Direct coupling of detergent purified human mGlu5 receptor to the heterotrimeric G proteins Gq and Gs

Open Access

Abstract

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