Suchergebnisse

The metabotropic glutamate (mGlu) receptors are class C G protein-coupled receptors (GPCRs) that modulate synaptic activity and plasticity throughout the mammalian brain. Signal transduction is initiated by glutamate binding to the venus flytrap domains (VFT), which initiates a conformational change that is transmitted to the conserved heptahelical domains (7TM) and results ultimately in the activation of intracellular G proteins. While both mGlu1 and mGlu5 activate Gαq G-proteins, they also increase intracellular cAMP concentration through an unknown mechanism. To study directly the G protein coupling properties of the human mGlu5 receptor homodimer, we purified the full-length receptor, which required careful optimisation of the expression, N-glycosylation and purification. We successfully purified functional mGlu5 that activated the heterotrimeric G protein Gq. The high-affinity agonist-PAM VU0424465 also activated the purified receptor in the absence of an orthosteric agonist. In addition, it was found that purified mGlu5 was capable of activating the G protein Gs either upon stimulation with VU0424465 or glutamate, although the later induced a much weaker response. Our findings provide important mechanistic insights into mGlu5 G protein-dependent activity and selectivity. © 2018 The Author(s). ; The authors would like to acknowledge Laurent Prézeau, Julie Kniazeff, Philippe Rondard and Cyril Goudet for helpful discussion and Chris Tate for a critical reading of the manuscript. We are grateful to Eric Trinquet (Cisbio, France) for providing us the SNAP tag full-length mGlu5 expression plasmid, as well as to Arpege and FFP platforms at the IGF. Karine Rottier was supported by FRM "ingenieur de Recherche" program and Chady Nasrallah is supported by ATIP grant (2014–2016) and the University of Montpellier, Postodocotral scientist program (2016–2018). Joan Font and Amadeu Llebaria acknowledge MINECO (PCIN-2013–017 C03-01 and CTQ2014-57020-R), the Catalan Government (2014SGR109 and 2014CTP0002) and to ERANET Neuron project 'LIGHTPAIN'for support. Guillaume Lebon acknowledges Program ATIP (2013–2016), the CNRS, INSERM and the University of Montpellier for their support. ; Peer reviewed

Metabotropic glutamate receptors (mGluRs) are dimeric G-protein-coupled receptors activated by the main excitatory neurotransmitter, L-glutamate. mGluR activation by agonists binding in the venus flytrap domain is regulated by positive (PAM) or negative (NAM) allosteric modulators binding to the 7-transmembrane domain (7TM). We report the cryo-electron microscopy structures of fully inactive and intermediate-active conformations of mGlu5 receptor bound to an antagonist and a NAM or an agonist and a PAM, respectively, as well as the crystal structure of the 7TM bound to a photoswitchable NAM. The agonist induces a large movement between the subunits, bringing the 7TMs together and stabilizing a 7TM conformation structurally similar to the inactive state. Using functional approaches, we demonstrate that the PAM stabilizes a 7TM active conformation independent of the conformational changes induced by agonists, representing an alternative mode of mGlu activation. These findings provide a structural basis for different mGluR activation modes. ; We would like to thank Joël Bockaert, Chris Tate, Philippe Marin, and Richard Henderson for careful reading and for their comments on the manuscript. We would like to thank SLS PSI XA06 microfocus beamline scientist (Villigen; Switzerland), MRC Laboratory Molecular Biology EM and computing core facilities (Cambridge, UK), and the platform of pharmacology Arpeges of the Institut de Génomique Fonctionnelle (Montpellier, France). This project was funded by the ATIP grant from CNRS. C.N. was funded by the Post-doctoral program from Montpellier University. A.E.B. was supported by the Labex EpiGenMed (program "Investissements d'avenir," ANR-10-LABX-12-01), L.B. was supported by Ligue Contre le Cancer. J.F.-I., X.G.-S., J.C., and A.L. were supported by Ministerio de Ciencia e Innovación, Agencia Estatal de Investigación and ERDF-FEDER European Fund (project CTQ2017-89222-R) and by the Catalan government (2017 SGR 1604). J.-L.B. was supported by ANR (ANR-17-CE11-0011). J.-P.P. was supported by the Fondation Recherche Médicale (FRM team: DEQ20170326522), grants from ANR (DynaMuR2, ANR-18-CE11-0004; Lanthslider, ANR-17-CE11-0046). G.C. and K.R.V. were supported by Medical Research Council grant MC-U105184322. K.R.V. acknowledges SERB, India for the Ramanujan Fellowship (RJN-094/2017), DBT B-Life grant DBT/PR12422/MED/31/287/2014, and the support of the Department of Atomic Energy, Government of India, Government of India, under Project Identification No. RTI4006. G.L. was supported by ANR grants AT2R-TRAAK Bioanalgesics (ANR-17-CE18-0001), DynaMuR2 (ANR-18-CE11-0004), SWITCH-ON (ANR-20-CE11-0019), and Fondation Recherche Médicale (ING20121226326). ; Peer reviewed