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Ghrelin receptor conformational dynamics regulatethe transition from a preassembled to an activereceptor:Gq complexMarjorie Damiana, Sophie Marya, Mathieu Maingota, Cline MKadmia, Didier Gagnea, Jean-Philippe Leyrisa,1,Sverine Denoyellea, Grald Gaibeletb, Laurent Gavaraa, Mauricio Garcia de Souza Costac, David Perahiac,Eric Trinquetd, Bernard Mouillacb, Sgolne Galandrine, Cline Galse, Jean-Alain Fehrentza, Nicolas Floqueta,Jean Martineza, Jacky Mariea, and Jean-Louis Banresa,2
aCNRS UMR 5247 and Facult de Pharmacie, Institut des Biomolcules Max Mousseron, Universit Montpellier 1 et 2, 34093 Montpellier Cedex 05, France;bCNRS UMR 5203, INSERM U661, and Institut de Gnomique Fonctionnelle, Universit Montpellier 1 et 2, 34094 Montpellier Cedex 05, France; cCNRS UMR8113 and Laboratoire de Biologie et de Pharmacologie Applique, Ecole Normale Suprieure de Cachan, F-94235 Cachan, France; dCisbio Bioassays, 30200Codolet, France; and eUMR 1048, Institut des Maladies Mtaboliques et Cardiovasculaires, 31432 Toulouse Cedex 4, France
Edited by Robert J. Lefkowitz, Howard Hughes Medical Institute, Duke University Medical Center, Durham, NC, and approved December 8, 2014 (received forreview August 1, 2014)
How G protein-coupled receptor conformational dynamics controlG protein coupling to trigger signaling is a key but still open ques-tion. We addressed this question with a model system composedof the purified ghrelin receptor assembled into lipid discs. Combin-ing receptor labeling through genetic incorporation of unnaturalamino acids, lanthanide resonance energy transfer, and normalmode analyses, we directly demonstrate the occurrence of twodistinct receptor:Gq assemblies with different geometries whoserelative populations parallel the activation state of the receptor.The first of these assemblies is a preassembled complex with thereceptor in its basal conformation. This complex is specific of Gqand is not observed with Gi. The second one is an active assemblyin which the receptor in its active conformation triggers G proteinactivation. The active complex is present even in the absence ofagonist, in a direct relationship with the high constitutive activityof the ghrelin receptor. These data provide direct evidence of amechanism for ghrelin receptor-mediated Gq signaling in whichtransition of the receptor from an inactive to an active conforma-tion is accompanied by a rearrangement of a preassembled recep-tor:G protein complex, ultimately leading to G protein activationand signaling.
GPCR | G protein | preassembly | conformation dynamics | signaling
Gprotein-coupled receptors (GPCRs), one of the largest cellsurface receptor families, are involved in many cellular sig-naling processes (1). Based on this property, as well as their impor-tance as drug targets, the molecular aspects of GPCR functioninghave been extensively investigated. In particular, coupling toheterotrimeric G proteins has been the focus of numerous stud-ies. Indeed, delineating the molecular mechanisms responsiblefor receptor:G protein interaction is absolutely required to betterunderstand how signaling is controlled. Recent years have seenspectacular advances that have culminated in elucidation of the3D structure of the 2-adrenergic receptor:Gs complex (2).Nevertheless, the need for further progress remains, in particularto fully understand the dynamics of this interaction. This is acrucial question, given that how the receptor interacts with its Gprotein partner governs signaling, and thus biological and path-ophysiological responses.To date, two different models for GPCR:G protein interaction
have been proposed: collision coupling and preassembly. Origi-nally, it was proposed that receptors and G proteins couple bycollision (3, 4). One of the main features of this model is thatonly activated receptors interact with G proteins. Since then,alternative models of signaling have been developed. One ofthese, the preassembly model, proposes that the receptor and theG protein make a complex even in the absence of agonist (58).
Discriminating between the two models is crucial. Indeed, sig-naling outputs, such as the kinetics of G protein activation, willbe significantly different depending on whether the ligand-freereceptor is always in complex with its G protein or must first beactivated by the agonist to recruit the G protein and triggersignaling. Moreover, it has been shown that GPCR conforma-tional dynamics (911) and signaling in the absence of ligand arekey features of GPCR functioning (12). How receptor constitu-tive activity and conformational dynamics relate to their couplingto the G protein remains an open question.Here we used the purified ghrelin receptor GHS-R1a to an-
alyze the way in which this GPCR interacts with its G proteinpartners. Ghrelin is a neuroendocrine peptide hormone that actsthrough its cognate GPCR to control important biological pro-cesses, such as growth hormone secretion, food intake, and re-ward-seeking behaviors (13). Among the GPCRs, GHS-R1a hasbeen shown to have one of the highest basal Gq activation levelsboth in vitro (10, 14) and in vivo (15, 16). The physiologicalrelevance of GHS-R1a basal activity is substantiated by the occur-rence of a natural human mutation in the GHS-R1a gene (A204Esubstitution in the second extracellular loop of the receptor)
G protein-coupled receptors (GPCRs), one of the largest cellsurface receptor families, transmit their signals through thecoupling of intracellular partners, such as the G proteins.Knowing how this coupling occurs is essential, because it gov-erns the entire signaling process. To address this open question,we used a purified GPCR as a model towhich we applied variousstate-of-the-art biochemical and biophysical approaches. Bydoing so, we provide direct experimental evidence of a signalingmechanism in which receptor conformational changes are di-rectly linked to a rearrangement of a preassembled complexbetween the receptor and its cognate Gq protein. This shedslight on the way in which a GPCR interacts with G proteins totrigger signaling.
Author contributions: S.M., N.F., J. Marie, and J.-L.B. designed research; M.D., C.M., D.G.,J.-P.L., G.G., M.G.d.S.C., B.M., S.G., C.G., and N.F. performed research; M.M., S.D., L.G., E.T.,and J.-A.F. contributed new reagents/analytic tools; M.D., S.M., C.M., D.G., G.G., M.G.d.S.C.,D.P., B.M., C.G., J.-A.F., N.F., J. Martinez, J. Marie, and J.-L.B. analyzed data; and B.M., J.-A.F.,N.F., J. Martinez, J. Marie, and J.-L.B. wrote the paper.
The authors declare no conflict of interest.
This article is a PNAS Direct Submission.1Present address: Institut des Neurosciences de Montpellier, INSERM U1051, 34295 Mont-pellier Cedex 05, France.
2To whom correspondence should be addressed. Email: email@example.com.
This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.1073/pnas.1414618112/-/DCSupplemental.
www.pnas.org/cgi/doi/10.1073/pnas.1414618112 PNAS | February 3, 2015 | vol. 112 | no. 5 | 16011606
that dramatically decreases constitutive activity and is associ-ated with a short-stature phenotype (17). Along with its impor-tance in drug design, GHS-R1a is a prototype for peptide-activatedclass A GPCRs.To delineate the way in which the ghrelin receptor interacts
with G proteins, we used monomeric GHS-R1a reconstituted ina membrane-mimicking environment, lipid discs, and a combi-nation of innovative biochemical [labeling with unnatural aminoacid (UAA)] and biophysical [lanthanide resonance energy transfer(LRET) and normal mode (NM) analyses] approaches. By doingso, we provide the first direct evidence that ghrelin-mediated sig-naling involves a complex dialogue between the conformationaldynamics of the receptor and its ability to interact with the dif-ferent G protein subtypes to which it is coupled.
ResultsReceptor and Gq Labeling for LRET Measurements. Site-specific la-beling of the ghrelin receptor and its cognate Gq protein was firstrequired to monitor their interaction with LRET. The G proteinwas labeled on the free amino terminus of its q subunit with thedonor fluorophore (Lumi4-Tb) through a classical reaction withits N-hydroxysuccinimide (NHS) derivative at neutral pH (18).This allowed specific labeling at the Gq N terminus with 60%efficacy. Incomplete labeling does not affect LRET measure-ments, because only the emission of the acceptor originatingfrom energy transfer exclusively (sensitized emission) is mea-sured (19). Modification of the q subunit did not affect theability of the G protein trimer to become activated (SI Appendix,Fig. S1A).The purified GHS-R1a was labeled through UAA technology.
To this end, the pEVOL vector (20) was used to encode p-azido-L-phenylalanine (azidoF) into the ghrelin receptor sequence inresponse to a unique amber stop codon. AzidoF was inserted inthe cytoplasmic face of GHS-R1a, where it replaced F71 at thecytoplasmic tip of TM1 (SI Appendix, Fig. S2). This modificationdid not affect either ligand binding or G protein activation (SIAppendix, Fig. S3). The azidoF-containing receptor was assem-bled as a monomer into lipid discs (10) and then labeled with thefluorescence acceptor (Alexa Fluor 488) using the strain-pro-moted alkyne-azide cycloaddition reaction (21, 22). Approxi-mately 90% of labeling was achieved under these conditions,whereas no labeling was observed with the unmodified receptor(SI Appendix, Fig. S4).
LRET-Monitored GHS-R1a:Gq Interaction. LRET measu