1 s2.0 S0091679X15002010 Main

ARTICLE IN PRESS
Resonance Energy
Transfer-Based
Approaches to Study
GPCRs
Mohammed Akli Ayoub*, x
*Biologie et Bioinformatique des Syste`mes de Signalisation, Institut National de la Recherche
Agronomique, UMR85, Unite Physiologie de la Reproduction et des Comportements; CNRS,
UMR7247, Nouzilly, France
x
LE STUDIUM Loire Valley Institute for Advanced Studies, Orleans, France
E-mail: maayoub@tours.inra.fr
CHAPTER OUTLINE
Introduction ................................................................................................................ 2
1. RET Approaches ..................................................................................................... 3
2. Ligand Binding ....................................................................................................... 4
3. Ligand-Induced Conformational Changes ................................................................. 5
4. GPCReG Protein Interaction.................................................................................... 7
5. Second Messenger Production .............................................................................. 11
6. b-Arrestin Recruitment ......................................................................................... 12
7. Receptor Internalization ....................................................................................... 15
8. Receptor Oligomerization...................................................................................... 16
9. Summary and Outlook........................................................................................... 23
Acknowledgments ..................................................................................................... 23
References ............................................................................................................... 23
Abstract
Since their discovery, G protein-coupled receptors (GPCRs) constitute one of the most
studied proteins leading to important discoveries and perspectives in terms of their biology
and implication in physiology and pathophysiology. This is mostly linked to the remarkable
advances in the development and application of the biophysical resonance energy transfer
(RET)-based approaches, including bioluminescence and fluorescence resonance energy
transfer (BRET and FRET, respectively). Indeed, BRET and FRET have been extensively
applied to study different aspects of GPCR functioning such as their activation and regulation either statically or dynamically, in real-time and intact cells. Consequently, our view
Methods in Cell Biology, Volume 132, ISSN 0091-679X, http://dx.doi.org/10.1016/bs.mcb.2015.10.008
2016 Elsevier Inc. All rights reserved.
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Resonance Energy Transfer
on GPCRs has considerably changed opening new challenges for the study of GPCRs in
their native tissues in the aim to get more knowledge on how these receptors control the
biological responses. Moreover, the technological aspect of this field of research promises
further developments for robust and reliable new RET-based assays that may be compatible
with high-throughput screening as well as drug discovery programs.
INTRODUCTION
G protein-coupled receptors (GPCRs) constitute one of the largest cell surface receptor families mediating cellular signaling and are thereby involved in many physiological responses and pathophysiological situations. GPCRs constitute the site of
action of a large panel of natural mediators such as hormones and neurotransmitters
(Bockaert & Philippe Pin, 1999; Katritch, Cherezov, & Stevens, 2013; Lagerstrom &
Schioth, 2008). Therefore, fundamental research and pharmaceutical companies
have dedicated a particular interest for GPCRs being the target of 30e50% drugs
used to treat human diseases (Lundstrom, 2006; Schlyer & Horuk, 2006; Williams
& Hill, 2009; Wise, Gearing, & Rees, 2002). At the cellular and molecular levels, the
functioning of GPCRs is essentially orchestrated by their physical interaction with
and activation of the key signaling proteins known as heterotrimeric G proteins
(Bockaert, Homburger, & Rouot, 1987; Bourne, 1997; Gether, 2000; Gilman,
1987; Limbird, 1983; Moreira, 2014; Strange, 2008a). The initial model based on
the ternary complex comprising the ligand, the receptor, and the heterotrimeric G
protein can be considered too simplistic when considering all the recent advances
documenting that GPCRs also activate G protein-independent signaling pathways
such as those mediated by b-arrestins, Src, and proteins with PDZ domains. Moreover, recently new challenging concepts have emerged such as receptor oligomerization, allostery, receptor-G protein preassembly, biased signaling, orphan receptors,
and transactivation with receptor tyrosine kinases (Brady & Limbird, 2002;
Fredholm, Hokfelt, & Milligan, 2007; Garland, 2013; Gomes et al., 2016; Hermans,
2003; Katritch et al., 2013; Rajagopal, Rajagopal, & Lefkowitz, 2010; Strange,
2008b; Wootten, Christopoulos, & Sexton, 2013). These advances have been principally achieved through considerable progresses in developing the technologies used
to study GPCRs, in terms of their structure and complexes as well as their activation
and regulation. Among these methods, resonance energy transfer (RET)-based
approaches (bioluminescence and fluorescence resonance energy transfer (BRET
and FRET)) have significantly contributed to the major advances around GPCRs
leading our view toward this family of receptors being significantly evolved. In
fact, GPCRs constitute the research field of reference where RET-based assays are
elegantly used and are the subject of continual development and improvement in
terms of methodology and instrumentation (Ayoub & Pfleger, 2010; Conn, 2013;
Cottet et al., 2012; Hein, Frank, Hoffmann, Lohse, & Bunemann, 2005; Lohse,
Nuber, & Hoffmann, 2012; Nguyen & Daugherty, 2005; Pfleger & Eidne, 2006;
Pin, Ayoub, Maurel, Perroy, & Trinquet, 2008).
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1. RET approaches
In this chapter, the applications of RET-based technologies to study the different

aspects of GPCR function and regulation are described. This covers the process of
the activation of GPCRs followed by their desensitization and regulation going from
ligand binding, receptor conformation changes, receptor-G protein coupling and receptor oligomerization, to b-arrestin recruitment and receptor internalization and
recycling. The potential applications of RET in GPCR drug discovery are also
briefly discussed.
1. RET APPROACHES
RET approaches link the biophysical concepts of spectral overlap, distance, and proximity, in space and time, between an energy donor and an energy acceptor to the biological question of interest according to Forsters law (Forster, 1948; Pin et al., 2008;
Selvin, 2000). Thus, the changes in the distance between and/or orientation of the
donoreacceptor pair within the individual protein structure (intramolecular) or
proteineprotein/proteinesmall molecule complexes (intermolecular) constitute
the basis of the utilization of RET to study proteins in terms of their association/
dissociation as well as their activation, regulation, and trafficking in the cells (Lohse,
Bunemann, Hoffmann, Vilardaga, & Nikolaev, 2007). This becomes possible thanks
to the development of various resonance energy donors and energy acceptors
compatible with multiple BRET and FRET configurations and assays with improved
spectral properties and increased sensitivities. This includes multiple variants of
green fluorescent proteins (GFPs) and a large array of fluorescent dyes conjugated
to protein fragments, enzyme substrates, chemicals, ligands, or antibodies. These
fluorescent dyes are commonly used together for FRET assays or specifically combined with either the rare earth europium and terbium cryptates or chelates for timeresolved FRET (TR-FRET) and homogeneous time-resolved fluorescence (HTRF)
or various luciferases (Luc) and their substrates for BRET assays. In addition, the
development in the chemistry of RET was accompanied by exponential technical
advances leading to robust instrumentations dedicated to measure RET signals
with high sensitivity and constant reproducibility. Together, theses advances led to
get more insights (quantitatively and qualitatively) into different aspects of molecular and cellular biology, both in spatial and temporal terms, as well as in individual
cells or whole tissues (De, Loening, & Gambhir, 2007; Dragulescu-Andrasi, Chan,
De, Massoud, & Gambhir, 2011; Xu et al., 2007). For GPCRs and their functioning,
the utilization of RET enables the assessment of ligandereceptor, receptoreprotein,
or receptorereceptor complexes in both static and dynamic manners, in real-time
and live cells. These different levels of interactions involving GPCRs are described
below including ligand binding, ligand-induced conformational changes, homo- and
heteromerization, coupling with the heterotrimeric G proteins, modulation of intracellular second messengers, recruitment of b-arrestins, and receptor internalization
and recycling (Lohse et al., 2012; Salahpour et al., 2012). Thus, such advances in
RET-based technologies and their experimental advantages (flexibility, rapidity,
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miniaturization, etc.) open new perspectives in terms of their application in highthroughput screening (HTS) and drug discovery programs for the development of
new GPCR ligands and drugs (Cottet, Faklaris, et al., 2011; Degorce et al., 2009;
Heding, 2004; Milligan, 2004a; Robinson, Yang, & Zhang, 2014; Roda, Guardigli,
Pasini, & Mirasoli, 2003; Zwier et al., 2010).
2. LIGAND BINDING
Since the binding of a ligand on its specific receptor constitutes the first step in the
activation of GPCRs and their downstream signaling, the pharmacological characterization of any GPCR as well as the discovery of potential new ligands involves
the analysis of its ligand-binding properties. Thus, the utilization of highly selective
radioligands targeting GPCRs was the assay of choice for many years. However,
there are growing limitations on using radioactivity in the research laboratories
due to the issues of safety and disposal of radioactive waste issues. Therefore, there
is particular interest in the development of alternative nonradioactive approaches for
ligand binding, and consequently RET-based assays have emerged by labeling the
ligands and/or the receptors with luminescence and fluorescence dyes instead of
incorporation of classical isotopes (Cottet, Faklaris, et al., 2011; Stoddart,
Johnstone, et al., 2015; Stoddart, White, Nguyen, Hill, & Pfleger, 2015; Vernall,
Hill, & Kellam, 2013; Zwier et al., 2010). Indeed, many fluorescent GPCR-selective
ligands compatible with RET-based assays have been synthesized (Stoddart,
Johnstone, et al., 2015; Stoddart, White, et al., 2015; Vernall et al., 2013; Zwier
et al., 2010). Consequently, RET-based assays can be used as a readout of ligand
binding, using a RET compatible donoreacceptor pair between the ligand and its
receptor by assessing either a direct binding of the fluorescently labeled ligand to
the receptor or competition of the binding of the fluorescently labeled ligand by
another unlabeled ligand as illustrated in Figure 1.
Early during the emergence of FRET, the possibility of using FRET to investigate ligand binding of cell surface receptors was reported (Louie & Tromberg,
1998). For GPCRs, one of the pioneer RET-based binding assay works was conducted on the muscarinic M1 receptor (M1R) using GFP-fused receptor and nonfluorescent ligands using FRET quenching (Tahtaoui et al., 2005). In this study, binding
properties (Kd) and kinetic parameters (Kon/Koff) of ligand binding were elegantly
determined for some M1R ligands (Tahtaoui et al., 2005). In addition, a homogeneous ligand-binding assay based on TR-FRET was reported on human complement
5a receptor (C5aR) tagged with hemagglutinin (HA) epitope at the N-terminus (Hu,
Zhou, Hamman, & Liu, 2008). In this study, C5a binding was assessed by measuring
TR-FRET signal between C5a labeled with terbium chelate (energy donor) and
HA-C5aR labeled with anti-HA antibody conjugated with Alexa Fluor-488 (energy
acceptor) (Hu et al., 2008). Later, other RET-based ligand-binding assays using
various FRET configurations have been reported on other GPCRs including
b2-adrenergic (b2AR) (Martikkala et al., 2009), cannabinoid CB2 (Sexton et al.,
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3. Ligand-Induced conformational changes
RET
D
D
RET Signal
(A)
L
Binding of
fluorescent ligand
Ligand Concentration
RET
RET decrease
Competition of binding
of fluorescent ligand
RET Signal
(B)
FIGURE 1 LigandeG protein-coupled receptor interaction assessed by resonance energy

transfer (RET)-based assays.
(A) RET signals are measured between the energy donor (D)-fused receptor (R) and the
energy acceptor (A)-conjugated ligand (L). Saturation binding curves are generated by
increasing the concentration of the ligand allowing the determination of Kd values of the
fluorescent ligand. (B) Competition of the RET signals measured between the donor-fused
receptor (DeR) and the acceptor-conjugated ligand (AeL) by an unlabeled ligand (L0 ). RET
decrease is plotted as function of the increasing doses of L0 allowing the determination of Ki
values of the unlabeled ligand.
2011), vasopressin/oxytocin (Albizu et al., 2010), adenosine A3 (Stoddart et al.,

2012), and dopamine D1/3 (Hounsou et al., 2015) receptors. In most studies, RET
data were very consistent with those obtained using the classical radioligand-binding
method. The first study reporting ligandeGPCR interactions using BRET has just
been reported by Stoddart, Johnstone, et al. (2015) where a new generation of
BRET donor (luciferase), NanoLuc, was fused to the N-terminus of various GPCRs
including b2AR, adenosine A1/3, and angiotensin AT1 receptors. The ligand-binding
properties (Kon/Koff, Kd, and Ki) were determined by measuring the BRET increase
between the NanoLuc-tagged receptor and its selective ligand labeled with redshifted probes for the first time at the cell surface (Stoddart, Johnstone, et al.,
2015; Stoddart, White, et al., 2015).
3. LIGAND-INDUCED CONFORMATIONAL CHANGES

As a consequence of ligand binding, conformational changes of the different domains
within the receptor constitute an important aspect of research on GPCRs. To gain insights into structural and conformational modifications upon GPCR activation
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fluorescence- and luminescence-based techniques have been extensively used. The

first studies were based on fluorescence lifetime analysis on purified receptors using
covalent fluorescence labeling of single cysteine residues localized in different environments of the receptor protein. This revealed the existence of different conformations of the b2AR whether the receptor was bound or not to its agonist (Gether, Lin, &
Kobilka, 1995; Ghanouni, Gryczynski, et al., 2001; Ghanouni, Steenhuis, et al.,
2001; Neumann, Wohland, Whelan, Zare, & Kobilka, 2002). Moreover, the changes
in the intrinsic fluorescence of tryptophan residues or covalently incorporated probes
were also used in many occasions as readout of ligandereceptor interaction
(Damian, Mary, Martin, Pin, & Baneres, 2008; El Moustaine et al., 2012; Rahmeh
et al., 2012). Subsequently, RET-based approaches have been adapted to develop
various conformational sensors of GPCRs expressed in cells taking advantage of
the biophysical properties of these techniques based on the changes in the distance
and/or orientation of the energy donor and energy acceptor moieties (Zhang,
Campbell, Ting, & Tsien, 2002). The RET-based assays introduce both the energy
donor and acceptor partners into the same receptor protein as illustrated in
Figure 2(A) to assess intramolecular changes in RET signals as a consequence of
ligand-promoted conformational changes in the receptor. Of course, GPCRs constitute the model of choice for the application of such approaches due to their structure
of seven transmembrane domains linked by three extracellular and three intracellular
loops and ended by a relatively flexible C-terminal tail. Thus, ligand binding is
thought to affect dramatically such surface and molecular organization leading to
receptor activation and its interaction and coupling with the heterotrimeric G proteins as well as other partners such as GPCR kinases (GRKs) and b-arrestins. The
most important studies concern those reported on the a1-adrenergic (a1AR)
(Vilardaga, Bunemann, Krasel, Castro, & Lohse, 2003; Zurn et al., 2009), b2AR
(Granier, Kim, Fung, Bokoch, & Parnot, 2009), parathyroid hormone (PTHR)
(Castro, Nikolaev, Palm, Lohse, & Vilardaga, 2005; Vilardaga et al., 2003), and
adenosine A1 (Hoffmann et al., 2005) receptors using FRET, as well as the
ODR-10 GPCR in Caenorhabditis elegans studied by BRET upon its activation
by femtomolar levels of volatile ligands (Dacres et al., 2011). The study on PTH
revealed a two-step ligand-binding process with a very fast first phase (milliseconds)
followed by a slow second one (seconds) (Castro et al., 2005; Vilardaga et al., 2003).
Interestingly, the RET-based approaches have also been applied in the context of
GPCR homo- and heterodimers where each energy partner is fused to one protomer
at a specific domain within the dimer (Figure 2(B)). Indeed, in addition to their use
to reveal receptor dimerization (see below), RET was also used to detect conformational changes of GPCR dimers upon ligand binding. This is the case with FRET on
a1AR/m-opioid heterodimers (Vilardaga et al., 2008) and mGluR1 homodimers
(Tateyama, Abe, Nakata, Saito, & Kubo, 2004), as well as HTRF technology applied
to mGluR2 homodimers (Doumazane et al., 2013). Furthermore, BRET has been
used to study ligand-induced conformational changes within melatonin (MT1R/
MT2R) (Ayoub et al., 2002; Ayoub, Levoye, Delagrange, & Jockers, 2004; Journe
et al., 2014), vasopressin V2 (V2R) (Granier et al., 2004), and chemokine
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4. GPCReG protein interaction
R
L
Ligand-Induced
conformational changes
within the monomer
RET Signal
(A)
D
Low RET
Basal RET
D
Dimer
RET decrease
A
L
Ligand-Induced
conformational changes
within the dimer
RET Signal
(B)
RET increase
FIGURE 2 Detection of ligand-induced conformational changes on G protein-coupled receptors

(GPCRs) using resonance energy transfer (RET)-based assays.
(A) In the case of GPCR monomers, both donor and acceptor are fused to the receptor at
different positions (here C-terminus and the third intracellular loop), and changes in the
intramolecular RET signals (here shown as an increase) are measured upon ligand binding.
Sigmoidal doseeresponse curves are generated by increasing the concentrations of the
ligand allowing the determination of EC50 values reflecting the potency of the ligand to
activate and induce conformational changes on the receptor. (B) In the case of GPCR dimers,
the donor and acceptor are fused to two different protomers (here shown at the N-terminus),
and changes in the intermolecular RET signals (here shown as a decrease) are measured
upon ligand binding. Sigmoidal doseeresponse curves are generated by increasing the
concentrations of the ligand allowing the determination of EC50 values reflecting the potency
of the ligand to induce conformational changes within the receptor dimer.
CXCR4 (Percherancier et al., 2005) receptor homo- and heterodimers. In these

studies, RET signals specifically measured from dimer species have been shown
to either increase or decrease upon ligand binding allowing the assessment of
ligand-promoted conformational changes on the dimers. In addition, these assays
have been shown to be useful for the profiling of various agonist and antagonist
ligands acting on heterodimers versus homodimers as shown for MT1R/MT2R heterodimers (Ayoub et al., 2004).
4. GPCReG PROTEIN INTERACTION

The question on how GPCReG protein coupling occurs has been widely studied
over the years. The initial model explaining the functioning of the GPCReG protein
complex has evolved considerably (Bourne, 1997; Limbird, 2004; Moreira, 2014;
Strange, 2008b) and new concepts have emerged such as constitutive activity
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(Seifert & Wenzel-Seifert, 2002), precoupling and/or preassembly (Ayoub et al.,

2007; Ayoub, Trinquet, Pfleger, & Pin, 2010; Gales et al., 2006; Leff & Scaramellini,
1998; Nobles, Benians, & Tinker, 2005; Qin, Dong, Wu, & Lambert, 2011), multiple
coupling (Hamm, 1998; Hermans, 2003; Simon, Strathmann, & Gautam, 1991),
functional selectivity (Kenakin, 2012; Rajagopal et al., 2010; Urban et al., 2007),
and intracellular G protein activation (Irannejad et al., 2013; Koelle, 2006). The
physical interaction and the functional coupling of GPCRs with their cognate heterotrimeric G proteins were initially studied using the GTPgS-binding assay
(Senogles et al., 1987; Stadel, Shorr, Limbird, & Lefkowitz, 1981; Tian, Duzic,
Lanier, & Deth, 1994) and biochemical techniques (Neumann et al., 2002; Smith
& Limbird, 1981), and recently through crystallographic analysis (Kobilka &
Schertler, 2008; Moreira, 2014; Rasmussen et al., 2011). However, the detailed analysis of the activation/deactivation processes of GPCReG protein complexes in realtime and in live cells was very limited for a long time, until the development of RET
techniques (Ayoub, Al-Senaidy, & Pin, 2012; Hein et al., 2005; Lohse et al., 2012;
Pin et al., 2008; Vilardaga et al., 2009). RET-based assays enable assessment of the
intimate changes (increase or decrease) in the proximity between GPCRs and G proteins and/or their conformational modifications upon activation. For this, molecular
fusions with energy donors and acceptors are fused to the receptors (mostly at their
C-terminus) and/or the different G protein subunits (Ga and Gbg). These fusion proteins are then transiently coexpressed, and measurements of RET signals carried
out, in real-time and live cells, before and upon stimulation with the GPCRselective agonist of interest as described in Figure 3 and previously described
(Ayoub, Al-Senaidy, et al., 2012). The first studies were based on FRET measurements between the different Ga and Gbg subunits upon GPCR activation using
different variants of GFPs as FRET donors and acceptors. Indeed, the pioneer
work was reported in Dictyostelium discoideum measuring in real time the dissociation between Ga and Gbg directly in live cells upon chemoattractant application
(Janetopoulos & Devreotes, 2002; Janetopoulos, Jin, & Devreotes, 2001). Then
the following RET studies on the assessment of G protein activation through the
measurements of BRET and FRET signals between the Ga and Gbg subunits
were reported in various mammalian cell lines and yeast coexpressing unmodified
GPCRs (Figure 3(A)) (Ayoub et al., 2009; Ayoub, Landomiel, et al., 2015; Azpiazu
& Gautam, 2004; Bellot et al., 2015; Digby, Lober, Sethi, & Lambert, 2006; Gibson
& Gilman, 2006; Yi, Kitano, & Simon, 2003). As a result, the idea of nondissociation of the Gabg trimer upon GPCR activation was proposed and supported by the
evidence that such activation induces conformational changes of the complexes that
translate into either an increase or decrease in RET signals between G protein subunits (Ayoub, Al-Senaidy, et al., 2012; Azpiazu & Gautam, 2004; Breton, Lagace, &
Bouvier, 2010; Digby et al., 2006; Gales et al., 2006; Gibson & Gilman, 2006;
Yi et al., 2003). The next advances in the application of RET to investigate
GPCReG protein interaction and coupling are characterized by the ability to measure FRET and BRET signals directly between the GPCRs themselves and their
cognate G proteins (Ga and Gbg) as well as the dynamics of their complexes
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4. GPCReG protein interaction
(A)
L
L
G G/
D
Dissociation
of G protein complex
G/
RET decrease
RET
(B)
L
L
G protein
Formation of
R/G protein complex
G protein
D
RET
(C)
L
L
G protein
Low RET
Conformational changes
within R/G protein complex
D
RET increase
FIGURE 3 G protein-coupled receptor (GPCR)eG protein interaction assessed by resonance

energy transfer (RET)-based assays.
(A) The changes in the basal intermolecular RET signals (here shown as a decrease)
measured between the different G protein subunits fused to the donor (Ga-D) and receptor
(Gb/g-A) are assessed as a consequence of their physical dissociation upon ligand-induced
activation of the unmodified GPCR. (B) The increase in the intermolecular RET signals
between the donor-fused receptor (ReD) and the acceptor-fused G protein (either a, b, or
g subunits) (G protein-A) is measured upon ligand binding and GPCR activation. (C) The
changes in the basal RET signals (here shown as an increase) measured between the donorfused receptor (ReD) and the acceptor-fused G protein (G protein-A), either a, b, or g
subunits, are assessed reflecting the ligand-induced conformational changes within the
preassembled GPCReG protein complexes.
(Figure 3(B) and (C)) (Ayoub, Al-Senaidy, et al., 2012; Ayoub et al., 2007, 2010;
Ayoub, Zhang, et al., 2015; Bellot et al., 2015; Gales et al., 2005, 2006; Hasbi
et al., 2007; Hein et al., 2005; Nobles et al., 2005; Qin et al., 2011). Real-time
kinetics of the activation of GPCReG protein complexes were reported in live cells
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10
showing a rapid and transient activation in some cases and slow and stable physical
association in others as previously discussed (Ayoub, Al-Senaidy, et al., 2012; Lohse
et al., 2012). Moreover, constitutive receptoreG protein association was reported in
many situations while agonist-induced interaction was shown for some GPCReG
protein pairs (Figure 3(C)) (Ayoub, Al-Senaidy, et al., 2012; Lohse et al., 2012).
Consequently, the RET-based studies opened an interesting debate on the physical
and functional GPCReG protein interaction and its dynamics analyzed by RETbased approaches challenging the classical model of agonist-inducing GPCReG
protein interaction and coupling (Ayoub, Al-Senaidy, et al., 2012; Bourne, 1997;
Leff, 1995; Strange, 2008b). Indeed, the model of free collision is contrary
to the concepts of precoupling and preassembly (Ayoub, Al-Senaidy, et al.,
2012; Ayoub et al., 2007, 2010; Gales et al., 2005, 2006; Hasbi et al., 2007; Hein
et al., 2005; Nobles et al., 2005; Philip, Sengupta, & Scarlata, 2007; Qin et al.,
2011). In this context, some studies reported an increase in BRET or FRET signals
between the receptors and G proteins exclusively upon agonist stimulation as shown
for a2AR-G1/g2 (Kuravi, Lan, Barik, & Lambert, 2010), a1AR-Gai (Hein et al.,
2005), ghrelin-Gaq (Damian et al., 2015), and protease-activated receptor
(PAR1/2)-Ga12 complexes (Ayoub & Pin, 2013; Ayoub et al., 2010). However, in
other situations significant and specific basal RET signals between the receptors
and G proteins were measured even in the absence of receptor activation as shown
for a1AR (Nobles et al., 2005), adenosine A2 (Nobles et al., 2005), muscarinic M3
(Qin et al., 2011), thrombin PAR1 (Ayoub, Al-Senaidy, et al., 2012; Ayoub et al.,
2007, 2010), trypsin PAR2 (Ayoub & Pin, 2013), a2AR (Breton et al., 2010; Gales
et al., 2005, 2006; Nobles et al., 2005), d-opioid (Audet et al., 2008), CXCR4 and
CXCR7 (Levoye, Balabanian, Baleux, Bachelerie, & Lagane, 2009), ghrelin
(Damian et al., 2015), and bradykinin (Philip et al., 2007) receptors and their
different cognate G proteins. Receptor activation was shown to either increase or
decrease the basal RET signals between the GPCRs and their cognate G proteins.
In some cases, the basal RET signals measured were correlated to the constitutive
activity of the receptors as shown for the preassembly between the ghrelin receptor
and Gaq protein (Damian et al., 2015). However, the basal RET signals measured
from the preassembled GPCReG protein complexes were mostly independent of
any constitutive activity of the receptors or basal activation of the G proteins studied.
For instance, treatments with GPCR-selective antagonists as well as pertussis toxin
(on receptoreGai/o complexes) have been shown to completely abolish the agonistinduced BRET increase between the receptor and Ga subunit, while such treatments
had no effect on the basal BRET signals (Ayoub et al., 2007, 2010; Gales et al.,
2006). Moreover, the agonist-promoted RET changes have been shown to be sensitive to the position where the energy donor and acceptor were inserted into the receptor and/or the G protein (Ayoub et al., 2007, 2010; Gales et al., 2006).
Together, these observations support the notion of agonist-induced conformational
changes within the preassembled GPCReG protein complexes as previously discussed (Ayoub, Al-Senaidy, et al., 2012; Lohse et al., 2012). These observations
are consistent with the old biochemical studies reporting the stable association
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5. Second messenger production
between various GPCRs and G proteins independently of the activation state of the
receptors (Lachance, Ethier, Wolbring, Schnetkamp, & Hebert, 1999; Roka et al.,
1999; Seifert & Wenzel-Seifert, 2002; Senogles et al., 1987). The discordances
raised regarding the preassembly or not between GPCRs and G proteins may depend
on the cellular system and the RET-based assay used. More importantly, the preassembly may constitute a mechanism of regulation of the multiple coupling and
signaling of GPCRs with the occurrence and circumstances of preassembly depending on the Ga protein considered. Indeed, PAR1 has been shown to form preassembled complexes with Gai1/o but not Ga12 (Ayoub et al., 2009, 2010), and ghrelin
receptor has been reported to preassemble with Gaq but not Gai (Damian et al.,
2015), where both assembly was assessed by BRET and FRET techniques. Similarly,
a given G protein may have different association modes with different GPCRs as
nicely demonstrated for Ga12 which interacted with PAR1 and PAR2 in an
agonist-dependent manner while being able to exist in preassembled complexes
with other GPCRs such as vasopressin V1a and muscarinic M3 receptors (Ayoub
& Pin, 2013; Ayoub et al., 2010). Therefore, the population of PAR1/2 preassembled
with Gai1 may not be able to couple with Ga12 and vice versa. Moreover, the desensitization properties may be different depending upon whether the receptor is preassembled or not as shown with PAR1, where the preassembled PAR1-Gai1 complexes
recruit b-arrestins but the induced PAR1-Ga12 complexes did not. In native tissues,
the preassembly or not between GPCRs and their cognate G proteins may be determined by their expression levels and compartmentalization in the cells, as well as
their interactions with other membrane or cytosolic partners such GPCR oligomerization. Conceptually, these RET-based studies challenge the classical dogma of
GPCReG protein coupling and revive the interesting debate regarding the nature
as well as the properties of such molecular interaction and its role in GPCR signaling
and regulation. The concept of preassembly raises intriguing questions regarding
GPCR functioning and illustrates the complexity of GPCReG protein coupling,
signaling, and regulation. Therefore, further investigations on this aspect should
consider other new concepts such as GPCR heterodimerization and their biased
signaling, with the aim to generating new evidence for the reality of such preassembly
and its impact on physiology and pathophysiology involving GPCRs.
5. SECOND MESSENGER PRODUCTION

The development of RET-based assays to investigate GPCR signaling constitutes
another important advance in the field, being used as alternative approaches to the
classical biochemical and radioactive-based assays. Indeed, many BRET and FRET
assays and sensors have been developed to assess the production of intracellular
second messengers, such as cyclic adenosine monophosphate (cAMP), inositol
phosphate (IP), and calcium (Ca2), and to detect the activation of important downstream signaling proteins such as the extracellular signal-regulated kinases (ERK1/2)
(Klarenbeek & Jalink, 2014; Robinson et al., 2014; Salahpour et al., 2012; Xu et al.,
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12
2013; Zhang et al., 2002). The first category of these assays comprises the molecular
RET sensors where the second messenger sensor is genetically double fused to energy donor and acceptor (either BRET or FRET), and transfected in cells, and intramolecular RET changes (increase or decrease) are then assessed in real-time and live
cells as a consequence of the changes in the intracellular concentrations and/or binding of the second messenger on the sensor following GPCR activation (Figure 4(A)).
This has been mostly used for the measurement of cAMP by FRET (DiPilato, Cheng,
& Zhang, 2004; Klarenbeek, Goedhart, van Batenburg, Groenewald, & Jalink, 2015;
Klarenbeek & Jalink, 2014) and BRET (Ayoub, Landomiel, et al., 2015; Barak et al.,
2008; Comps-Agrar, Kniazeff, Brock, Trinquet, & Pin, 2012; Jiang et al., 2007;
Monnier et al., 2011), Ca2 release (Ayoub, Landomiel, et al., 2015; Gorokhovatsky
et al., 2004; Naumann, Kampff, Prober, Schier, & Engert, 2010; Whitaker, 2010),
ERK1/2 phosphorylation (Harvey et al., 2008; Xu et al., 2013), and Rho-GTPase
activation (Nakamura, Aoki, & Matsuda, 2005; Nakamura, Kurokawa, Kiyokawa,
& Matsuda, 2006). The second category of RET assays is mostly based on HTRF
technology, where intermolecular FRET signals are measured between energy donor
and acceptor using various combinations of fluorescently labeled antibodies and/or
purified second messengers compatible with HTRF technology (Figure 4(B))
(Degorce et al., 2009; Gabriel et al., 2003; Norskov-Lauritsen, Thomsen, &
Brauner-Osborne, 2014; Pin et al., 2008). Thus, for cAMP and IP1 assays the
changes in the production of second messengers upon GPCR activation results in
the variation of the amplitude of RET signals between the purified donor- and
acceptor-conjugated antibodies and reagents added into the cell samples. In contrast,
ERK1/2 activation is assessed by an increase of the RET signals between the donorand acceptor-conjugated antibodies recognizing different motifs of the active
ERK1/2 as described in Figure 4(B) (Ayoub et al., 2014). Thus, various HTRF-based
kits have been developed and commercialized by CisBio Bioassays company for the
investigation of GPCR signaling in terms of cAMP (Armstrong, Seeber, Ayoub,
Feldman, & Pfleger, 2013; Ayoub et al., 2009; Ayoub, Landomiel, et al., 2015;
Ayoub et al., 2007; Norskov-Lauritsen et al., 2014), IP (Ayoub, Angelicheva,
et al., 2012; Ayoub et al., 2009; Trinquet, Bouhelal, & Dietz, 2011; Trinquet
et al., 2006), and ERK1/2 signaling pathways (Ayoub et al., 2014; Jia, Quinn, Gagnon, & Talanian, 2006). These assays are now widely used and many other scientific
publications reported their utilization to study GPCR pharmacology and signaling.
The rapidity of the RET-based assays and their miniaturization in 96-, 384-, and
1536-well plates constitute the big asset for their application in both fundamental
research and HTS programs to screen new ligands and pharmacologically profile
GPCRs and their multiple signaling pathways.
6. b-ARRESTIN RECRUITMENT
It is well accepted that b-arrestins play a major role in not only GPCR desensitization and internalization but also their signaling (Carman & Benovic, 1998; DeWire,
ARTICLE IN PRESS
6. b-Arrestin recruitment
(A)
L
L
G protein
Formation and activation

of R/G protein complex
Binding to the sensor
Intracellular production
of second messengers
(cAMP, Ca2+)
Sensor
G protein
Sensor
RET decrease
Basal RET
(B)
L
L
G protein
Formation and activation

of R/G protein complex
G protein
cAMP or IP1
Basal RET
RET
decrease
pERK1/2
Intracellular production
of second messengers
RET
FIGURE 4 G protein-coupled receptor (GPCR)-induced modulation of intracellular second

messengers assessed by resonance energy transfer (RET)-based assays.
(A) In the case of a cytoplasmic sensor for intracellular second messengers (cAMP, Ca2,
etc.), both donor and acceptor are fused the molecular sensor at different positions (mostly
N- and C-terminus), and changes in the intramolecular RET signals (here shown as a
decrease) are measured as a consequence of changes in the cytoplasmic concentrations of
the second messenger upon ligand binding and GPCReG protein activation. (B) For the
assessment of intracellular concentrations of the second messengers (cAMP, IP1, pERK1/2,
etc.), specific antibodies recognizing the different forms of the second messengers and
conjugated to donor and acceptor are used. The changes in the cytoplasmic concentrations
of the second messenger upon GPCR activation is measured either par competition (for
cAMP and IP1) or increase (pERK1/2) of the RET signals between the two donor- and
acceptor-conjugated antibodies.
Ahn, Lefkowitz, & Shenoy, 2007; Lefkowitz & Shenoy, 2005; Luttrell & Lefkowitz,
2002; Reiter & Lefkowitz, 2006). Therefore, the study of the physical association
between GPCRs and b-arrestins constitutes an important field of research and investigation both in vitro and in vivo. One of the important features of the physical
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14
interaction between GPCRs and b-arrestins is of course its temporal and spatial
dynamic, since the recruitment of the cytosolic pools of b-arrestins to the plasma
membrane receptors is thought to be initiated by receptor activation and mostly
mediated through receptor phosphorylation by specific GRKs at the receptor
C-terminus (Gurevich & Gurevich, 2004; Reiter & Lefkowitz, 2006; Shenoy &
Lefkowitz, 2011). The first fluorescence-based assays to investigate GPCRebarrestin association were based on confocal microscopy using GFP-fused b-arrestins
allowing the assessment of their membrane translocation upon GPCR activation
(Barak, Ferguson, Zhang, & Caron, 1997; Evans et al., 2001; Ferguson & Caron,
2004; Heding et al., 2000; Holliday, Holst, Rodionova, Schwartz, & Cox, 2007;
Laporte, Oakley, Holt, Barak, & Caron, 2000; Lin & Trejo, 2013; Shenoy &
Lefkowitz, 2003). However, the major advances in BRET and FRET technologies
to study GPCRs also covered b-arrestins as key components in GPCR signaling
and regulation (Lohse et al., 2012). Thus, various RET-based assays have been
developed to monitor GPCR-promoted b-arrestin recruitment in real-time and live
cells using either BRET (Heding, 2004; Kaczor et al., 2014; Kamal et al., 2009;
Kocan, Dalrymple, Seeber, Feldman, & Pfleger, 2010; Kocan & Pfleger, 2009;
Pfleger, Dalrymple, Dromey, & Eidne, 2007; Vrecl, Jorgensen, Pogacnik, & Heding,
2004), HTRF (Ayoub et al., 2010), or FRET (Krasel, Bunemann, Lorenz, & Lohse,
2005; Violin, Ren, & Lefkowitz, 2006; Wehbi et al., 2013) technologies. The most
used approaches consist of the fusion of both the receptor and b-arrestin to energy
donor and acceptor, and the increase of intermolecular RET signals reflecting
b-arrestin recruitment is measured in real-time and live cells upon receptor activation (Figure 5(A)). These different assays were used in different contexts to assess
agonist-induced b-arrestin recruitment mediated by many GPCRs including neuropeptide Y (Berglund, Schober, Statnick, McDonald, & Gehlert, 2003), PAR1/2
(Ayoub et al., 2009; Ayoub et al., 2007; Ayoub & Pin, 2013; Ayoub et al., 2010),
follicle-stimulating hormone (FSHR) and luteinizing hormone (LHR) (Ayoub,
Landomiel, et al., 2015), vasopressin type 2 (V2R) (Armstrong et al., 2013; Bertrand
et al., 2002; Kocan et al., 2009; Tenenbaum et al., 2009), b2AR (Krasel et al., 2005;
Vrecl et al., 2004), neurokinin type 1 (NK1R) (Vrecl et al., 2004), chemokine
(CCR5, CXCR4, and CXCR2) (See, Seeber, Kocan, Eidne, & Pfleger, 2011),
CCR2 (Ayoub, Zhang, et al., 2015), angiotensin II type 1 (AT1R) (Ayoub, Zhang,
et al., 2015; Porrello et al., 2011), orexin (OX1/2R) (Dalrymple, Jaeger, Eidne, &
Pfleger, 2011; Jaeger, Seeber, Eidne, & Pfleger, 2014), bradykinin (See et al.,
2011), melatonin (MT1) (Levoye, Dam, Ayoub, Guillaume, Couturier, et al.,
2006), glucagon-like-1 (GLP-1R) (Jorgensen, Martini, Schwartz, & Elling, 2005),
and PTHR (Wehbi et al., 2013) receptors. Alternatively, another RET-based
approach to assess b-arrestin recruitment has been used which measures intramolecular BRET changes (increase or decrease) within a b-arrestin double brilliance,
where both energy donor and acceptor are genetically fused to b-arrestin (Charest,
Terrillon, & Bouvier, 2005). In this configuration, the b-arrestin sensor is coexpressed with the unmodified GPCR of interest, and agonist-induced b-arrestin
recruitment is then assessed as changes in the conformation of b-arrestin following
ARTICLE IN PRESS
7. Receptor internalization
(A)
L
L
Formation
of R/-arrestin complex
-arrestin
D
RET
-arrestin
(B)
L
L
Formation
of R/-arrestin complex
-arrestin
-arrestin
RET decrease
within R/-arrestin complex
Basal RET
FIGURE 5 G protein-coupled receptor (GPCR)earrestin interaction assessed by resonance
energy transfer (RET)-based assays.
(A) The increase in the intermolecular RET signals between the donor-fused receptor (ReD)
and acceptor-fused b-arrestins (b-arrestin-A) is measured upon ligand binding and GPCR
activation. (B) In the case of a cytoplasmic b-arrestin sensor (double brilliance), both donor
and acceptor are fused the molecular sensor at different positions (N- and C-terminus), and
the changes in the intramolecular RET signals (here shown as a decrease) are measured
reflecting the plasma membrane translocation of b-arrestin sensor upon GPCR activation.
its association with the activated receptor (Figure 5(B)). This assay has been reported for the association of b-arrestin with various GPCRs such as V2R, d-opioid,
CCR5, and AT1R (Charest et al., 2005; Shukla et al., 2008).
7. RECEPTOR INTERNALIZATION
Following their activation, GPCRs are known to undergo desensitization followed
by the internalization process in clathrin-coated pits as a consequence of receptor
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16
phosphorylation by specific GRKs and their association with b-arrestins (Carman &
Benovic, 1998; DeWire et al., 2007; Lefkowitz & Shenoy, 2005; Luttrell &
Lefkowitz, 2002; Reiter & Lefkowitz, 2006). Like for b-arrestin recruitment the classical approaches to assess such GPCR trafficking are based on confocal microcopy
and cell surface ELISA using fluorescently fused or labeled receptors. More recently,
RET-based assays have been proposed as alternative approaches to monitor
GPCR internalization in real-time and live cells and miniaturized formats (96- and
384-well plates) compatible with HTS using astute adaptation of both BRET and
TR-FRET techniques. BRET-based assays consist of the assessment of the agonistpromoted changes in the basal BRET signal specifically measured at the intracellular
face of the plasma membrane between the C-terminally energy donor-fused receptor
(GPCR-Luc) and an energy acceptor-fused membrane marker (GFP-KRas protein)
(Figure 6(A)). In this configuration, the vicinity between the two partners is significantly decreased upon agonist activation due to the reduction in receptors at the
plasma membrane, which is then measured as a decrease in the basal BRET signal
between GPCR-Luc and GFP-KRas as described in Figure 6(A). This assay has
been successfully applied to investigate the internalization of many GPCRs
including b2AR (Lan, Kuravi, & Lambert, 2011), OX2R (Jaeger, Seeber, et al.,
2014), bile acid receptor (TGR5R) (Jensen et al., 2013), and FSHR and LHR
(Ayoub, Landomiel, et al., 2015). The second RET-based assay for GPCR internalization uses TR-FRET technology where the receptor of interest is N-terminally
fused to a relatively small fluorescent protein and labeled with specific dyes compatible with the extracellular TR-FRET process with free-energy acceptor molecules
added to the cells (Figure 6(B)). This assay has been elegantly validated on
OX1R and cannabinoid CB1 (Ward, Pediani, & Milligan, 2011), vasopressin V1a
(Faklaris et al., 2015) as well as GLP-1R (Roed et al., 2015, 2014) receptors.
8. RECEPTOR OLIGOMERIZATION
Since the discovery of GPCRs, their oligomerization constitutes undoubtedly one of
the major concepts that has profoundly impacted our view on this family of membrane proteins. Indeed, early literature in 1980s and 1990s proposed the concept
of receptorereceptor interaction as a mechanism of integration of GPCR signaling
in the central nervous system (Agnati, Ferre, Lluis, Franco, & Fuxe, 2003; Fuxe &
Agnati, 1985; Fuxe, Marcellino, Guidolin, Woods, & Agnati, 2008). From the
2000s, a particular interest has been given to the study of the oligomerization of
GPCRs and its impact on their functioning. Consequently, over the years interesting
findings have been obtained regarding the role of such organization in the maturation, activation, G protein coupling, downstream signaling, and regulation of GPCRs
(Angers, Salahpour, & Bouvier, 2002; Bai, 2004; Bouvier, 2001; Bulenger, Marullo,
& Bouvier, 2005; Devi, 2001; Ferre et al., 2009; Gomes et al., 2016; Jordan &
Devi, 1999; Milligan, 2004b; Prinster, Hague, & Hall, 2005; Szidonya, Cserzo, &
Hunyady, 2008; Terrillon & Bouvier, 2004b; Vischer, Castro, & Pin, 2015).
ARTICLE IN PRESS
8. Receptor oligomerization
(A)
L
Protein X
Ligand - induced
receptor internalization
Protein X
A
Endosome
Basal RET
RET decrease
(B)
Basal RET
A
D
Free RET acceptor
A
A
Ligand - induced
receptor internalization
RET decrease
Endosome
FIGURE 6 G protein-coupled receptor (GPCR) internalization assessed by resonance energy

(A) The decrease in the intermolecular basal RET signals between the donor-fused receptor
(ReD) and a plasma membrane protein marker (Protein X) fused to the acceptor (Protein X-A) is
measured as a consequence of GPCR internalization upon its activation. (B) The decrease in the
intermolecular basal RET signals between the donor-fused receptor (ReD) and free acceptor
(A) fluorescent molecules is measured as readout of GPCR internalization upon activation.
The major advances in the field concern the heterodimerization between the different
subfamilies of GPCRs including the orphan receptors and its impact on the functioning of GPCRs, as well as its implication in diverse physiological and pathophysiological models (Devi, 2001; Ferre et al., 2009; Gomes et al., 2016; Gomes, Gupta,
& Devi, 2013; Jordan & Devi, 1999; Levoye, Dam, Ayoub, Guillaume, & Jockers,
2006; Prinster et al., 2005; Vischer et al., 2015). Therefore, heterodimerization would
constitute one of the mechanisms of diversifying and finely regulating GPCR
signaling as multiple signaling pathways are possible through a limited number of
receptors expressed within a given organism. Importantly, the reality of oligomers
in native tissues has been elegantly demonstrated for many GPCRs in different
models (Albizu et al., 2010; Baba et al., 2013; Kaupmann et al., 1998; Rivero-Muller
et al., 2010; Vischer et al., 2015; Waldhoer et al., 2005).
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18
From the methodological point of view, in addition to the classical pharmacological and biochemical techniques, RET-based approaches have contributed considerably to the emergence of GPCR oligomerization and its investigation directly in live
cells. Indeed, different BRET and FRET assays have been developed and used over
the years, including their simple and combined application using fluorescent and
luminescent proteins, fluorescently conjugated antibodies as well as their combination with fluorescent ligands, and complementation-based approaches (Angers,
Salahpour, & Bouvier, 2001; Angers et al., 2000; Ayoub & Pfleger, 2010; Carriba
et al., 2008; Cottet, Albizu, et al., 2011; Cottet et al., 2012; Doumazane et al.,
2011; Faklaris et al., 2015; Gandia et al., 2008; Gomes et al., 2016; Hamdan,
Percherancier, Breton, & Bouvier, 2006; James, Oliveira, Carmo, Iaboni, & Davis,
2006; Johnstone & Pfleger, 2012; Kaczor & Selent, 2011; Kent, McAlpine, Sabetnia,
& Presland, 2007; Overton & Blumer, 2002; Pfleger, Seeber, & Eidne, 2006; Pin
et al., 2008; Robitaille, Heroux, Baragli, & Hebert, 2009). The most used configurations of RET-based assay consist of the fusion of the two protomers of the
GPCR dimer (homo- or heterodimer) with the energy donor and acceptor at either
their C-terminus (Figure 7(A)) or N-terminus (Figure 7(B)) and the measurement
of BRET and FRET signals directly in intact cells. Genetic fusion of the fluorescent
and luminescent proteins to the receptors of interest is required in most of the cases,
although sometimes alternative covalent labeling is used (TR-FRET/Tag-Lite). In
addition, fluorescently conjugated antibodies compatible with TR-FRET and recognizing the small tags (Flag, HA, myc) were also used to investigate GPCR oligomerization (Maurel et al., 2008, 2004). These approaches were applied in either the
absence or presence of GPCR activation and the appropriate controls (Ayoub &
Pfleger, 2010), and in most cases receptor activation had no effect on the dimer-specific RET signals, supporting the notion of constitutive and agonist-independent
GPCR oligomerization (Angers et al., 2002; Bai, 2004; Bouvier, 2001; Bulenger
et al., 2005; Devi, 2001; Gomes et al., 2016; Gurevich & Gurevich, 2008; Jordan
& Devi, 1999; Milligan, 2004b; Prinster et al., 2005; Rovira, Pin, & Giraldo,
2010; Szidonya et al., 2008; Terrillon & Bouvier, 2004b). Thus, the ligand-induced
BRET changes (increase) observed with some GPCR homo- and heterodimers have
been interpreted to reflect conformational changes within the constitutive dimers
rather than changes in its dimerization state (Figure 7(C)) as shown for MT1R/
MT2R (Ayoub et al., 2002, 2004) and CXCR4 (Percherancier et al., 2005). These
conclusions are somehow consistent with the extracellular TR-FRET changes
(decrease) using a sensor for glutamate binding on the well-established constitutive
mGluR2 dimers (Doumazane et al., 2013). However, the FRET decrease measured
on the somatostatin receptor (SSTR2) homodimer (Grant, Collier, & Kumar, 2004)
and BRET increase within the heterodimer formed by m-opioid and cholecystokinin
(CCK2) receptors (Zheng et al., 2009) were associated with the dissociation and association of the complexes, respectively. However, recent studies have challenged
some of the applications of the BRET technique and the different controls used to
reveal the oligomerization of GPCRs (Bouvier, Heveker, Jockers, Marullo, &
Milligan, 2007; James et al., 2006; Lan et al., 2015; Szalai et al., 2014).
ARTICLE IN PRESS
(A)
R2
R1
Dimerization
R1/R2
RET
RET
(B)
R1
(C)
Dimerization
R1/R2
R1/R2 dimer
R1/R2 dimer
R2
R1
R2
Basal RET
L1
L2
Binding of L1 and L2
within R1/R2 dimer
L2
L1
RET increase
FIGURE 7 G protein-coupled receptor (GPCR) oligomerization assessed by resonance energy

To investigate the homo- and heterodimerization of GPCRs, the two protomers (R1 and R2)
are fused with the donor (R1-D) and acceptor (R2-A) at either C-terminus (A) or N-terminus
(B) of R1 and R2, and the changes in the intermolecular RET signals are measured as
consequence of their physical interaction/proximity. (C) The effect of receptor activation on
GPCR dimerization is also examined by assessing the changes in the basal intermolecular
RET signals (here shown as an increase) measured within the constitutive R1/R2 dimers
reflecting the conformational changes induced by ligand binding.
Consequently, the homodimerization of some GPCRs such as b2AR has been questioned (James et al., 2006; Lan et al., 2015). Furthermore, the development of recent
fluorescence and luminescence approaches based on protein fragment complementation led to their combination with the classical BRET and FRET technologies. The
complementation of two protein moieties of luciferase and/or variants of GFP genetically fused to the receptors of interest allows specific BRET or FRET signals to be
measured as a consequence of receptor homo- or heterodimerization (Ciruela,
Vilardaga, & Fernandez-Duenas, 2010; Hebert, Gales, & Rebois, 2006; Molinari,
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ARTICLE IN PRESS
20
Casella, & Costa, 2008; Rebois et al., 2008; Robitaille et al., 2009). The RET signals
can be measured through the complementation of the energy donor and/or acceptor
within the different protomers forming the oligomers using BRET, FRET, or both
combined (Faklaris et al., 2015). This has been used to provide evidence for the
following GPCR interactions: CB1R/dopamine D2R/adenosine A2AR (Carriba
et al., 2008), AT1R/AT2R (Porrello et al., 2011), mGluR5/dopamine D2R/adenosine
A2aR (Cabello et al., 2009), and different homo- and heteromers of mGluRs
(Doumazane et al., 2011). In addition, the heterodimerization between two or
more GPCRs has also been investigated by RET-based approaches in a liganddependent manner as described in Figure 8. The first configuration measured RET
signals between the complemented dimer protomers and their related interacting
protein (i.e., G proteins, b-arrestins) upon receptor activation and this has been reported on AT1R/a2cAR (Bellot et al., 2015), CCR2/CXCR4 (Armando et al.,
2014), dopamine D1R/D2R (Urizar et al., 2011), a1AR/m-opioid (Vilardaga et al.,
2008), and adenosine A2aR/dopamine D2R (Bonaventura et al., 2015) heterodimers
as well as the adenosine A2aR homodimer (Gandia et al., 2008) (Figure 8(A)). The
second assay is based on the GPCR-heteromer identification technology (GPCRHIT) recently developed to detect GPCR heterodimers in a ligand-dependent format
(Ayoub & Pfleger, 2010; Johnstone & Pfleger, 2012, 2015; See et al., 2011). This
assay can be applied using an intracellular interacting molecule or a fluorescently
labeled ligand (Jaeger, Armstrong, Hill, & Pfleger, 2014). GPCR-HIT using an intracellular interacting molecule was successfully applied to reveal CCR2/CCR5 (See
et al., 2011), CCR2/CXCR4 (See et al., 2011; Armando et al., 2014), AT1R/
AT2R (Porrello et al., 2011), a1AR/CXCR2 (Mustafa et al., 2012), GLP1R/GIPR
(Schelshorn et al., 2012), CXCR3/CXCR4 (Watts et al., 2013), and AT1R/CCR2
(Ayoub, Zhang, et al., 2015) heteromers (Figure 8(B)). Importantly, these two
RET configurations not only revealed new GPCR heterodimers but also shed
more light into their mechanisms of activation and/or regulation in terms of G protein coupling and b-arrestin recruitment (Armando et al., 2014; Ayoub, Zhang, et al.,
2015; Bellot et al., 2015; Mustafa et al., 2012; Terrillon & Bouvier, 2004a; Urizar
et al., 2011).
The recent advances in TR-FRET and Tag-Lite technologies using fluorescent
ligands allowed labeling the extracellular domains of GPCRs without disturbing their
function as well as the detection of homo- and heterodimers at the cell surface
(Albizu et al., 2010; Cottet, Albizu, et al., 2011; Cottet et al., 2013, 2012;
Doumazane et al., 2013; Faklaris et al., 2015; Hounsou et al., 2015). The TR-FRET
approach with the fluorescent ligands is mostly based on two different ligand-binding
assays as described in Figure 9(A) and (B). The combination of fluorescent agonists/
antagonists selective for the two GPCRs of interest is used to measure RET signal as
consequence of ligand binding on the heterodimers. This approach has been used in a
seminal work by Albizu et al. (2010) revealing for the first time the existence of
GPCR dimers in native tissues as shown for vasopressin V1a and oxytocin receptors
expressed in the mammary glands. In addition, the utilization of fluorescent ligands to
track the dimers brought interesting conclusions regarding the binding properties of
ARTICLE IN PRESS
(A)
R2
R1
Dimerization
R1/R2
L2
A2
A1
L2-induced
R1/R2/protein X complex
Protein X
R2
Protein X
RET
(B)
R1
L2
Dimerization
R1/R2
L2
L2
L2-induced
D
Protein X
Protein X
A
RET
FIGURE 8 G protein-coupled receptor (GPCR) oligomerization assessed by ligand-dependent

resonance energy transfer (RET)-based assays.
(A) In the complementation assay, the two protomers (R1 and R2) are fused with the two
moieties (R1-A1 and R2-A2) of the acceptor at their C-terminus, and an interacting protein
(i.e., G proteins, b-arrestins, etc.) is fused with the energy donor (Protein X-D). The
increase in the intermolecular RET signals between the complemented R1/R2 dimer and
protein X is measured as a consequence of R1-A1/R2-A2 interaction and the recruitment
of Protein X-D to the activated dimer upon the binding of R2-selective ligand (L2). (B)
In GPCR-heteromer identification technology configuration, only one protomer is fused with
the donor at its C-terminus (R1-D), and the interacting protein X is fused with the acceptor
(Protein X-A). The increase in the intermolecular RET signals between R1-D and protein
X-A is measured upon the activation of unmodified R2 by its selective ligand (L2)
demonstrating physical interaction between R1 and R2 and the recruitment of Protein X to
the activated R1/R2 dimer.
dimers as well as ligandedimer stoichiometry when agonists and antagonists were

used (Albizu et al., 2010). Alternatively, the ligand-binding form of the GPCRHIT assay can be utilized (Jaeger, Armstrong, et al., 2014), by the binding of donor
or acceptor fluorescent ligands on one GPCR protomer combined with the labeling
(covalent or not) of the energy donor or acceptor fused to the N-terminus of the other
protomer within the GPCR dimer as described in Figure 9(C). This is exemplified by
the study of the D1/D3 heteromer (Hounsou et al., 2015). The combination of
different Tag-Lite labeling based on SNAP-tag, CLIP-tag, and HALO-tag with the
fluorescent ligands with various spectral properties implies more possibilities for
multiplexing TR-FRET assays and simultaneous detection of the heterodimerization
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ARTICLE IN PRESS
22
(A)
D
L1
L2
R2
R1
RET
Dimerization
R1/R2
RET
(B)
R1
R2
L2
Dimerization
R1/R2
Binding of L2
(C)
D
TR-FRET
L1
R2
R1
L2
Dimerization
R1/R2
Protein X
L1/L2-induced
Protein X
D A
BRET
FIGURE 9 Detection of G protein-coupled receptor oligomerization at the cell surface using

resonance energy transfer (RET)-based assays with fluorescent ligands.
(A) In the case of RET between two fluorescent ligands, the two protomers (R1 and R2) are
unmodified and labeled with their selective fluorescently conjugated ligands (D-L1 for R1 and
A-L2 for R2). The increase in the intermolecular RET signals measured between the two
bound ligands is assessed as a consequence of R1/R2 interaction at the cell surface.
(B) Alternatively, RET can be measured between one protomer N-terminally fused to the
donor (D-R1) and protomer R2-selective ligand conjugated with the acceptor (A-L2). The
increase in the intermolecular RET signals measured between D-R1 and the A-L2 bound on
R2 is assessed to reveal R1/R2 interaction at the cell surface. (C) The RET configuration in
(A) can be combined with that in Figure 8(B) where the cell surface specific time-resolved
fluorescence resonance energy transfer (TR-FRET) signals measured on R1/R2 dimers upon
the binding of their selective fluorescent ligands (D-L1 and A-L2) are correlated with the
intracellular bioluminescence resonance energy transfer (BRET) signals measured between
the R1-D0 protomer and protein-A0 as a consequence of the recruitment of the latter to the
R1/R2 dimer.
ARTICLE IN PRESS
References
between various GPCRs coexpressed in the same cell (Doumazane et al., 2011;
Faklaris et al., 2015). Interestingly, the utilization of fluorescent ligands to measure
the extracellular TR-FRET signals on GPCR heterodimers opens a possibility for
its combination with BRET measurements at the cytoplasmic side. Consequently,
conformational changes or association of the heterodimers with their signaling or
regulatory proteins (i.e., G proteins, b-arrestins) can be simultaneously measured
upon the binding of the fluorescent ligands (Figure 9(C)). Such multiplexed
TR-FRET/BRET assays would allow the correlation of the heterodimerization
between two GPCRs with its functional consequences in terms of activation, desensitization, and internalization of the heterodimers.
9. SUMMARY AND OUTLOOK

In summary, RET-based technologies have substantially contributed to the recent advances in GPCRs. Their applications have been evolved from the detection of receptorereceptor interactions (oligomerization) to dissecting, in real-time and live cells,
the molecular mechanisms of activation/deactivation, functional coupling, intracellular signaling, and trafficking of complexes involving GPCRs. Since the development of RET-based approaches, the major advances in their application to GPCRs
concern the discovery of various functional and physical interactions between
GPCRs with specific pharmacological and/or signaling properties as well as implication in physiology and pathophysiology. More importantly, the new generation of
RET-based assays using TR-FRET with fluorescent ligands has been shown to be
powerful for the detection of GPCR oligomers in their native contexts. Also, the
development of fluorescent ligands compatible with BRET and TR-FRET allows
the assessment of ligand-binding properties opening a new era for the development
of robust ligand-binding assays compatible with HTS programs. Furthermore, the
RET-based technologies enable henceforth the assessment of the pharmacology of
GPCRs as well as their function in quantitative and qualitative ways by considering
the recent concepts such as heterodimerization and biased signaling.
ACKNOWLEDGMENTS
I would like to thank LE STUDIUM Loire Valley Institute for Advanced Studies, AgreenSkills Plus, and ARD 2020 Biomedicament program for their support. Special thanks to
A/Prof. Kevin Pfleger for his comments on and review of this chapter.
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ARTICLE IN PRESS

Resonance Energy Transfer

In this chapter, the applications of RET-based technologies to study the different

Resonance Energy Transfer

3. Ligand-Induced conformational changes

FIGURE 1 LigandeG protein-coupled receptor interaction assessed by resonance energy

2011), vasopressin/oxytocin (Albizu et al., 2010), adenosine A3 (Stoddart et al.,

3. LIGAND-INDUCED CONFORMATIONAL CHANGES

Resonance Energy Transfer

fluorescence- and luminescence-based techniques have been extensively used. The

4. GPCReG protein interaction

FIGURE 2 Detection of ligand-induced conformational changes on G protein-coupled receptors

CXCR4 (Percherancier et al., 2005) receptor homo- and heterodimers. In these

4. GPCReG PROTEIN INTERACTION

Resonance Energy Transfer

(Seifert & Wenzel-Seifert, 2002), precoupling and/or preassembly (Ayoub et al.,

4. GPCReG protein interaction

FIGURE 3 G protein-coupled receptor (GPCR)eG protein interaction assessed by resonance

Resonance Energy Transfer

5. Second messenger production

5. SECOND MESSENGER PRODUCTION

Resonance Energy Transfer

Formation and activation

Binding to the sensor

Formation and activation

FIGURE 4 G protein-coupled receptor (GPCR)-induced modulation of intracellular second

Resonance Energy Transfer

Resonance Energy Transfer

Free RET acceptor

FIGURE 6 G protein-coupled receptor (GPCR) internalization assessed by resonance energy

Resonance Energy Transfer

FIGURE 7 G protein-coupled receptor (GPCR) oligomerization assessed by resonance energy

Resonance Energy Transfer

FIGURE 8 G protein-coupled receptor (GPCR) oligomerization assessed by ligand-dependent

dimers as well as ligandedimer stoichiometry when agonists and antagonists were

Resonance Energy Transfer

FIGURE 9 Detection of G protein-coupled receptor oligomerization at the cell surface using

9. SUMMARY AND OUTLOOK

Resonance Energy Transfer

protein complex measured by bioluminescence resonance energy transfer in living cells.

Resonance Energy Transfer

in native tissues: application to GPCR oligomerization. Methods in Molecular Biology,

Resonance Energy Transfer

metabotropic glutamate receptors. FASEB Journal: Official Publication of the Federation

Resonance Energy Transfer

Resonance Energy Transfer

Limbird, L. E. (1983). Beta-adrenergic stimulation of adenylate cyclase and alpha-adrenergic

Resonance Energy Transfer

spectroscopy. Proceedings of the National Academy of Sciences of the United States of

Resonance Energy Transfer

Seifert, R., & Wenzel-Seifert, K. (2002). Constitutive activity of G-protein-coupled receptors:

Resonance Energy Transfer

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