTR3-

specific ligands.

Integration of signalling pathways through direct

receptor interactions is not confined to subtypes of the

SSTR, as SSTR5 is able to hetero-dimerize with the

dopamine D2 receptor [143] as previously discussed.This heterodimeric interaction appears to be induced by

agonists of either receptor, with the conformational

state of one receptor appearing to modulate the other.

However, hetero-dimerization does not always appear

to affect ligand binding and coupling, for example these

do not appear to be affected by SSTR2A/l-opioidreceptor hetero-dimerization [135]. Instead, these

receptors appear to modulate phosphorylation anddesensitization of each other.

7.5. Opioid receptors

Opioids are known to have a wide range of biological

effects including their most well characterized roles in

pain control [179]. Opioids can also modulate various

aspects of the immune and endocrine systems, includingthe release of GnRH and luteinizing hormone [44,95].

All observed effects of opioids are mediated through

three opioid receptors—mu ðlÞ, kappa ðjÞ, and delta

ðdÞ.A number of early pharmacological studies provided

the first indirect evidence for the probable existence of

dimerization between opioid receptor family members.

Additional pharmacological properties have been ob-served that were originally thought to be due to the

presence of an undiscovered opioid receptor family

member. However, no such sequences have been found

in the human genome and it is now widely thought that

the differences observed between in vivo and cloned re-

ceptor pharmacology can be explained by hetero-di-

merization between the three known opioid receptors. In

support of this, single opioid receptor subtype knockoutmice showed alterations in the functioning of the other

remaining opioid receptors [81]. Clear evidence for

opioid receptor homo- and heterodimeric or oligomeric

interactions came from more recent biochemical studies

demonstrating an interaction between the j and d, butnot l [74] and between l and d opioid receptors [46,49].

Biophysical techniques have also been used to investi-

gate opioid receptor interactions. BRET has been usedto detect d [51,102] and l [51] opioid receptor homo-

oligomers and TR-FRET has also been employed to

demonstrate that the d opioid receptor homodimers are

actually present at the cell surface [102].

Hetero-dimerization of the opioid receptors may also

help to explain the previously observed pharmacological

properties and synergy between various opioid analogs

in vivo. It was demonstrated that selective j and d ag-onists had a reduced affinity for the j–d heterodimeric

complex, but that a combination of selective agonists

could act synergistically at the j–d heterodimer to po-

tentiate signalling [74]. Hetero-dimerization may also

play a role in regulating the trafficking properties of

these receptors, as non-selective opioid agonist etor-

phine, which can induce internalization of d but not jopioid receptor, did not cause internalization of d whenco-expressed with j [74]. Similarly, the l–d heterodimer

displays reduced affinities for l and d selective ligands

but increased affinity for others [46]. Furthermore,

combinations of l and d selective agonists act syner-

gistically at the heterodimer to potentiate signalling [49].

Such potentiation of l opioid receptor signalling by dselective drugs has been previously observed in many

studies and hetero-dimerization provides a potentialmechanism for this cross-modulation. Clinically, this

synergy translates into lower doses of morphine being

required in the presence of sub-analgesic doses of d-re-ceptor specific ligands, and this may have consequences

for the development of morphine tolerance.

It is likely that the conformational changes occurring

in these receptors as a result of hetero-dimerization are

responsible for the altered ligand binding properties.Such conformational changes could also potentially al-

ter the efficiency and nature of the G-protein coupling,

as observed for the l–d heterodimeric complex. In

contrast to individually expressed l and d receptors, co-

expressed receptors continued to signal in the presence

of pertussis toxin, most likely due to the interaction of

the heterodimeric complex with a different G-protein


[46]. To date, a heterodimeric interaction between the land j opioid receptors has not been detected, although

there is a large amount of evidence for synergy between

l and j mediated-pathways [75].

Recent studies also indicate that opioid receptors

can interact with other GPCRs. The d and j opioid

receptors are both capable of interacting with the b2-adrenergic receptor as demonstrated by co-immuno-

precipitation and BRET [76,102]. Although neitherheterodimer showed altered ligand binding, changes in

receptor internalization and MAPK signalling were

observed [76]. The in vivo importance of this interaction

is still not clear, although studies have shown that low

doses of selective opioids can inhibit norepinephrine-

mediated functions [133,183]. Hetero-dimerization may

provide a possible mechanism for this, as opioid ligands

could alter b2-adrenergic receptor responsiveness byaltering its internalization through formation of a

heterodimeric complex with an opioid receptor.

Co-immunoprecipitation studies have shown that the

l opioid receptor can form complexes with the so-

matostatin receptor SSTR2A and that these complexes

have novel properties [135]. The SSTR2A specific ligand

was able to induce phosphorylation, desensitization and

internalization of both the SSTR2A and l opioid re-ceptors. Conversely, the l ligand induced cross-phos-

phorylation and desensitization, but not internalization,

of the SSTR2A receptor present in the heterodimer. It is

thought that ligand binding to one receptor causes a

conformational change in the other allowing access by

G-protein coupled receptor kinases (GRKs) and b-ar-restins. Stimulation with the l ligand may not expose all

the phosphorylation sites on the SSTR2A required forb-arrestin binding. In contrast, the conformational

changes that occur upon binding the SSTR2A ligand

appear sufficient to allow b-arrestin binding. Therefore,

cross-phosphorylation occurs, but not cross-internali-

zation. The SSTR2A/l opioid receptor heterodimer

may have functional relevance in vivo as the two

receptors are co-expressed and functionally linked in

pain-processing pathways [157].Cross-internalization of receptors due to hetero-di-

merization has been described in several reports

[65,88,144]. In particular, the concept is potentially im-

portant in the regulation of l opioid receptor trafficking

and morphine tolerance. Morphine, unlike the l agonist

DAMGO, can activate the l opioid receptor without

promoting internalization. However, when treatments

with sub-analgesic doses of l agonist DAMGO incombination with morphine were carried out on neurons

from rat spinal cords, morphine-bound l opioid recep-

tor endocytosis was observed [62]. Furthermore, rats co-

administered sub-analgesic doses of DAMGO together

with morphine produced less tolerance than those trea-

ted with morphine alone, which may have important

implications for the treatment of chronic pain.

8. A role for heterodimerization in vivo?

Despite previous reports providing evidence for

GPCR oligomerization and the notion that cross-talk at

the level of receptors may explain some of the known

physiological interactions occurring within the neuro-

endocrine system, it has been suggested that this phe-

nomenon is an artefactual observation of protein

overexpression in cell culture systems and is of littlerelevance in vivo [137,148]. The majority of studies on

GPCR dimerization have been performed in re-

combinant systems, with evidence of whether dimers or

oligomers exist in vivo being speculated upon based on

pharmacological, biochemical, and biophysical evi-

dence. As receptor function may be modulated by the

dimeric or oligomeric state of the receptor, it will be

important to determine the organization of receptorsnot only in vitro under different conditions and stimuli,

but also in vivo in the receptors� native membranes.

Studies supporting an in vivo role for oligomerization

are those that have been able to detect oligomerization

when receptors are expressed at physiological levels.

Furthermore, several studies have shown heterodimer

formation between endogenously expressed receptors

using co-immunoprecipitation, such as the AT1 andbradykinin B2 receptors [2], adenosine A1 and P2Y1

receptors [182] and the metabotropic glutamate 1 and 5

receptors [43].

A recent study on the hetero-dimerization of the

angiotensin AT1R and the bradykinin B2 receptor

provided the first direct evidence that hetero-dimeriza-

tion can play an important role in vivo, demonstrating

that this interaction plays a critical role in preeclampsia[4]. This finding demonstrates that hetero-dimerization

can indeed have important clinical implications for the

development of novel and improved treatment strategies

for GPCR-associated diseases.

Preeclampsia is one of the most serious complications

in pregnancy in the Western world. Characterized by

hypertension, preeclamptic women are hypersensitive to

the pressor effects of angiotensin II (ATII). Abdallaet al. [4] have demonstrated that this sensitivity directly

correlates with a rise in AT1R/bradykinin B2 receptor

heterodimers both in the platelets and omental vessels of

the placenta in preeclamptic women, when compared to

normotensive pregnant women. The increase in hetero-

dimer formation results in increased receptor/G-protein

activity. As rises in markers of oxidative stress (such

as H2O2) are characteristic in both groups of womenduring pregnancy, the authors investigated the effect

of H2O2 on homo- and heterodimer formation. Whilst

H2O2 could induce homo-oligomerization of AT1 re-

ceptors, resulting in a decreased response to ATII,

constitutive AT1R/B2R hetero-oligomers were unaf-

fected and the increased response to ATII re-

mained. Thus, the increase in heterodimer formation in


preeclamptic women means that the protective feedbackmechanism of oxidative stress in controlling blood

pressure is ineffective in these women, resulting in the

hypertension observed. This is the first example of

hetero-oligomerization playing a direct role in human

disease.

9. Pharmaceutical and clinical implications of dimeriza-tion

GPCR hetero-dimerization may represent a way of

diversifying the response from GPCRs expressed in a

particular cell type, thereby having important implica-

tions for drug design and administration of pharma-

ceuticals used to treat a wide range of GPCR-associated

diseases. The formation of heterodimers represents apotential set of novel drug targets that is yet to be ex-

ploited, with the link between drug discovery and olig-

omerization yet to be made. The challenge for the

pharmaceutical industry will be to understand the im-

plications of receptor oligomerization. A way forward is

to apply this knowledge in the design of new drugs and

therapeutic regimes, rather than selecting drugs tested

only by analyzing receptors in isolation. The pharma-cology of the individual receptor expressed in a cell

line may be quite different from its pharmacology in its

native state.

Targeting or harnessing oligomerization events

opens up a variety of novel avenues for modulating or

discovering new methods of treatment, as exemplified

by the findings on the l opioid receptor. New ap-

proaches to treating disease may simply be developedby using available drugs in different combinations to

activate or inhibit the novel properties of heterodimers.

This may ultimately lead to more effective treatment

strategies with fewer side-effects. For instance octreo-

tide, a somatostatin agonist, may cause unwanted side

effects by activation of the dopaminergic system due to

formation of somatostatin/dopamine hetero-oligomers

[143]. Thus, screening for agonists that have a greateror lower affinity for the hetero-oligomer, or using drugs

in combination, will greatly aid in the intervention and

treatment of a variety of diseases that involve the

GPCR family.

The search will be on to find agents that promote or

disrupt oligomerization, or selectively induce hetero-di-

merization over homo-dimerization (or vice versa) as

these may be of significant clinical value. This screeningprocess would be greatly facilitated by the development

of sensitive high throughput screening assays based

upon measuring oligomerization, perhaps using reso-

nance energy transfer techniques such as BRET. Pep-

tides that mimic certain dimerization interfaces may

represent one possible strategy. Several studies have al-

ready used synthetic peptides representing transmem-

brane domains to reduce or inhibit receptor function[63]. The use of heterodimeric ligands may represent one

mechanism to specifically target the heterodimer. These

could theoretically either block or stimulate the novel

functional properties of the heterodimer without sig-

nificantly affecting the function of the individual recep-

tors [47]. As hetero-oligomer formation can result in

altered pharmacology it provides a target for drug dis-

covery in its own right. Consequently, future drug dis-covery programs must consider heterodimeric receptors

as novel targets for the screening of potential drug

candidates.

10. Conclusions

Even though oligomerization of GPCRs is notconsidered to be a biological myth, researchers in

molecular biology, physiology, or even the pharma-

ceutical industry have not completely shifted their view

in how these receptors are studied and depicted in

models. Cross-talk at the level of receptors may explain

some of the known physiological interactions occurring

within the neuroendocrine system. With regard to

GPCR dimerization it is important to understand notonly the effect of these interactions on receptor func-

tion, but perhaps more importantly the physiological

and pathophysiological consequences of dimerization,

and the relevance for new drug development. Future

studies should be aimed at investigating the role of

receptor oligomerization in physiological systems, as

well as further unravelling its molecular basis and

mechanism of regulation.The drug-discovery process for novel GPCR com-

pounds could be transformed by the information gen-

erated from detailed knowledge of receptor

oligomerization. This in turn will increase knowledge of

the number of signalling molecules, pathways and re-

ceptor domains that regulate oligomerization, which can

then be used as targets for rational drug design. The

hope is that, from a detailed understanding of theseprocesses, more incisive approaches to treatment will

evolve to exploit the phenomenon of oligomerization.

Acknowledgments

This work has been supported by the following grants

from the National Health and Medical ResearchCouncil (Project Grant 212065). K.E. and K.K. are

supported by NHMRC Principle Research Fellow and

Peter Doherty Post-doctoral Fellowships, respectively.

K.P. is supported by a WAIMR Post-doctoral Fellow-

ship. We also thank Ms. Lisa Beavis, Ms. Ruth Seeber,

and Dr. Uli Schmidt for help with the preparation of the

manuscript.


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