CHAPTER FIVE

The Dynamics of GPCROligomerization and TheirFunctional ConsequencesRory Sleno, Terence E. H�ebert1McGill University, Montreal, QC, Canada1Corresponding author e-mail address: terence.hebert@mcgill.ca

Contents

1. What Does Structural Biology Tell us About GPCR Dimers? 1432. The Dynamic Nature of GPCR Oligomerization: Stability, Instability, and

Metastability 1433. Trafficking of Signaling Complexes 1444. GPCR Dimers Per Se May Not Always Be Stably Associated 1455. Ligand-Induced Receptor Trafficking and GPCR Dimerization 1476. Larger Metastable Entities: Allostery Is the New Cooperativity 1477. Structural Asymmetries in GPCR Oligomers? 1498. Asymmetries in GPCR Oligomers Translate Into Signaling Consequences 1519. Conformational Profiling of GPCRs in an Oligomeric Context 156

10. Conformational Asymmetry in the AT1R/FP Pair 15711. Implications of Metastable GPCR Complexes for Drug Discovery 160Acknowledgments 163References 163

Abstract

The functional importance of G protein-coupled receptor (GPCR) oligomerizationremains controversial. Although obligate dimers of class C GPCRs are well accepted,the generalizability of this phenomenon is still strongly debated with respect to otherclasses of GPCRs. In this review, we focus on understanding the organization anddynamics between receptor equivalents and their signaling partners in oligomericreceptor complexes, with a view toward integrating disparate viewpoints into a unifiedunderstanding. We discuss the nature of functional oligomeric entities, and howasymmetries in receptor structure and function created by oligomers might have impli-cations for receptor function as allosteric machines and for future drug discovery.

International Review of Cell and Molecular Biology, Volume 338 # 2018 Elsevier Inc.ISSN 1937-6448 All rights reserved.https://doi.org/10.1016/bs.ircmb.2018.02.005

141

G protein-coupled receptor (GPCR) dimers and oligomers have been a

subject of interest for more than 20 years. Most if not all GPCRs can

form dimers and possibly higher order structures in heterologous expres-

sion systems (see Bulenger et al., 2005; Ferre, 2015; Ferre et al., 2014;

Franco et al., 2016; Gomes et al., 2016; H�ebert and Bouvier, 1998;

Kleinau et al., 2016; Marsango et al., 2017; Milligan, 2009; Prezeau

et al., 2010; Prinster et al., 2005 for review). However, it is also clear that

GPCRs can signal as monomeric proteins when reconstituted into

proteoliposomes (Whorton et al., 2007, 2008). The critical issue is to

determine the functional roles of such dimers and oligomers under native

conditions in cells and tissues, and how they might be approached as ther-

apeutic targets (Albizu et al., 2010). Work over the past few years has

supported notion that GPCR signaling complexes facilitate rapid and

specific signaling. Thus, in one sense, receptors per se can be viewed as

scaffolds for formation of specific “hardwired” signaling complexes or sig-

naling hubs. Such complexes may be distinct for individual receptor

monomers, homodimers, or heterodimers leading to unique signaling out-

puts for different receptor complexes. On the other hand, the notion that

such hubs represent conformationally dynamic nodes in larger signaling

arrays has not been considered carefully.

Many excellent reviews have been published in recent years describing

GPCR dimerization in detail, technical approaches to identifying and

characterizing dimers (see earlier) and attempting to develop standards

by which dimers or oligomers might be accepted as bona fide (Gomes

et al., 2016). At the same time, there are reports which challenge the exis-

tence of stable GPCR dimers or the existence of receptor dimers at all. Per-

haps such Manichean views of GPCR dimerization and oligomerization

are no longer helpful. Here, we want to focus on two particular features

of GPCR organization: (1) the dynamic nature of receptor oligomers that

may exist on different timescales and be driven by agonists or allosteric

modulators and (2) the idea that different asymmetric arrangements of

receptors associated with their signaling partners into larger signaling arrays

may add considerable granularity to the dynamics of such signaling net-

works. We would argue that these two core features of GPCR oligomers

and their associated signaling machinery reflect their nature as allosteric

machines which are sensitive to a large number of inputs in the cell.

Finally, we consider the implications of these features for drug discovery

going forward.

142 Rory Sleno and Terence E. H�ebert

1. WHAT DOES STRUCTURAL BIOLOGY TELL US ABOUTGPCR DIMERS?

There have been a small number of GPCR crystal structures that show

the presence of GPCR homodimers including the CXCR4 chemokine

receptor (Wu et al., 2010) and the μ- and κ-opioid receptors (Manglik

et al., 2012; Wu et al., 2012). In the particular case of CXCR4, ligand-

bound dimers were detected in five independent structures. The interface

between the two monomeric units included transmembrane domain

(TM) V and TMVI in CXCR4, between TMI, TMII, and TMVII in

the κ-opioid receptor and between TMV and TMVI in the μ-opioid recep-tor (reviewed in Katritch et al., 2013). These results highlight how the inter-

faces between receptor protomers vary depending on the homodimer

studied, a point we will come back to later. Such distinct interfaces in GPCR

homodimers probably means that heterodimer interfaces will also be as var-

ied, if not more. This becomes an evenmore important issue in the context of

receptor oligomers. To illustrate this, in a structure of oligomeric turkey

β1-adrenergic receptors, two dimer interfaces were identified—one interface

involving TMI, TMII, the C-terminal H8, and extracellular loop 1 was iden-

tified and another interface involved TMIV, TMV, intracellular loop 2, and

extracellular loop 2 (Huang et al., 2013). A recent review has discussed what

the implications of these structures are on our conception of the receptor/G

protein interface (Cordomi et al., 2015). While GPCRs can form dimeric or

even oligomeric structures, the vast majority of crystal structures (reviewed in

Katritch et al., 2012, 2013; Lu and Wu, 2016) showed little evidence for

GPCR dimers, at least under the conditions required to purify and reconsti-

tute these receptors for structural studies. As mentioned earlier, GPCRs can

signal as monomeric proteins when reconstituted into proteoliposomes

(Whorton et al., 2007, 2008). We would argue that the cellular context, lost

when GPCRs are purified, likely plays a key role in understanding the true

nature and relevance of receptor dimerization and oligomerization in vivo.

2. THE DYNAMIC NATURE OF GPCR OLIGOMERIZATION:STABILITY, INSTABILITY, AND METASTABILITY

Many studies have suggested that dimers are fairly stable entities, being

assembled during receptor biosynthesis. There is evidence that receptor

143Asymmetric GPCR Signaling Mediated by Metastable Oligomeric Complexes

dimerization is required for efficient surface localization of a number of

GPCRs including the β2AR (Dupr�e et al., 2006; Salahpour et al., 2004)

and the α1BAR (Lopez-Gimenez et al., 2007; reviewed in Milligan,

2010). Further, our work over the past few years supported the idea that

GPCR dimers initially form in the ER (Dupr�e and H�ebert, 2006; reviewedin Dupr�e et al., 2009). Further, these dimers could initially interact with

their G protein and effector partners in the ER and were subsequently traf-

ficked to the cell surface. Given the need for fidelity in GPCR signaling,

such arrangements could represent a mechanism to assure rapid and specific

signaling. In this sense, receptors and certain effector molecules can be

viewed as scaffolds for formation of specific hardwired signaling complexes

or signaling hubs.

Assembly of GPCR signaling complexes can occur during biosynthesis

as discussed earlier, rather than as transient responses to agonist stimulation

at the plasma membrane. GPCRs, as monomers, dimers, and oligomers,

could certainly act as scaffolds for formation and function of distinct hard-

wired signaling hubs leading to unique phenotypic outputs depending on

the composition and cellular context of such complexes. For example, a

number of studies have demonstrated association, copurification, or

coimmunoprecipitation of receptors with G proteins (reviewed in P�etrinand H�ebert, 2011; Rebois and H�ebert, 2003). Stable or metastable (more

on that later) interactions of GPCRs with effector partners can be viewed

as a mechanism to assure rapid and specific signaling. Our own studies of

the ontogeny of such signaling complexes suggest that complexes of either

β1AR or β2AR (Lavine et al., 2002) assemble with effector partners such as

adenylyl cyclase (Baragli et al., 2008; Dupr�e et al., 2007) and Kir3 channels

(David et al., 2006; Rebois et al., 2006; Robitaille et al., 2009). Similar pre-

assembled signaling complexes have recently been identified for M3 musca-

rinic receptors and Gαq (Qin et al., 2011). PDZ ligand-bearing GPCRs may

also facilitate formation of specific dimer-based complexes, depending on

what PDZ proteins are recruited to each protomer (Camp et al., 2015).

3. TRAFFICKING OF SIGNALING COMPLEXES

Signaling complexes formed during receptor biosynthesis have been

shown to be insensitive to dominant negative versions of Rab1 or Sar1

(but not Rabs 2, 6, or 11) constructs (Dupr�e et al., 2006, 2007), which reg-

ulate anterograde receptor trafficking (reviewed in Dong et al., 2007; Dupr�eand H�ebert, 2006). Rabs and Sar1 are monomeric G proteins demonstrated

to be important for vesicular transport to and from different cellular

144 Rory Sleno and Terence E. H�ebert

membrane compartments (Zerial and McBride, 2001). A recent study has

also shown that cell surface transport of α2B-adrenergic receptors from the

ER to the Golgi apparatus also depends on Rab 43 (Li et al., 2017). Interest-

ingly, our data also showed that Gα subunits are assembledwith nascent recep-

tor/Gβγ/effector complexes either in ER export sites or in the Golgi since

this interaction was blocked by dominant negative Sar1 and Rab 1 (Dupr�eet al., 2006, 2007). Using this traffic block-based approach, we also showed

that larger receptor oligomers could also be assembled in the ER. Using

a combination of bioluminescence resonance energy transfer (BRET) and

protein fragment complementation assays (PCA) to study the interactions

between two β2AR constructs tagged with each half of split Venus, we

detected an interaction with β2AR-Rluc, suggesting the presence of a larger

receptor homooligomer (P�etrin and H�ebert, 2012). Interestingly, we also

detected a larger array when we use two AT1R constructs tagged with each

half of split Venus with the β1AR-Rluc or β2AR-Rluc in a heterooligomeric

context (P�etrin and H�ebert, 2012). None of these interactions were sensitive

to block with dominant negative versions of either Rab1, suggesting both

homo- and heterooligomers also form in the ER as an early event in receptor

biogenesis. This says nothing about future stability, however, once such com-

plexes reach the plasma membrane and receptors are stimulated by ligands,

but it does provide a means of delivering the relevant signaling machinery

to cellular destinations in a coordinated manner.

4. GPCR DIMERS PER SE MAY NOT ALWAYS BESTABLY ASSOCIATED

In contrast, several recent reports have suggested that dimers for various

GPCRs either do not exist or exist in an equilibrium with their monomeric

forms at the plasma membrane on distinct timescales. Resonance energy

transfer (RET) has been an enabling technology used to characterize GPCR

oligomers, and such experiments have been used extensively to support the

notion of stable receptor dimerization (Angers et al., 2000; Calebiro et al.,

2013; Dorsch et al., 2009; Mercier et al., 2002; Pfleger and Eidne, 2006).

However, some authors have performed controls which have led them to

reinterpret the data to suggest that dimers are dynamic structures on a milli-

second timescale at best or fleeting or nonexistent at worst (Felce et al., 2014,

Gavalas et al., 2013, James et al., 2006, Kawano et al., 2013, Lan et al., 2011,

2015; reviewed in Felce et al., 2018).

Using antibody cross-linking and fluorescence recovery after photo-

bleaching (FRAP), it was demonstrated that dimerization of D2 dopamine

145Asymmetric GPCR Signaling Mediated by Metastable Oligomeric Complexes

receptors was a highly dynamic process. For example, when one partner in the

dimerwas immobilized and photobleached, the other dimer partner remained

mobile (Fonseca andLambert, 2009). In another study, using a combinationof

labeled ligands and total internal reflectance fluorescence (TIRF)microscopy,

it was demonstrated that M1 muscarinic receptors existed in an equilibrium

between monomeric and dimeric forms where rapid interconversion could

be detected using single-particle tracking (Hern et al., 2010). Similar findings

were obtainedusing single-particle trackingof theN-formyl peptide receptor,

again using a fluorescently labeled ligand and TIRF microscopy (Kasai et al.,

2011). Indeed, there is evidence from several reports that suggest agonist occu-

pation further increases dimer stability. It will be interesting to pursue such

studies using heterodimeric receptors or arrays of larger complexes, rather than

simply homodimers. This becomes important in light of recent studies

demonstrating allosteric interactions between GPCRs in both contexts. As

discussed later, it is especially critical in the case of receptor heterodimers.

Do these single-particle tracking approaches exclude the possibility of

receptor oligomers as such? The data could be interpreted in several ways,

one of which would imply allosteric interactions in the context of receptor

oligomers, rather than a simple monomer:dimer equilibrium. One consid-

eration is that there could be an equilibrium between dimers and larger

structures. Using quantum-dot tagging of neurokinin-1 receptor, a recent

study demonstrated that a considerable plasticity occurs, depending on

the presence or absence of agonist with respect to receptor clustering,

suggesting an organizational plasticity dependent on cell state (Veya et al.,

2015). Another recent study using spatial intensity distribution analysis

(SpIDA) showed that the distribution of M1 muscarinic receptors mono-

mers vs dimers/oligomers was sensitive to agonist, the latter being more

prevalent following agonist stimulation (Pediani et al., 2016). Similar results

were obtained when studying D2 dopamine receptors using single-molecule

tracking where a lifetime of 68 ms was measured under control conditions

which was increased with agonist stimulation (Kasai et al., 2017). Using an

interesting technique combining TIRF microscopy with immunoprecipita-

tion (SiMPull) has shown that slightly more than half of the β2AR expressed

in HEK 293 cells was in a dimeric form and a similar proportion of

heterodimers was seen when the β1AR and β2AR were coexpressed (Jain

et al., 2011). It has been suggested that dimerization and/or oligomerization

stabilizes GPCRs in their functional states, preserving their functional life-

times (Ge et al., 2017; Jastrzebska et al., 2015). Different GPCRs may be

more or less stable as monomers, dimers, or oligomers (Calebiro et al., 2013).

146 Rory Sleno and Terence E. H�ebert

Other approaches using super resolution microscopy have also demon-

strated a similar dynamic structural arrangement for a number of GPCR

oligomers (reviewed in Scarselli et al., 2016). This latter review also high-

lights the idea that larger GPCR signaling complexes may in part be

influenced by the dynamic cytoskeleton or by scaffolding proteins such as

PDZ proteins or A-kinase anchoring proteins as well. The use of single-

particle tracking methods to study GPCR oligomerization has recently been

the subject of a number of detailed reviews (Briddon et al., 2018; Calebiro

and Sungkaworn, 2018; Pediani et al., 2017). If there is a message in all of

these studies, it is that receptor oligomerization must be viewed from mul-

tiple vantage points to be interpreted in the living cell context.

5. LIGAND-INDUCED RECEPTOR TRAFFICKINGAND GPCR DIMERIZATION

Oneway to reconcile these disparate observations might be to imagine

that the specificity of cellular signaling might be engineered by assembly of

larger receptor-based complexes in the ER during biosynthesis as we have

discussed earlier. However, once targeted to the cell surface, such complexes

may be under a different set of constraints that permits them the conforma-

tional flexibility inherent in GPCR signaling. There is some evidence that

receptor/effector complexes cotraffic when the receptor is internalized as for

the opioid-like receptor 1 (and opioid receptors as heterodimers) and

N-type calcium channels (Altier, 2012; Altier et al., 2006; Evans et al.,

2010) or between the β2AR and L-type calcium channels (Flynn and

Altier, 2013). They may also dissociate when receptors internalize in

response to sustained agonist signaling. For example, using BRET it was

demonstrated that β2AR become dissociated when the receptors are inter-

nalized (Lan et al., 2011). However, some of the alterations in BRET signals

could also be attributed to conformational changes rather than a loss of

interaction per se.

6. LARGER METASTABLE ENTITIES: ALLOSTERYIS THE NEW COOPERATIVITY

A number of studies have suggested that GPCRs can form higher

order complexes in addition to homo- or heterodimers. PCA have been

used to expand our knowledge regarding GPCR oligomerization. Recon-

stitution of split luciferase (Gaussia or Renilla) and split GFP constructs has

147Asymmetric GPCR Signaling Mediated by Metastable Oligomeric Complexes

shown that dimers of β2AR (Rebois et al., 2008) andD2 dopamine receptors

(Guo et al., 2008) can be detected, complementing immunopurification and

RET approaches, and most importantly, these approaches can be combined

to detect and examine larger complexes. A number of investigators have used

three partner PCA/RET to show that higher order complexes of GPCRs

such as the A2A-adenosine receptor homo- and heterooligomers with CB1

cannabinoid/D2 dopamine receptors (Carriba et al., 2008; Gandia et al.,

2008; Vidi et al., 2008a,b; reviewed in Cordomi et al., 2015; George

et al., 2014) and CXCR4multimers (Hamatake et al., 2009) can be detected.

One might imagine an organizational paradigm where “snapshots” reveal

considerable dynamism between individual components of GPCR signaling

arrays but a global “metastability” that is built during biosynthesis of the var-

ious components of an allosteric complex (Fig. 1).

Studies of ligand binding cooperativity (Ma et al., 2007, 2008), to many

investigators, remain the strongest evidence that allostery exists between

E1

E3

E2

E1

R1

R2

R2

R1

G

G R1

R1

R2

R2

G

G

R1

R2

R1

R2

G

G

R1

R2

G

Actin filaments

Fig. 1 Snapshots of dynamic GPCR signaling arrays. Individual components of GPCR sig-naling complexes comprised of multiple receptor equivalents, G proteins, effector mol-ecules, and other associated partners such as the cytoskeleton. Any given interactionmay be transient but the stability of the larger complex is maintained by many shiftinginteractions which contribute to metastability. The stars indicate how a labeled partnermight shift in a metastable complex when tracking with single molecule approaches.Subcomplexes might be or seem less stable at any one time than a receptor mosaic.Each small part of themosaic may add to the overall affinity and stability of the complexover time. E, represents an effector molecule; G, indicates heterotrimeric G protein; R,indicates receptor.

148 Rory Sleno and Terence E. H�ebert

functional equivalents in GPCR homo- and heterodimers and oligomers.

Of course, ligand-binding experiments provided initial evidence that

GPCRs were multimeric proteins, with allostery providing a mechanism

to explain cooperativity measured between different equivalents of ligand

(De Lean et al., 1980). The subsequent discovery that G proteins were crit-

ical allosteric regulators of receptors, turned the attention of the field toward

receptor–G protein interactions for many years, leading to the development

of the ternary model and then the extended ternary model. Even when the

ternary model gave way to the extended ternary model to accommodate

ligand-independent signaling and inverse agonism, the notion of coopera-

tivity between ligands did not come back into the discussion. However,

more careful ligand-binding studies explicitly suggested that oligomeric

arrangements of receptors could explain cooperativity between receptors

and G proteins and cooperativity between equivalents of receptor ligands

(Chidiac et al., 1997; Green et al., 1997; Ma et al., 2007; Sohy et al.,

2009). Indeed, the cooperativity, or more precisely, the allostery between

ligand-binding sites in receptor homodimers, explicitly defined as such

could still be measured even when G protein partners were removed from

receptor preparations (Peterson et al., 1984; Wreggett and Wells, 1995).

Cooperativity in ligand binding at the M2 muscarinic receptor dimer rec-

onstituted with G protein was lost when a monomeric version of receptor

was studied (Redka et al., 2013). Interactions between orthosteric and allo-

steric ligands can be detected in M2 muscarinic receptor monomers but

those seen in receptor oligomers were richer and more complex, likely

reflecting their broader functional roles in the cell (Shivnaraine et al.,

2016). This argues that allostery is manifested between the receptors them-

selves through physical contact as part of a dimer.

7. STRUCTURAL ASYMMETRIES IN GPCR OLIGOMERS?

FRET approaches have suggested similar higher order structures for

the M2 muscarinic receptor and the β2AR (Fung et al., 2009; Pisterzi

et al., 2010). Using spectral deconvolution and fluorescence lifetime imag-

ing, it was shown that M2 receptor homotetramers are likely to be in a

rhomboid orientation, rather than a simple square array of receptor mono-

mers (Pisterzi et al., 2010; Fig. 2A, left panel). Such potential structural

asymmetries may have dramatic impacts on signaling complex organization

and thus functional outputs. Homodimers or even homotetramers in a

square array have, by definition, fewer possibilities for asymmetric

149Asymmetric GPCR Signaling Mediated by Metastable Oligomeric Complexes

arrangements compared to rhomboid-shaped homotetramers, where struc-

tural asymmetries can be introduced with respect to how the entire receptor,

G protein, effector complex is arranged (Fig. 2A, right panel). In the case of

heterotetramers, the potential of either square or rhomboid arrangements for

distinct allosteric interactions between receptors, G proteins, and effectors is

R1

R1

R2

R2

G

G R1

R2

R1

R2

G

G

R1

R1

R1

R1

G

G

A

B

R1

R1

R1 R1

G

G

R1

R2

R1 R2

G

G R2

R2

R1 R1

G

G

R1

R1

R2 R2

G

G R2

R1

R1 R2

G

G

Fig. 2 Asymmetric organization of receptor homo- and heterooligomers. (A) Differentviews of receptor homotetramers in square or rhomboid configurations. In the rhom-boid configuration, potential structural asymmetries with respect to organization ofthese complexes become evident. Thus, how receptors are organized and assembledwith the interacting proteins might be controlled in the cell to produce distinct signal-ing architectures. (B) GPCR heterotetramers increase the organizational complexity fur-ther. The assembly of heterodimers and heterotetramers provides a much larger scopefor the assembly of distinctly regulated allosteric signaling machines in either square orrhomboid orientations. Even in the “square” configuration (top), a number of asymmetriesbecome possible with respect to how the signaling complex is organized, which againbecome greater in the “rhomboid” configuration (bottom). These differential arrange-ments may be manifested by ligand binding cooperativity between receptor equivalentsand in how this information is transmitted to interacting proteins.

150 Rory Sleno and Terence E. H�ebert

even greater (Fig. 2B). Similar findings were obtained using single-particle

tracking approaches examining the rhomboid “shape” of adenosine A1/A2

receptor heterodimers (Navarro et al., 2016). This latter study also showed

that the receptor tetramer was in complex with two Gα subunits, Gαs andGαi, adding considerable conformational possibilities to such a signaling

array. Thus, structural asymmetries in GPCRs may translate into steric con-

straints that play out into the organization of signaling complexes and ulti-

mately in their function as allosteric machines. In fact, a recent study

suggested that ghrelin receptor significantly alters D2 dopamine receptor sig-

naling, presumably via heterodimerization in brain regions which never see

ghrelin, suggesting a function for the apo-receptor as a pure allosteric mod-

ulator, rather than a signaling receptor in these cells (Kern et al., 2012). Such

observations might explain why heterodimers have been difficult to detect in

standard drug screens—where one receptor might only be an allosteric mod-

ulator of its partner, rather than signaling on its own.

8. ASYMMETRIES IN GPCR OLIGOMERS TRANSLATEINTO SIGNALING CONSEQUENCES

GPCRs that signal as monomers have markedly restricted allosteric

possibilities for modulation, inherent even in receptor dimers, likely relying

more on standard molecular cross talk mediated by protein kinases and other

enzymes as part of regulatory pathways (Fig. 3A). One obvious functional

advantage of dimers, more easily understood in the context of heterodimers,

is that they can act on each other via bidirectional allosteric interactions,

which may or may not depend on ligand occupation or the presence of par-

ticular signaling partners (Fig. 3B). However, more complex interactions

may also result from structural asymmetries depending on the relative ori-

entation and position of signaling partners, and the conformational space

sampled by each of the partners (Fig. 3C). The first identified asymmetric

receptor heterodimer was the GABA-B receptor, which consists of two sub-

units, one of which binds ligand and the other which transmits the signal to

the G protein (Jordan et al., 2001; McVey et al., 2001). This particular

receptor complex is unique in that one subunit does not bind ligand but allo-

sterically modulates the other and vice versa (reviewed in Barki-Harrington

et al., 2003; Karla et al., 2010; McGraw et al., 2006). Other class C receptor

heterodimers likely have similar pathways for conformational cross talk

given their similar organizational plans. Although in those cases, where each

dimer has two ligand-binding sites, one can wonder which site becomes the

151Asymmetric GPCR Signaling Mediated by Metastable Oligomeric Complexes

signal-driving stimulus and which functions as an allosteric site. For exam-

ple, in mGlu2/4 heterodimers (reviewed in Kammermeier, 2012), G pro-

tein coupling was mediated exclusively by mGlu4 heptahelical domain—

allostery was mediated by the extracellular ligand-binding domains and

information transfer occurred from the heptahelical domain of mGlu2

(Liu et al., 2017). In these studies, the authors used SNAP-tagged receptors,

engineered to be functionally distinct, an approach similar to the Javitch

G

R2E

G

R1E

G

R1 R1

G

R1 R2

G

R1 R2

Allosteric Signaling

G

R1 R2

Signaling Allosteric

Cross talk

A

B

C

Fig. 3 Allostery in GPCRmonomers and oligomers. (A) Monomeric GPCR signaling com-plexes have limited possibilities for interreceptor allosteric modulation but can certainlyregulate each other’s activity via molecular cross talk mediated by second messenger-activated protein kinases. (B) Allosteric communication in homo- and heterodimers witha shared G protein where information flow can go in a bidirectional manner betweenthe different partners. However, the allosteric possibilities are greatest in theheterodimer. (C) Assembly of GPCR heterodimers which interact in distinct ways witha shared G protein can be assembled in different orientations such that in one case,R1 signals and R2 is a nonsignaling allosteric modulator (whether occupied by ligandor not) of R1. The converse arrangement is also possible. Receptor homodimers mightbe asymmetrically organized with respect to their G protein and effector partners butthis is unlikely to have functional consequences per se since cooperative effectsbetween the receptor equivalents could be sensed in the same way.

152 Rory Sleno and Terence E. H�ebert

group (see later). This is something we will come back to later. There is a

specificity to these organizational paradigms in class C GPCRs, for example,

signaling asymmetries were not detected using LRET for combinations of

mGlu1 and mGlu4 (Moreno Delgado et al., 2017). Functional evidence

for these effects in vivo were found using mGlu4 knockout mice was also

noted in this study.

Questions about such asymmetries have been raised for other classes of

GPCRs as well. In a seminal article, the Javitch group showed that the two-

receptor equivalents in the context of a D2 dopamine receptor homodimer

are organized asymmetrically with respect to their G protein partners (Han

et al., 2009) such that occupation by ligand of one receptor activates the

receptor and occupation of the other modulates signaling allosterically. Fur-

ther, they predicted using molecular modeling that the actual interfaces

between each receptor equivalent and a shared G protein were different.

In the context of a homodimer this may not be as important as either recep-

tor can serve each role and the asymmetry may not be detectable, but cer-

tainly such differences might be revealed as positive or negative

cooperativity in ligand binding. However, such allosteric interactions are

likely to be of even greater importance in GPCR heterodimers and heter-

ooligomers which could also be exploited pharmacologically to control

receptor function (Lane et al., 2014). Using a similar approach, functional

histamine H3 receptor and A2A-adenosine receptor heterooligomers were

detected both in recombinant systems and in rat striatum (Marquez-

Gomez et al., 2018). These authors show that heterodimerization altered

the functional selectivity of the H3 receptor.

This notion adds an entirely unappreciated wrinkle to signaling from

heterodimers though, when we consider how asymmetry might play out

in terms of bidirectional allosteric receptor regulation. We examined this

possibility more directly in class A GPCRs, where we demonstrated that

the angiotensin II receptor type I (AT1R) and the receptor for prostaglandin

F2α (FP) could form heterodimeric complexes in both HEK 293 and vas-

cular smooth muscle cells (VSMCs), the latter where both receptors are

again expressed endogenously. AT1R and FP represent important real

and potential targets at the core of many biological functions. AT1R is a pri-

mary target in the treatment of hypertension with AT1R antagonists of the

sartan family being widely prescribed (Borghi and Rossi, 2015). A role for

FP has also been shown in the regulation of blood pressure where its block-

ade has been suggested to reduced blood pressure (Yu et al., 2009). FP is

involved in parturition with enhanced PGF2α signaling initiating labor

153Asymmetric GPCR Signaling Mediated by Metastable Oligomeric Complexes

via stimulation of smooth muscle contraction in the myometrium (Jenkin,

1992; Mejia et al., 2015). The AT1R is also expressed in the myometrium

with increased levels measured during pregnancy (Bird et al., 1997; Cox

et al., 1993; Yamaleyeva et al., 2013). Examining this putative receptor com-

plex may yield novel drug targets in these tissues. An understanding as to how

these two receptors communicate at a structural level could facilitate rational

drug design and may suggest strategies to approach other GPCR oligomers.

AT1R and FP in VSMC could be copurified together using immuno-

precipitation combined with photoaffinity labeling and by acceptor photo-

bleaching FRET in HEK 293 cells as well (Goupil et al., 2015). Experiments

conducted in abdominal aorta rings measuring contraction revealed that

PGF2α-dependent activation of FP potentiated Ang II-induced contrac-

tion, whereas FP antagonists had the opposite effect. Similarly, PGF2α-mediated vasoconstriction was symmetrically regulated when using either

an AT1R agonist or antagonist. We also showed that their shared down-

stream pathway involving PKC was modulated in a similar fashion by dual

occupancy of each receptor by its cognate ligand. Ang II-mediated vasocon-

striction in the abdominal artery was potentiated by threshold concentra-

tions of PGF2α, as was the effect of PGF2α by Ang II. However,

occupancy by two different FP antagonists also resulted in inhibition of

Ang II-mediated contraction, an effect that cannot simply be explained

by stimulation of second messenger-mediated cross talk. Similar results were

obtained when we pretreated cells with L158,809, the AT1 antagonist when

measuring FP-mediated contraction (Goupil et al., 2015).

Going further, we also observed asymmetrical responses in the

heterodimers when following binding to their respective agonists (or in some

cases regulated simply by the presence of the partner receptor).We examined

a number of integrated phenotypic responses, including receptor-mediated

MAPK activation and DNA and protein synthesis in HEK 293 cells and

in VSMC. With respect to signaling in VSMC, we showed that occupation

of AT1R with an antagonist L158,809 strongly potentiated ERK1/2 activa-

tion by FP, an effect that was not reciprocated by occupation of FP with a

specific antagonist AS604872 when measuring Ang II-mediated ERK1/2

signaling (Goupil et al., 2015; summarized in Fig. 4A). In order to further

characterize the effects of stimulating the AT1R/FP dimer, we used [3H]-

thymidine incorporation as a DNA synthesis marker (indicative of cell pro-

liferation), and [3H]-leucine incorporation as a protein synthesis marker. We

pretreated VSMC with L158,809 to determine if it could potentiate [3H]-

thymidine incorporation following PGF2α stimulation. PGF2α alone elicited

154 Rory Sleno and Terence E. H�ebert

FP AT1R

A

FP AT1R

C

FPsensor

AT1R

E

FP AT1R

sensor

G

FP AT1R

B

FP AT1R

D

FPsensor

AT1R

F

FP AT1R

sensor

H

Agonist Agonist

Antagonist Antagonist

Antagonist Antagonist

Agonist Agonist

Gαq activation/contraction Gα

q activation/contraction

Growth ERK1/2 Growth ERK1/2

Gαq/11

Gαq/11

Fig. 4 Signaling and conformational asymmetries in GPCR dimers. Occupation of either(A) FP (red) or (B) AT1R (blue) with their respective antagonists reduced aortic contractileand G protein-mediated responses promoted by agonist stimulation of the other proto-mer. (C) Ang II-mediated MAPK signaling remained unchanged in the face of antagonistoccupancy of FP. However, occupation of FP with antagonist inhibited cell growthinduced by Ang II. (D) Finally, occupation of AT1R with an antagonist strongly potenti-ated FP-dependent MAPK signaling, but had no effect on PGF2α-induced cell growth.Conformational information is transmitted asymmetrically between protomers of theAT1R/FP heterooligomer and is dependent on the G protein (lower panel). (E)+ (G) Inthe absence of Gαq/11/12/13, ligand binding to the sensor receptor can elicit a conforma-tional (black arrow) change in FP, though blunted for the AT1R (black bar), while ligandbinding to the partner protomer leads to no sensed rearrangement of the sensor recep-tor (black bar). (F)+ (H) When Gαq is present, full responses to ligand binding sensorreceptors are observed (black arrows), while ligand binding to the partner protomer onlyinduce a conformational change in FP driven by AT1R (black arrow) but not fromAT1R toFP (black bar).

a small increase in [3H]-thymidine, which could be inhibited by AS604872,

but not by L158,809 pretreatment. Similar results were obtained with [3H]-

leucine incorporation, and L158,809 had a slight potentiating effect on

PGF2α-induced protein synthesis. However, AS604872, the FP antagonist,

was as potent as L158,809 in inhibiting both Ang II-induced [3H]-thymidine

and [3H]-leucine incorporation (Goupil et al., 2015), showing again a striking

asymmetry in the regulation of cellular responses integrated via the receptor

heterodimer.

Previous studies have demonstrated the AT1R heterodimerizes with

CB1 cannabinoid receptors, for example, also results in altered signaling pro-

files compared to the parent receptors (Dai et al., 2009) and similar results

have recently been shown for AT1R/apelin, AT1R/α2CAR, AT1R/

CB1 cannabinoid, and AT1R/β2AR receptor heterodimers (Barki-

Harrington et al., 2003; Bellot et al., 2015; Haspula and Clark, 2017;

Siddiquee et al., 2013; Toth et al., 2017). These results clearly indicate that

fuller examination of signaling profiles is required to understand both sym-

metries and asymmetries for the AT1R/FP pair and likely for many receptor

heterodimers. In fact, GPCRs like the AT1R seem to act as dimer “hubs,”

interacting with multiple GPCR partners (reviewed in Takezako et al.,

2017) which suggest that as we understand more and more regarding signal-

ing pathways downstream of a given receptor, the more cause we have for

examining the effects of putative partner receptors on how they might

augment, interfere, or bias such responses.

9. CONFORMATIONAL PROFILING OF GPCRsIN AN OLIGOMERIC CONTEXT

Although many studies have been able to demonstrate ligand effects on

putative receptor dimers, it has been difficult to tease out information regard-

ing the effects of one receptor on another in a directional sense to evaluate

signaling asymmetries more systematically, especially in the context of GPCR

heterodimers. Several fluorescence-based RET (FRET) approaches have

been developed that can capture intramolecular rearrangements in GPCRs

in response to agonist. These biosensors make use of small fluorescent mol-

ecules, such as fluorescein biarsenical hairpin binders (FlAsH), as the acceptor

and report on ligand binding-associated conformational rearrangements in

multiple GPCRs (Bourque et al., 2017; Devost et al., 2017; Maier-

Peuschel et al., 2010; Ziegler et al., 2011; Z€urn et al., 2009). Such biosensorshave also been used to examine conformational dynamics of GPCRs that

156 Rory Sleno and Terence E. H�ebert

form receptor oligomers. Initially, interactions within a class C homodimer of

mGluR1 and a class A heterodimer of α2A-adrenergic and μ-opioid receptorswere profiled (Hlavackova et al., 2012; Vilardaga et al., 2008). Once again,

the insights gained through the study of class C GPCRs accepted as obligate

dimers highlight the value of using such approaches to study other receptor

pairs. Indeed, the latter article shed some light on conformational cross talk in

a putative class A receptor heterodimer. These authors demonstrated that

morphine, targeting the μ-opioid receptor, affected the conformation of

the α2A-adrenergic receptor in the presence of its ligand norepinephrine. Thiseffect was shown to be G protein independent and still detectable in isolated

membranes, presumably stripped of downstream signaling partners suggesting

a simplemechanism of dimerizationmediated through direct GPCR/GPCR

contact.

10. CONFORMATIONAL ASYMMETRY IN THEAT1R/FP PAIR

We recently adapted this approach to study the AT1R/FP

heterodimer described earlier, engineering FlAsH tags and Renilla luciferase

into both AT1R and FP and coexpressing them with their untagged coun-

terparts (Sleno et al., 2017).We demonstrated a surprising asymmetric trans-

mission of conformational information between protomers of the putative

AT1R/FP heterodimer (summarized in Fig. 4B). The AT1R-induced con-

formational rearrangement in FP was dependent on both expression and

activatibility of Gαq and also suggested the possible involvement of the prox-

imal Gαq-effector PLC, highlighting again, the notion that signaling com-

plexes containing GPCRs, G proteins, and effectors are the core unit of

receptor organization. This latter observation was consistent with reports

showing that PLCβ is stably associated with Gαq as well (Dowal et al.,

2006). Next, we demonstrated that the AT1R-driven conformational

change in FP was predominantly independent of a key distal downstream

receptor signaling pathway that both receptors presumably share, activation

of PKC. This again suggests that the transmission of information occurs at

the level of the membrane, propagated via a shared G protein as part of a

heterodimeric allosteric signaling complex. However, using a CRISPR cell

line deleted for several Gα subunits, we showed that even in the absence of

Gαq, the AT1R/FP heterodimer remained intact, supporting the idea that

although Gαq subunits are not critical to the assembly of the receptor

heterodimer, they are important conduits of allostery between the two

157Asymmetric GPCR Signaling Mediated by Metastable Oligomeric Complexes

receptors. It remains to be seen whether other G protein heterotrimers that

could partner with either or both receptors might also serve this role in the

absence of Gαq/11/12/13. We had shown earlier that Gβγ subunits were crit-ical in the formation of GPCR dimers and their associated signaling com-

plexes (Dupr�e et al., 2006, 2009). Our data here suggest that a functional

Gαq acted as an allosteric conduit, connecting the two receptors once assem-

bled into a signaling complex. One interesting finding was that β-arrestin-biased AT1R ligands (Zimmerman et al., 2012) also demonstrated a

dependence on Gαq although they elicited no Gαq activation per se. This

added further support to the notion that Gαq plays a key structural role

enabling conformational cross talk between receptors, regardless of the

nature of the bound ligand and that G proteins are still important even for

so-called G protein-independent signaling. Therefore, we feel that we dem-

onstrated a novel mechanism in which allosteric interactions can transmit

information between protomers of a GPCR heterodimer. It bears recalling

that the nature of the AT1R/FP complex seems to be distinct from the

demonstrated independence of the G proteins for conformational cross talk

between the μ-opioid and α2A-adrenergic receptor complex (Vilardaga et al.,

2008), suggesting that heterodimer-specific arrangements are possible.

As discussed earlier, within the AT1R/FP complex each receptor was

capable of modulating the functional output of the other through asymmet-

ric allosteric interactions (Goupil et al., 2015). Similar asymmetric structural

arrangements have been noted in luteinizing hormone oligomers (Jonas

et al., 2015), rhodopsin (Mishra et al., 2016), mGluR2/3 heterodimers

(Levitz et al., 2016), and leukotriene B4 receptor dimers (Damian et al.,

2006). These studies support the notion that individual protomers in a

receptor dimer may interact with a shared G protein through distinct inter-

faces (see also Han et al., 2009), suggesting that structural asymmetries may

translate into functional or conformational asymmetries. Our results here

further strengthen the case for functional AT1R/FP heterodimeric com-

plexes and provide insight into the mechanism by which the two receptors

communicate. Though the precise functional consequences of AT1R-

induced change in FP conformation are yet to be determined, it also appears

to be asymmetric in nature. We observed this asymmetry in allosteric com-

munication between receptors, with AT1R modulating FP (when the FP

was tagged and coexpressed with untagged AT1R) but not the converse

(when conformational biosensors of AT1R (Devost et al., 2017) were used

with untagged FP). Further, the AT1R to FP conformational cross talk in

the heterodimer may be biased toward Gαq/11, as no effect was observed

158 Rory Sleno and Terence E. H�ebert

when we altered Gα12/13 or Gαi function or levels. This could represent a

coupling preference of the heterodimer or it may be possible that our biosen-

sors are sensitive to conformations driven by particular G proteins coupled to

the heterodimer. Capitalizing on such signal bias and asymmetric conforma-

tional cross talk may provide novel venues for targeting heterodimers,

ignored in most current drug discovery programs (Goupil et al., 2012,

2013; Khoury et al., 2014). Aswe have demonstrated previously, ligand bind-

ing to AT1R can modulate the functional output of FP (Goupil et al., 2015).

Since both AT1R and FP couple to Gαq, it is difficult to explore the func-

tional effect of the AT1R-induced conformational effects on FP, as they share

a number of common signaling outputs. It is also important to acknowledge

that there is also the possibility that the induced conformation may be silent

with respect to signaling (Kenakin and Miller, 2010). A larger understanding

of unique and shared receptor signaling outputs may help settle this question.

We showed that at least as regards receptor conformation, Ang II could

be considered as a biased ligand for FP, while the converse is not true for

PGF2α and the AT1R. Regardless of the mechanisms, the use of conforma-

tional biosensors could be used to identify new conformational and allosteric

connections between known and orphan GPCRs, without the requirement

for knowledge about how downstream signaling or receptor trafficking is

altered. This may help identify novel targets for drug discovery as ligands

for one receptor may in fact act as allosteric modulators of heterodimer part-

ners and provide several new vantage points from which to understand

receptor dynamics. We also think that such approaches will foster the devel-

opment of receptor screens that are portable from cell type to cell type

regardless of a priori knowledge about downstream signaling.

These findings indicate that formation of the AT1R/FP dimer again cre-

ated a novel allosteric signaling unit that showed both symmetrical and

asymmetrical responses, depending on the signaling or phenotypic output

measured. A similar picture could also emerge for the AT1R/purinergic

P2Y6 receptor (P2Y6R) pair (Nishimura et al., 2016). In this case, it is clear

that the presence of P2Y6R affected AT1R signaling but the converse was

not explored in detail. The AT1R seems to dimerize with many different

GPCR partners; thus, this has implications for the use of drugs to modulate

one or both receptors in a putative dimer pair in the clinical setting.

We also showed similar effects in a putative heterodimer that forms

between the β2AR and the oxytocin receptor (Wrzal et al., 2012a,b).

The β2AR/OTR pair was an allosteric dimer in myometrial cells, again

which express both receptors endogenously. Specifically, occupation of

159Asymmetric GPCR Signaling Mediated by Metastable Oligomeric Complexes

the β2AR binding site by either agonist, antagonist, or inverse agonist damp-

ened signaling through OTR via mechanisms that cannot simply involve

second messenger-mediated cross talk. Similar results were seen for

β2AR-mediated signaling in the case of antagonist- or inverse agonist-

occupied OTR. The presence of the OTR in either myometrial cells or

in HEK 293 cells altered β2AR signaling output providing credence to

the notion that the dimeric complex forms a unique signaling entity. The

βAR are also likely signaling hubs about which many oligomeric complexes

can be built.

11. IMPLICATIONS OF METASTABLE GPCR COMPLEXESFOR DRUG DISCOVERY

We feel that the notion of dynamic, yet metastable GPCR signaling

complexes, has tremendous implications for the formation and function of

receptor heterodimers, in that multiple asymmetrical arrangements become

possible depending on the relative orientation of each monomer to the

G protein and possibly effector molecules. Thus, in one arrangement, proto-

mer 1 is the signaling receptor and protomer 2 is an allosteric modulator that

does not necessarily generate a signaling output of its own and the converse is

true when the system is organized the other way around effectively gener-

ating two distinct signaling entities containing the same pair of receptors

(Fig. 3C). However, in heterooligomers, structural asymmetries in

receptor/G protein assembly may have dramatic consequences for signaling

(Fig. 2). As discussed, this idea greatly increases the potential organizational

complexity of GPCR signaling and further suggests that determinants of sig-

naling complex assembly might be of paramount importance in initially

defining signaling specificity in a given tissue, cellular, or subcellular com-

partment. Further, it suggests perhaps why heterodimers may have been dif-

ficult to detect in vivo since one receptor might in fact be silent with respect

to signaling and thus missed in standard drug screens. That arrangement can

be reversed if the complex is assembled or arranged differently—i.e., even

with the same set of interacting partners, signaling output could be quite dis-

tinct. Not only are these considerations important for therapeutic efficacy,

but may also predict and explain numerous off-target effects of currently used

drugs. We need to assess the consequences for cellular signaling when recep-

tors dimerize where one receptor may be silent with respect to signaling, the

structural basis for such potential asymmetries in signaling and to understand

mechanisms involved in how such complexes might be assembled.

160 Rory Sleno and Terence E. H�ebert

The potential organizational complexity of GPCR signaling is greatly

increased in the context of asymmetric heterodimers and suggests that

understanding the determinants of signaling complex assembly will be of

paramount importance in defining signaling specificity at any given moment

in particular tissues, cellular, or subcellular compartments (Milligan, 2007,

2009). This has tremendous implications for the formation of receptor

heterodimers and heterooligomers, in that multiple asymmetrical arrange-

ments might be possible depending on the relative orientation of each

monomer to the G protein and possibly effector. Further diversity is added

when we consider heterotetramers which can (1) have different numbers of

each component subunit and (2) several distinct potential arrangements of

those subunits. Important questions remaining include how and where

heterotetramers can form, in what order subunits are added, in what stoichi-

ometry, and how signaling partners are added. As we have seen, receptor

complexes can contain multiple receptors, what some authors have termed

as receptor mosaics (Agnati et al., 2010). These mosaics have been demon-

strated to be regulated by allosteric interactions between the receptor equiv-

alents which comprise them (Bonaventura et al., 2015; Ferre et al., 2016).

GPCR dimers, both homodimers such as CCR7 (Hauser et al., 2016) and

heterodimers (Navarro et al., 2016), likely act as hubs about which signaling

complexes are organized. Also, if there are direct interactions between

GPCRs and other receptor classes, might these structural asymmetries be

important in their function as well?

The role of preassembly during biosynthesis has important implications

for the formation of asymmetric, receptor-based signaling complexes.

Asymmetry in the context of GPCR heterodimers can be viewed in mul-

tiple ways—either structural, functional, or a combination of the two. Func-

tional asymmetry can be defined as differences in signaling mediated by a

receptor heterodimer, where occupancy of one receptor alters signaling

via the other and this relationship may differ depending on how the recep-

tors are stimulated—that is the asymmetry need not be necessarily reciprocal.

To understand functional asymmetries, we first need to more extensively

characterize signaling pathways downstream of putative heterodimers. Teas-

ing out the determinants of such assemblies will be critical for understanding

what complexes are formed in a given context and may provide mechanistic

insight into how asymmetric arrays are built. One simple way to use this sys-

tem is to test the notion that the timing of synthesis, or the order of assembly,

of key signaling components associated with a given GPCR heterodimer or

heterooligomer determines which receptor becomes the signaling receptor

161Asymmetric GPCR Signaling Mediated by Metastable Oligomeric Complexes

and which becomes the allosteric modulator. Approaches such as FlAsH–BRET profiling may allow us to tease apart such asymmetries with a view

toward making dimers druggable in a more consistent fashion. They may

also guide studies which aim to develop bitopic ligands for particular GPCR

dimer pairs (reviewed in Gomes et al., 2016).

Considerations of allosteric vs signaling roles might be especially impor-

tant in that we now understand that GPCRs do not act as simple switches

that turn single signaling pathways “on” or “off.” Instead, individual recep-

tors or receptor complexes engage multiple signaling cascades and individual

ligands can have differential efficacies toward specific subsets of these signal-

ing effectors. Such ligand-biased signaling or functional selectivity offers

interesting opportunities to identify and develop compounds with increased

selectivity and improved safety profiles. Despite years of important investi-

gation, the mechanistic basis of biased signaling through GPCRs remains

incompletely characterized. It has been assumed that different receptors

“select” downstream signaling pathways in response to different ligands

and that occupation of the ligand-binding site might alter or stabilize unique

receptor conformations. This may be an oversimplification of what occurs in

the context of the living cell, however. It may be possible that assembly or

colocalization of receptor homo- and heterodimeric/oligomeric complexes

is a more likely basis for distinct cellular responses to particular ligands. One

strategy might involve targeting assembly of signaling complexes to actually

provide a more “selective” set of biased assembly modulators compared with

current approaches designed to find biased ligands. However, much work

remains to identify the molecular determinants of signaling complex assem-

bly in the interim, especially in the context of GPCR oligomers.

Understanding GPCR oligomeric arrays and the interactions therein

might also be exploitable. Certainly, G protein and effectors (as well as other

aspects of cellular context) could also have allosteric impacts on ligand bind-

ing, as parts of larger complexes, with particular networks of allostery

depending on which proteins interact during different phases of signal trans-

duction (Figs. 1–3). All of these notions are consistent with highly organizedand metastable arrays of GPCR signaling molecules. Individual contacts

between members of such arrays may come and go, but the broader stability

of the larger complex is preserved under a range of conditions. Such a vision

of metastable GPCR oligomeric signaling complexes allows us to imagine

how parts might work independently as classically definedmonomeric recep-

tors; how individual dimers might be transient and sensitive to ligand and/or

162 Rory Sleno and Terence E. H�ebert

cellular conditions; and how depending on the technique used might present

a different view of the overall stoichiometry of such complexes at any given

time in any given cell. One thing that is clear is that once we remove these

players from the cellular environment for structural studies, for example, we

reduce this complexity dramatically. This may be why fewer dimers are

detected under conditions used to crystallize receptors and suggest that in

cellulo structural approaches such as cryo-EM might be quite useful going

forward.

ACKNOWLEDGMENTSThis work was supported by grants from the Canadian Institutes of Health Research to

T.E.H. (MOP-130309). R.S. was awarded scholarships from the McGill CIHR Drug

Development Training Program.

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171Asymmetric GPCR Signaling Mediated by Metastable Oligomeric Complexes