Post on 08-Oct-2020
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.
REFERENCESAgnati, L.F., Guidolin, D., Albertin, G., Trivello, E., Ciruela, F., Genedani, S.,
Tarakanov, A., Fuxe, K., 2010. An integrated view on the role of receptor mosaics atperisynaptic level: focus on adenosine A2A, dopamine D2, cannabinoid CB1, and meta-botropic glutamate mGlu5 receptors. J. Recept. Signal Transduct. 30, 355–369.
Albizu, L., Cottet, M., Kralikova, M., Stoev, S., Seyer, R., Brabet, I., Roux, T., Bazin, H.,Bourrier, E., Lamarque, L., Breton, C., Rives, M.L., Newman, A., Javitch, J.,Trinquet, E., Manning, M., Pin, J.P., Mouillac, B., Durroux, T., 2010. Time-resolvedFRET between GPCR ligands reveals oligomers in native tissues. Nat. Chem. Biol.6, 587–594.
Altier, C., 2012. GPCR and voltage-gated calcium channels (VGCC) signaling complexes.Subcell. Biochem. 63, 241–262.
Altier, C., Khosravani, H., Evans, R.M., Hameed, S., Peloquin, J.B., Vartian, B.A.,Chen, L., Beedle, A.M., Ferguson, S.S., Mezghrani, A., Dubel, S.J., Bourinet, E.,Mcrory, J.E., Zamponi, G.W., 2006. ORL1 receptor-mediated internalization ofN-type calcium channels. Nat. Neurosci. 9, 31–40.
Angers, S., Salahpour, A., Joly, E., Hilairet, S., Chelsky, D., Dennis, M., Bouvier, M., 2000.Detection of β2-adrenergic receptor dimerization in living cells using bioluminescenceresonance energy transfer (BRET). Proc. Natl. Acad. Sci. U.S.A. 97, 3684–3689.
Baragli, A., Grieco, M.-L., Trieu, P., Villeneuve, L.R., H�ebert, T.E., 2008. Heterodimers ofadenylyl cyclases 2 and 5 show enhanced functional responses in the presence of Gαs.Cell. Signal. 20, 480–492.
Barki-Harrington, L., Luttrell, L.M., Rockman, H.A., 2003. Dual inhibition of β-adrenergicand angiotensin II receptors by a single antagonist: a functional role for receptor-receptorinteraction in vivo. Circulation 108, 1611–1618.
Bellot, M., Galandrin, S., Boularan, C., Matthies, H.J., Despas, F., Denis, C., Javitch, J.,Mazeres, S., Sanni, S.J., Pons, V., Seguelas, M.H., Hansen, J.L., Pathak, A., Galli, A.,Senard, J.M., Gales, C., 2015. Dual agonist occupancy of AT1-R-α2C-ARheterodimers results in atypical Gs-PKA signaling. Nat. Chem. Biol. 11, 271–279.
Bird, I.M., Zheng, J., Cale, J.M., Magness, R.R., 1997. Pregnancy induces an increase inangiotensin II type-1 receptor expression in uterine but not systemic artery endothelium.Endocrinology 138, 490–498.
163Asymmetric GPCR Signaling Mediated by Metastable Oligomeric Complexes
Bonaventura, J., Navarro, G., Casado-Anguera, V., Azdad, K., Rea, W., Moreno, E.,Brugarolas, M., Mallol, J., Canela, E.I., Lluis, C., Cortes, A., Volkow, N.D.,Schiffmann, S.N., Ferre, S., Casado, V., 2015. Allosteric interactions between agonistsand antagonists within the adenosineA2A receptor-dopamineD2 receptor heterotetramer.Proc. Natl. Acad. Sci. U.S.A. 112, E3609–18.
Borghi, C., Rossi, F., 2015. Role of the renin-angiotensin-aldosterone system and its phar-macological inhibitors in cardiovascular diseases: complex and critical issues. High BloodPress. Cardiovasc. Prev. 22, 429–444.
Bourque, K., P�etrin, D., Sleno, R., Devost, D., Zhang, A., H�ebert, T.E., 2017. Distinct con-formational dynamics of three G protein-coupled receptors measured using FlAsH-BRET biosensors. Front. Endocrinol. (Lausanne) 8, 61.
Briddon, S.J., Kilpatrick, L.E., Hill, S.J., 2018. Studying GPCR pharmacology in membranemicrodomains: fluorescence correlation spectroscopy comes of age. Trends Pharmacol.Sci. 39, 158–174.
Bulenger, S., Marullo, S., Bouvier, M., 2005. Emerging role of homo- andheterodimerization in G protein-coupled receptor biosynthesis and maturation. TrendsPharmacol. Sci. 26, 131–137.
Calebiro, D., Sungkaworn, T., 2018. Single-molecule imaging of GPCR interactions.Trends Pharmacol. Sci. 39, 109–122.
Calebiro, D., Rieken, F., Wagner, J., Sungkaworn, T., Zabel, U., Borzi, A., Cocucci, E.,Zurn, A., Lohse, M.J., 2013. Single-molecule analysis of fluorescently labeledG protein-coupled receptors reveals complexes with distinct dynamics and organization.Proc. Natl. Acad. Sci. U.S.A. 110, 743–748.
Camp, N.D., Lee, K.-S., Wacker-Mhyre, J.L., Kountz, T.S., Park, J.-M., Harris, D.-A.,Estrada, M., Stewart, A., Wolf-Yadlin, A., Hague, C., 2015. Individual protomers ofa G protein-coupled receptor dimer integrate distinct functional modules. Cell Discov.1, 15011.
Carriba, P., Navarro, G., Ciruela, F., Ferre, S., Casado, V., Agnati, L., Cortes, A., Mallol, J.,Fuxe, K., Canela, E.I., Lluis, C., Franco, R., 2008. Detection of heteromerization ofmore than two proteins by sequential BRET-FRET. Nat. Methods 5, 727–733.
Chidiac, P., Green, M.A., Pawagi, A.B., Wells, J.W., 1997. Cardiac muscarinic receptors.Cooperativity as the basis for multiple states of affinity. Biochemistry 36, 7361–7379.
Cordomi,A.,Navarro,G.,Aymerich,M.S., Franco,R., 2015. Structures forGprotein-coupledreceptor tetramers in complex with G proteins. Trends Biochem. Sci. 40, 548–551.
Cox, B.E., Ipson, M.A., Shaul, P.W., Kamm, K.E., Rosenfeld, C.R., 1993. Myometrialangiotensin II receptor subtypes change during ovine pregnancy. J. Clin. Invest.92, 2240–2248.
Dai, S., Hall, D.D., Hell, J.W., 2009. Supramolecular assemblies and localized regulation ofvoltage-gated ion channels. Physiol. Rev. 89, 411–452.
Damian, M., Martin, A., Mesnier, D., Pin, J.P., Baneres, J.L., 2006. Asymmetric conforma-tional changes in a GPCR dimer controlled by G proteins. EMBO J. 25, 5693–5702.
David, M., Richer, M., Mamarbachi, A.M., Villeneuve, L.R., Dupr�e, D.J., H�ebert, T.E.,2006. Interactions between GABA-B1 receptors and Kir 3 inwardly rectifying potassiumchannels. Cell Signal. 18, 2172–2181.
De Lean, A., Stadel, J.M., Lefkowitz, R.J., 1980. A ternary complex model explains theagonist-specific binding properties of the adenylate cyclase-coupled β-adrenergic recep-tor. J. Biol. Chem. 255, 7108–7117.
Devost, D., Sleno, R., P�etrin, D., Zhang, A., Shinjo, Y., Okde, R., Aoki, J., Inoue, A.,H�ebert, T.E., 2017. Conformational profiling of the AT1 angiotensin II receptor reflectsbiased agonism,G protein coupling, and cellular context. J. Biol. Chem. 292, 5443–5456.
Dong, C., Filipeanu, C.M., Duvernay, M.T., Wu, G., 2007. Regulation of G protein-coupled receptor export trafficking. Biochim. Biophys. Acta 1768, 853–870.
164 Rory Sleno and Terence E. H�ebert
Dorsch, S., Klotz, K.N., Engelhardt, S., Lohse, M.J., Bunemann, M., 2009. Analysis ofreceptor oligomerization by FRAP microscopy. Nat. Methods 6, 225–230.
Dowal, L., Provitera, P., Scarlata, S., 2006. Stable association between Gαq and phospholi-pase Cβ1 in living cells. J. Biol. Chem. 281, 23999–24014.
Dupr�e, D.J., H�ebert, T.E., 2006. Biosynthesis and trafficking of seven transmembrane recep-tor signalling complexes. Cell Signal. 18, 1549–1559.
Dupr�e, D.J., Robitaille, M., Ethier, N., Villeneuve, L.R., Mamarbachi, A.M., H�ebert, T.E.,2006. Seven transmembrane receptor core signaling complexes are assembled prior toplasma membrane trafficking. J. Biol. Chem. 281, 34561–34573.
Dupr�e, D.J., Baragli, A., Rebois, R.V., Ethier, N., H�ebert, T.E., 2007. Signalling complexesassociated with adenylyl cyclase II are assembled during their biosynthesis. Cell Signal.19, 481–489.
Dupr�e, D.J., Robitaille, M., Rebois, R.V., H�ebert, T.E., 2009. The role of Gβγ subunits inthe organization, assembly, and function of GPCR signaling complexes. Annu. Rev.Pharmacol. Toxicol. 49, 31–56.
Evans, R.M., You, H., Hameed, S., Altier, C., Mezghrani, A., Bourinet, E.,Zamponi, G.W., 2010. Heterodimerization of ORL1 and opioid receptors and its con-sequences for N-type calcium channel regulation. J. Biol. Chem. 285, 1032–1040.
Felce, J.H., Knox, R.G., Davis, S.J., 2014. Type-3 BRET, an improved competition-basedbioluminescence resonance energy transfer assay. Biophys. J. 106, L41–L43.
Felce, J.H., Davis, S.J., Klenerman, D., 2018. Single-molecule analysis of G protein-coupledreceptor stoichiometry: approaches and limitations. Trends Pharmacol. Sci. 39, 96–108.
Ferre, S., 2015. The GPCR heterotetramer: challenging classical pharmacology. TrendsPharmacol. Sci. 36, 145–152.
Ferre, S., Casado, V., Devi, L.A., Filizola, M., Jockers, R., Lohse, M.J., Milligan, G.,Pin, J.P., Guitart, X., 2014. G protein-coupled receptor oligomerization revisited: func-tional and pharmacological perspectives. Pharmacol. Rev. 66, 413–434.
Ferre, S., Bonaventura, J., Tomasi, D., Navarro, G., Moreno, E., Cortes, A., Lluis, C.,Casado, V., Volkow, N.D., 2016. Allosteric mechanisms within the adenosine A2A-dopamine D2 receptor heterotetramer. Neuropharmacology 104, 154–160.
Flynn, R., Altier, C., 2013. A macromolecular trafficking complex composed of β2-adrenergic receptors, A-kinase anchoring proteins and L-type calcium channels.J. Recept. Signal Transduct. Res. 33, 172–176.
Fonseca, J.M., Lambert, N.A., 2009. Instability of a class a G protein-coupled receptor olig-omer interface. Mol. Pharmacol. 75, 1296–1299.
Franco, R., Martinez-Pinilla, E., Lanciego, J.L., Navarro, G., 2016. Basic pharmacologicaland structural evidence for class A G protein-coupled receptor heteromerization. Front.Pharmacol. 7, 76.
Fung, J.J., Deupi, X., Pardo, L., Yao, X.J., Velez-Ruiz, G.A., Devree, B.T., Sunahara, R.K.,Kobilka, B.K., 2009. Ligand-regulated oligomerization of β2-adrenoceptors in a modellipid bilayer. EMBO J. 28, 3315–3328.
Gandia, J., Galino, J., Amaral, O.B., Soriano, A., Lluis, C., Franco, R., Ciruela, F., 2008.Detection of higher-order G protein-coupled receptor oligomers by a combinedBRET-BiFC technique. FEBS Lett. 582, 2979–2984.
Gavalas, A., Lan, T.H., Liu, Q., Correa Jr., I.R., Javitch, J.A., Lambert, N.A., 2013. Segre-gation of family A G protein-coupled receptor protomers in the plasma membrane. Mol.Pharmacol. 84, 346–352.
Ge, B., Lao, J., Li, J., Chen, Y., Song, Y., Huang, F., 2017. Single-molecule imaging revealsdimerization/oligomerization of CXCR4 on plasma membrane closely related to itsfunction. Sci. Rep. 7, 16873.
George, S.R., Kern, A., Smith, R.G., Franco, R., 2014. Dopamine receptor heteromericcomplexes and their emerging functions. Prog. Brain Res. 211, 183–200.
165Asymmetric GPCR Signaling Mediated by Metastable Oligomeric Complexes
Gomes, I., Ayoub, M.A., Fujita, W., Jaeger, W.C., Pfleger, K.D.G., Devi, L.A., 2016.G protein-coupled receptor heteromers. Annu. Rev. Pharmacol. Toxicol. 56, 403–425.
Goupil, E., Laporte, S.A., H�ebert, T.E., 2012. Functional selectivity in GPCR signaling:understanding the full spectrum of receptor conformations. Mini Rev. Med. Chem.12, 817–830.
Goupil, E., Laporte, S.A., H�ebert, T.E., 2013. GPCR heterodimers: asymmetries in ligandbinding and signalling output offer new targets for drug discovery. Br. J. Pharmacol.168, 1101–1103.
Goupil, E., Fillion, D., Cl�Ement, S., Luo, X., Devost, D., Sleno, R., P�etrin, D.,Saragovi, H.U., Thorin, E., Laporte, S.A., H�ebert, T.E., 2015. Angiotensin II typeI and prostaglandin F2α receptors cooperatively modulate signaling in vascular smoothmuscle cells. J. Biol. Chem. 290, 3137–3148.
Green, M.A., Chidiac, P., Wells, J.W., 1997. Cardiac muscarinic receptors. Relationshipbetween the G protein and multiple states of affinity. Biochemistry 36, 7380–7394.
Guo, W., Urizar, E., Kralikova, M., Mobarec, J.C., Shi, L., Filizola, M., Javitch, J.A., 2008.Dopamine D2 receptors form higher order oligomers at physiological expression levels.EMBO J. 27, 2293–2304.
Hamatake, M., Aoki, T., Futahashi, Y., Urano, E., Yamamoto, N., Komano, J., 2009.Ligand-independent higher-order multimerization of CXCR4, a G protein-coupledchemokine receptor involved in targeted metastasis. Cancer Sci. 100, 95–102.
Han, Y., Moreira, I.S., Urizar, E., Weinstein, H., Javitch, J.A., 2009. Allosteric communi-cation between protomers of dopamine class A GPCR dimers modulates activation. Nat.Chem. Biol. 5, 688–695.
Haspula, D., Clark, M.A., 2017. MAPK activation patterns of AT1R and CB1R in SHRversus Wistar astrocytes: evidence of CB1R hypofunction and crosstalk betweenAT1R and CB1R. Cell. Signal. 40, 81–90.
Hauser, M.A., Schaeuble, K., Kindinger, I., Impellizzieri, D., Krueger, W.A., Hauck, C.R.,Boyman, O., Legler, D.F., 2016. Inflammation-induced CCR7 oligomers form scaffoldsto integrate distinct signaling pathways for efficient cell migration. Immunity 44, 59–72.
H�ebert, T.E., Bouvier, M., 1998. Structural and functional aspects of G protein-coupledreceptor oligomerization. Biochem. Cell Biol. 76, 1–11.
Hern, J.A., Baig, A.H., Mashanov, G.I., Birdsall, B., Corrie, J.E., Lazareno, S., Molloy, J.E.,Birdsall, N.J., 2010. Formation and dissociation of M1 muscarinic receptor dimers seenby total internal reflection fluorescence imaging of single molecules. Proc. Natl. Acad.Sci. U.S.A. 107, 2693–2698.
Hlavackova, V., Zabel, U., Frankova, D., Batz, J., Hoffmann, C., Prezeau, L., Pin, J.P.,Blahos, J., Lohse, M.J., 2012. Sequential inter- and intrasubunit rearrangements duringactivation of dimeric metabotropic glutamate receptor 1. Sci. Signal. 5, ra59.
Huang, J., Chen, S., Zhang, J.J., Huang, X.Y., 2013. Crystal structure of oligomericβ1-adrenergic G protein-coupled receptors in ligand-free basal state. Nat. Struct.Mol. Biol. 20, 419–425.
Jain, A., Liu, R., Ramani, B., Arauz, E., Ishitsuka, Y., Ragunathan, K., Park, J., Chen, J.,Xiang, Y.K., Ha, T., 2011. Probing cellular protein complexes using single-moleculepull-down. Nature 473, 484–488.
James, J.R., Oliveira, M.I., Carmo, A.M., Iaboni, A., Davis, S.J., 2006. A rigorous exper-imental framework for detecting protein oligomerization using bioluminescence reso-nance energy transfer. Nat. Methods 3, 1001–1006.
Jastrzebska, B., Chen, Y., Orban, T., Jin, H., Hofmann, L., Palczewski, K., 2015. Disruptionof rhodopsin dimerization with synthetic peptides targeting an interaction interface.J. Biol. Chem. 290, 25728–25744.
Jenkin, G., 1992. Oxytocin and prostaglandin interactions in pregnancy and at parturition.J. Reprod. Fertil. Suppl. 45, 97–111.
166 Rory Sleno and Terence E. H�ebert
Jonas, K.C., Fanelli, F., Huhtaniemi, I.T., Hanyaloglu, A.C., 2015. Single molecule analysisof functionally asymmetric G protein-coupled receptor (GPCR) oligomers revealsdiverse spatial and structural assemblies. J. Biol. Chem. 290, 3875–3892.
Jordan, B.A., Trapaidze, N., Gomes, I., Nivarthi, R., Devi, L.A., 2001. Oligomerization ofopioid receptors with β2-adrenergic receptors: A role in trafficking and mitogen-activated protein kinase activation. Proc. Natl. Acad. Sci. U.S.A. 98, 343–348.
Kammermeier, P.J., 2012. Functional and pharmacological characteristics of metabotropicglutamate receptors 2/4 heterodimers. Mol. Pharmacol. 82, 438–447.
Karla, K.V.H., Michelle, R.T., Kymry, T.J., Samir, S.E.-D., Harish, R., Nael, A.M., 2010.A novel bioassay for detecting GPCR heterodimerization: transactivation of β2 adren-ergic receptor by bradykinin receptor. J. Biomol. Screen. 15, 251–260.
Kasai, R.S., Ito, S.V., Awane, R.M., Fujiwara, T.K., Kusumi, A., 2017. The class-A GPCRdopamine d2 receptor forms transient dimers stabilized by agonists: detection by single-molecule tracking. Cell Biochem. Biophys. https://doi.org/10.1007/s12013-017-0829-y.
Kasai, R.S., Suzuki, K.G., Prossnitz, E.R., Koyama-Honda, I., Nakada, C., Fujiwara, T.K.,Kusumi, A., 2011. Full characterization of GPCR monomer-dimer dynamic equilib-rium by single molecule imaging. J. Cell Biol. 192, 463–480.
Katritch, V., Cherezov, V., Stevens, R.C., 2012. Diversity and modularity of G protein-coupled receptor structures. Trends Pharmacol. Sci. 33, 17–27.
Katritch, V., Cherezov, V., Stevens, R.C., 2013. Structure-function of the G protein-coupled receptor superfamily. Annu. Rev. Pharmacol. Toxicol. 53, 531–556.
Kawano, K., Yano, Y., Omae, K., Matsuzaki, S., Matsuzaki, K., 2013. Stoichiometric anal-ysis of oligomerization of membrane proteins on living cells using coiled-coil labelingand spectral imaging. Anal. Chem. 85, 3454–3461.
Kenakin, T., Miller, L.J., 2010. Seven transmembrane receptors as shapeshifting proteins: theimpact of allosteric modulation and functional selectivity on new drug discovery.Pharmacol. Rev. 62, 265–304.
Kern, A., Albarran-Zeckler, R., Walsh, H.E., Smith, R.G., 2012. Apo-ghrelin receptorforms heteromers with DRD2 in hypothalamic neurons and is essential for anorexigeniceffects of DRD2 agonism. Neuron 73, 317–332.
Khoury, E., Clement, S., Laporte, S.A., 2014. Allosteric and biasedG protein-coupled receptorsignaling regulation: potentials for new therapeutics. Front. Endocrinol. (Lausanne). 5, 68.
Kleinau, G., Muller, A., Biebermann, H., 2016. Oligomerization of GPCRs involved inendocrine regulation. J. Mol. Endocrinol. 57, R59–R80.
Lan, T.H., Kuravi, S., Lambert, N.A., 2011. Internalization dissociates β2-adrenergic recep-tors. PLoS One 6, e17361.
Lan, T.H., Liu, Q., Li, C., Wu, G., Steyaert, J., Lambert, N.A., 2015. BRET evidence thatβ2 adrenergic receptors do not oligomerize in cells. Sci. Rep. 5, 10166.
Lane, J.R., Donthamsetti, P., Shonberg, J., Draper-Joyce, C.J., Dentry, S., Michino, M.,Shi, L., Lopez, L., Scammells, P.J., Capuano, B., Sexton, P.M., Javitch, J.A.,Christopoulos, A., 2014. A new mechanism of allostery in a G protein-coupled receptordimer. Nat. Chem. Biol. 10, 745–752.
Lavine, N., Ethier, N., Oak, J.N., Pei, L., Liu, F., Trieu, P., Rebois, R.V., Bouvier, M.,H�ebert, T.E., Van Tol, H.H., 2002. G protein-coupled receptors form stable complexeswith inwardly rectifying potassium channels and adenylyl cyclase. J. Biol. Chem.277, 46010–46019.
Levitz, J., Habrian, C., Bharill, S., Fu, Z., Vafabakhsh, R., Isacoff, E.Y., 2016. Mechanism ofassembly and cooperativity of homomeric and heteromeric metabotropic glutamatereceptors. Neuron 92, 143–159.
Li, C., Wei, Z., Fan, Y., Huang, W., Su, Y., Li, H., Dong, Z., Fukuda, M., Khater, M.,Wu, G., 2017. The GTPase Rab43 controls the anterograde ER-Golgi traffickingand sorting of GPCRs. Cell Rep. 21, 1089–1101.
167Asymmetric GPCR Signaling Mediated by Metastable Oligomeric Complexes
Liu, J., Zhang, Z., Moreno-Delgado, D., Dalton, J.A., Rovira, X., Trapero, A., Goudet, C.,Llebaria, A., Giraldo, J., Yuan, Q., Rondard, P., Huang, S., Liu, J., Pin, J.P., 2017. Allo-steric control of an asymmetric transduction in a G protein-coupled receptorheterodimer. Elife 6, pii: e26985. https://doi.org/10.7554/eLife.26985.
Lopez-Gimenez, J.F., Canals, M., Pediani, J.D., Milligan, G., 2007. The α1b-adrenoceptorexists as a higher-order oligomer: effective oligomerization is required for receptor mat-uration, surface delivery, and function. Mol. Pharmacol. 71, 1015–1029.
Lu, M., Wu, B., 2016. Structural studies of G protein-coupled receptors. IUBMB Life68, 894–903.
Ma, A.W., Redka, D.S., Pisterzi, L.F., Angers, S., Wells, J.W., 2007. Recovery of oligomersand cooperativity when monomers of the M2 muscarinic cholinergic receptor are rec-onstituted into phospholipid vesicles. Biochemistry 46, 7907–7927.
Ma, A.W., Pawagi, A.B., Wells, J.W., 2008. Heterooligomers of the muscarinic receptor andG proteins purified from porcine atria. Biochem. Biophys. Res. Commun. 374, 128–133.
Maier-Peuschel, M., Frolich, N., Dees, C., Hommers, L.G., Hoffmann, C., Nikolaev, V.O.,Lohse,M.J., 2010. A fluorescence resonance energy transfer-basedM2muscarinic recep-tor sensor reveals rapid kinetics of allosteric modulation. J. Biol. Chem. 285, 8793–8800.
Manglik, A., Kruse, A.C., Kobilka, T.S., Thian, F.S., Mathiesen, J.M., Sunahara, R.K.,Pardo, L., Weis, W.I., Kobilka, B.K., Granier, S., 2012. Crystal structure of the μ-opioidreceptor bound to a morphinan antagonist. Nature 485, 321–326.
Marquez-Gomez, R., Robins, M.T., Gutierrez-Rodelo, C., Arias, J.M., Olivares-Reyes,J.A., Van Rijn, R.M., Arias-Montano, J.A., 2018. Functional histamine H3 and aden-osine A2A receptor heteromers in recombinant cells and rat striatum. Pharmacol. Res.129, 515–525.
Marsango, S., Ward, R.J., Alvarez-Curto, E., Milligan, G., 2017. Muscarinic receptor oligo-merization. Neuropharmacology, pii: S0028-3908(17)30532-4. https://doi.org/10.1016/j.neuropharm.2017.11.023.
McGraw, D.W., Mihlbachler, K.A., Schwarb, M.R., Rahman, F.F., Small, K.M.,Almoosa, K.F., Liggett, S.B., 2006. Airway smooth muscle prostaglandin-EP1 receptorsdirectly modulate β2-adrenergic receptors within a unique heterodimeric complex.J. Clin. Invest. 116, 1400–1409.
McVey, M., Ramsay, D., Kellett, E., Rees, S., Wilson, S., Pope, A.J., Milligan, G., 2001.Monitoring receptor oligomerization using time-resolved fluorescence resonance energytransfer and bioluminescence resonance energy transfer. Human J. Biol. Chem.276, 14092–14099.
Mejia, R., Waite, C., Ascoli, M., 2015. Activation of Gq/11 in the mouse corpus luteum isrequired for parturition. Mol. Endocrinol. 29, 238–246.
Mercier, J.F., Salahpour, A., Angers, S., Breit, A., Bouvier, M., 2002. Quantitative assess-ment of β1- and β2-adrenergic receptor homo- and heterodimerization by biolumines-cence resonance energy transfer. J. Biol. Chem. 277, 44925–44931.
Milligan, G., 2007. G protein-coupled receptor dimerisation: molecular basis and relevanceto function. Biochim. Biophys. Acta 1768, 825–835.
Milligan, G., 2009. G protein-coupled receptor hetero-dimerization: contribution to phar-macology and function. Br. J. Pharmacol. 158, 5–14.
Milligan, G., 2010. The role of dimerisation in the cellular trafficking of G protein-coupledreceptors. Curr. Opin. Pharmacol. 10, 23–29.
Mishra, A.K., Gragg, M., Stoneman, M.R., Biener, G., Oliver, J.A., Miszta, P., Filipek, S.,Raicu, V., Park, P.S., 2016. Quaternary structures of opsin in live cells revealed byFRET spectrometry. Biochem. J. 473, 3819–3836.
Moreno Delgado, D., Moller, T.C., Ster, J., Giraldo, J., Maurel, D., Rovira, X., Scholler, P.,Zwier, J.M., Perroy, J., Durroux, T., Trinquet, E., Prezeau, L., Rondard, P., Pin, J.P.,2017. Pharmacological evidence for a metabotropic glutamate receptor heterodimer inneuronal cells. Elife 6, pii: e25233. https://doi.org/10.7554/eLife.25233.
168 Rory Sleno and Terence E. H�ebert
Navarro, G., Cordomi, A., Zelman-Femiak, M., Brugarolas, M., Moreno, E., Aguinaga, D.,Perez-Benito, L., Cortes, A., Casado, V., Mallol, J., Canela, E.I., Lluis, C., Pardo, L.,Garcia-Saez, A.J., McCormick, P.J., Franco, R., 2016. Quaternary structure of aG protein-coupled receptor heterotetramer in complexwithGi andGs. BMCBiol. 14, 26.
Nishimura, A., Sunggip, C., Tozaki-Saitoh, H., Shimauchi, T., Numaga-Tomita, T.,Hirano, K., Ide, T., Boeynaems, J.M., Kurose, H., Tsuda, M., Robaye, B.,Inoue, K., Nishida, M., 2016. Purinergic P2Y6 receptors heterodimerize with angioten-sin AT1 receptors to promote angiotensin II-induced hypertension. Sci. Signal. 9, ra7.
Pediani, J.D., Ward, R.J., Godin, A.G., Marsango, S., Milligan, G., 2016. Dynamic regu-lation of quaternary organization of the M1 muscarinic receptor by subtype-selectiveantagonist drugs. J. Biol. Chem. 291, 13132–13146.
Pediani, J.D., Ward, R.J., Marsango, S., Milligan, G., 2017. Spatial intensity distributionanalysis: studies of G protein-coupled receptor oligomerisation. Trends Pharmacol.Sci. 39, 175–186.
Peterson, G.L., Herron, G.S., Yamaki, M., Fullerton, D.S., Schimerlik, M.I., 1984. Purifi-cation of the muscarinic acetylcholine receptor from porcine atria. Proc. Natl. Acad. Sci.U.S.A. 81, 4993–4997.
P�etrin, D., H�ebert, T.E., 2011. Imaging-based approaches to understanding G protein-coupled receptor signalling complexes. Methods Mol. Biol. 756, 37–60.
P�etrin, D., H�ebert, T.E., 2012. The functional size of GPCRs—monomers, dimers or tet-ramers? Subcell. Biochem. 63, 67–81.
Pfleger, K.D., Eidne, K.A., 2006. Illuminating insights into protein-protein interactionsusing bioluminescence resonance energy transfer (BRET). Nat. Methods 3, 165–174.
Pisterzi, L.F., Jansma, D.B., Georgiou, J., Woodside, M.J., Chou, J.T., Angers, S., Raicu, V.,Wells, J.W., 2010. Oligomeric size of the m2 muscarinic receptor in live cells as deter-mined by quantitative fluorescence resonance energy transfer. J. Biol. Chem.285, 16723–16738.
Prezeau, L., Rives, M.L., Comps-Agrar, L., Maurel, D., Kniazeff, J., Pin, J.P., 2010. Func-tional crosstalk between GPCRs: with or without oligomerization. Curr. Opin.Pharmacol. 10, 6–13.
Prinster, S.C., Hague, C., Hall, R.A., 2005. Heterodimerization of G protein-coupledreceptors: specificity and functional significance. Pharmacol. Rev. 57, 289–298.
Qin, K., Dong, C., Wu, G., Lambert, N.A., 2011. Inactive-state preassembly of Gq-coupledreceptors and Gq heterotrimers. Nat. Chem. Biol. 7, 740–747.
Rebois, R.V., H�ebert, T.E., 2003. Protein complexes involved in heptahelical receptor-mediated signal transduction. Receptors Channels 9, 169–194.
Rebois, R.V., Robitaille, M., Gales, C., Dupr�e, D.J., Baragli, A., Trieu, P., Ethier, N.,Bouvier, M., H�ebert, T.E., 2006. Heterotrimeric G proteins form stable complexes withadenylyl cyclase and Kir3.1 channels in living cells. J. Cell Sci. 119, 2807–2818.
Rebois, R.V., Robitaille, M., P�etrin, D., Zylbergold, P., Trieu, P., H�ebert, T.E., 2008.Combining protein complementation assays with resonance energy transfer to detectmultipartner protein complexes in living cells. Methods 45, 214–218.
Redka, D.S., Heerklotz, H., Wells, J.W., 2013. Efficacy as an intrinsic property of the M2muscarinic receptor in its tetrameric state. Biochemistry 52, 7405–7427.
Robitaille, M., Ramakrishnan, N., Baragli, A., H�ebert, T.E., 2009. Intracellular traffickingand assembly of specific Kir3 channel/G protein complexes. Cell. Signal. 21, 488–501.
Salahpour, A., Angers, S., Mercier, J.F., Lagace, M., Marullo, S., Bouvier, M., 2004.Homodimerization of the β2-adrenergic receptor as a prerequisite for cell surfacetargeting. J. Biol. Chem. 279, 33390–33397.
Scarselli, M., Annibale, P., McCormick, P.J., Kolachalam, S., Aringhieri, S., Radenovic, A.,Corsini, G.U., Maggio, R., 2016. Revealing G protein-coupled receptor oligomeriza-tion at the single-molecule level through a nanoscopic lens: methods, dynamics andbiological function. FEBS J. 283, 1197–1217.
169Asymmetric GPCR Signaling Mediated by Metastable Oligomeric Complexes
Shivnaraine, R.V., Kelly, B., Sankar, K.S., Redka, D.S., Han, Y.R., Huang, F., Elmslie, G.,Pinto, D., Li, Y., Rocheleau, J.V., Gradinaru, C.C., Ellis, J., Wells, J.W., 2016. Allo-steric modulation in monomers and oligomers of a G protein-coupled receptor. Elife5, pii: e11685. https://doi.org/10.7554/eLife.11685.
Siddiquee, K., Hampton, J., Mcanally, D., May, L., Smith, L., 2013. The apelin receptorinhibits the angiotensin II type 1 receptor via allosteric trans-inhibition. Br. J. Pharmacol.168, 1104–1117.
Sleno, R., Devost, D., P�etrin, D., Zhang, A., Bourque, K., Shinjo, Y., Aoki, J., Inoue, A.,H�ebert, T.E., 2017. Conformational biosensors reveal allosteric interactions betweenheterodimeric AT1 angiotensin and prostaglandin F2α receptors. J. Biol. Chem.292, 12139–12152.
Sohy, D., Yano, H., De Nadai, P., Urizar, E., Guillabert, A., Javitch, J.A., Parmentier, M.,Springael, J.Y., 2009. Hetero-oligomerization of CCR2, CCR5, and CXCR4 and theprotean effects of “selective” antagonists. J. Biol. Chem. 284, 31270–31279.
Takezako, T., Unal, H., Karnik, S.S., Node, K., 2017. Current topics in angiotensin II type 1receptor research: focus on inverse agonism, receptor dimerization and biased agonism.Pharmacol. Res. 123, 40–50.
Toth, A.D., Gyombolai, P., Szalai, B., Varnai, P., Turu, G., Hunyady, L., 2017. Angiotensintype 1A receptor regulates β-arrestin binding of the β2-adrenergic receptor viaheterodimerization. Mol. Cell. Endocrinol. 442, 113–124.
Veya, L., Piguet, J., Vogel, H., 2015. Single molecule imaging deciphers the relation betweenmobility and signaling of a prototypical G protein-coupled receptor in living cells. J. Biol.Chem. 290, 27723–27735.
Vidi, P.A., Chemel, B.R., Hu, C.D., Watts, V.J., 2008a. Ligand-dependent oligomerizationof dopamine D2 and adenosine A2A receptors in living neuronal cells. Mol. Pharmacol.74, 544–551.
Vidi, P.A., Chen, J., Irudayaraj, J.M., Watts, V.J., 2008b. Adenosine A2A receptors assembleinto higher-order oligomers at the plasma membrane. FEBS Lett. 582, 3985–3990.
Vilardaga, J.P., Nikolaev, V.O., Lorenz, K., Ferrandon, S., Zhuang, Z., Lohse, M.J., 2008.Conformational cross-talk between α2A-adrenergic and μ-opioid receptors controls cellsignaling. Nat. Chem. Biol. 4, 126–131.
Whorton, M.R., Bokoch, M.P., Rasmussen, S.G., Huang, B., Zare, R.N., Kobilka, B.,Sunahara, R.K., 2007. A monomeric G protein-coupled receptor isolated in a high-density lipoprotein particle efficiently activates its G protein. Proc. Natl. Acad. Sci.U.S.A. 104, 7682–7687.
Whorton, M.R., Jastrzebska, B., Park, P.S., Fotiadis, D., Engel, A., Palczewski, K.,Sunahara, R.K., 2008. Efficient coupling of transducin to monomeric rhodopsin in aphospholipid bilayer. J. Biol. Chem. 283, 4387–4394.
Wreggett, K.A., Wells, J.W., 1995. Cooperativity manifest in the binding properties of puri-fied cardiac muscarinic receptors. J. Biol. Chem. 270, 22488–22499.
Wrzal, P.K., Devost, D., P�etrin, D., Goupil, E., Iorio-Morin, C., Laporte, S.A.,Zingg, H.H., H�ebert, T.E., 2012a. Allosteric interactions between the oxytocin receptorand the β2-adrenergic receptor in the modulation of Erk1/2 activation are mediated byheterodimerization. Cell. Signal. 24, 342–350.
Wrzal, P.K., Goupil, E., Laporte, S.A., H�ebert, T.E., Zingg, H.H., 2012b. Functional inter-actions between the oxytocin receptor and the β2-adrenergic receptor: implications forERK1/2 activation in human myometrial cells. Cell. Signal. 24, 333–341.
Wu, B., Chien, E.Y., Mol, C.D., Fenalti, G., Liu, W., Katritch, V., Abagyan, R.,Brooun, A., Wells, P., Bi, F.C., Hamel, D.J., Kuhn, P., Handel, T.M.,Cherezov, V., Stevens, R.C., 2010. Structures of the CXCR4 chemokine GPCR withsmall-molecule and cyclic peptide antagonists. Science 330, 1066–1071.
170 Rory Sleno and Terence E. H�ebert
Wu, H., Wacker, D., Katritch, V., Mileni, M., Han, G.W., Vardy, E., Liu, W.,Thompson, A.A., Huang, X.-P., Carroll, F.I., Mascarella, S.W., Westkaemper, R.B.,Mosier, P.D., Roth, B.L., Cherezov, V., Stevens, R.C., 2012. Structure of the humanκ-opioid receptor in complex with JDTic. Nature 485, 327–332.
Yamaleyeva, L.M., Neves, L.A., Coveleskie, K., Diz, D.I., Gallagher, P.E., Brosnihan, K.B.,2013. AT1, AT2, and AT(1-7) receptor expression in the uteroplacental unit of normo-tensive and hypertensive rats during early and late pregnancy. Placenta 34, 497–502.
Yu, Y., Lucitt, M.B., Stubbe, J., Cheng, Y., Friis, U.G., Hansen, P.B., Jensen, B.L.,Smyth, E.M., Fitzgerald, G.A., 2009. Prostaglandin F2α elevates blood pressure and pro-motes atherosclerosis. Proc. Natl. Acad. Sci. U.S.A. 106, 7985–7990.
Zerial, M., McBride, H., 2001. Rab proteins as membrane organizers. Nat. Rev. Mol. CellBiol. 2, 107–117.
Ziegler, N., Batz, J., Zabel, U., Lohse, M.J., Hoffmann, C., 2011. FRET-based sensors forthe human M1-, M3-, and M5-acetylcholine receptors. Bioorg. Med. Chem.19, 1048–1054.
Zimmerman, B., Beautrait, A., Aguila, B., Charles, R., Escher, E., Claing, A., Bouvier, M.,Laporte, S.A., 2012. Differential β-arrestin-dependent conformational signaling andcellular responses revealed by angiotensin analogs. Sci. Signal. 5, ra33.
Z€urn, A., Zabel, U., Vilardaga, J.-P., Schindelin, H., Lohse, M.J., Hoffmann, C., 2009.Fluorescence resonance energy transfer analysis of α2a-adrenergic receptor activationreveals distinct agonist-specific conformational changes. Mol. Pharmacol. 75, 534–541.
171Asymmetric GPCR Signaling Mediated by Metastable Oligomeric Complexes