Signalisation transmembranaire des récepteurs métabotropiques du glutamate – mGluNano

The metabotropic glutamate receptors (mGluRs), a particular family of G-protein coupled receptors (GPCRs), are activated by glutamate, the major excitatory neurotransmitter in the central nervous system. Among the eight types of mGluRs (8 genes encoding for mGluR1 to mGluR8 in human), they are not uniformly distributed throughout the brain, and some are found only in specific areas. As such, mGluRs are particularly interesting as drug targets for the treatment a whole range of neurological and psychiatric disorders (absence epilepsy, anxiety, depression, migraine, spasticity, Parkinson’s disease and pain). mGluRs are dimeric allosteric multidomain proteins composed of an extracellular Venus flytrap domain (VFT) where orthosteric agonists such glutamate and competitive antagonists bind, and a heptahelical transmembrane domain (7TM) common to all GPCRs and responsible for G-protein activation. Of major interest, allosteric modulators (AM) can directly act on 7TM to modify the level of receptor activation thus offering a large number of possible therapeutic applications. Positive allosteric modulators (PAM) are expected to enhance both potency and efficacy of the agonist-stimulated receptors, and to be better drugs than classical agonists since they enhance the activity of the receptor only when and where the natural agonist is present. Negative modulators (NAM) block the activation of G proteins by the receptor but they could activate G protein independent signaling pathway. One of the major and exciting challenges in mGluRs pharmacology is to understand the molecular bases of these AM, and to study how it is related to the conformational changes in the 7TM domains responsible for activation of G-protein and G-protein independent signaling. The aim of this proposal is to map the structural changes induced by either PAM or NAM using a combination of biophysics and biochemistry techniques. We propose for the first time : (i) to purify the 7TM of mGluRs; (ii) to reconstitute the purified 7TM as monomers or dimers in phospholipid bilayer nanodiscs; (iii) to perform site-specific labeling of one monomer or dimer with three different fluorophores compatible with time-resolved FRET; (iv) to evaluate the conformational changes in 7TM upon ligands binding. We will use mGluR2 and mGluR5 as a model since different classes of well-characterized AM are available for these receptors. The nanodisc system does not only constitute a native-like environnement for the mGluR but it also allows us to strictly control the stoechiometry of receptor either monomer or dimer. Preliminary results show that purified 7TM of mGluR2 can be reconstituted into nanodiscs and that particles containing either monomeric or dimeric 7TM activate G-protein. To map the structural changes induced by AM in the nanodics of 7TM in the Ångström range, we will develop an innovative double time-resolved FRET approach based on the site-specific orthogonal labeling with three fluorophores compatible with time-resolved FRET (terbium cryptate as a donor, and fluorescein and Alexa647 as acceptors). This program will be developed by three partners: Drs P. Rondard and S. Granier (Institut de Génomique Fonctionnelle, Montpellier) for mGluR 7TM biochemistry and biophysical analyses; Dr JL Banères (Montpellier) for nanodiscs system and biophysical analyses; CisBio for the development of chemical tools for FRET. In summary, this program is at the crossroad of chemistry, photophysics, biochemistry and biophysics. We expect to obtain a comprehensive picture of the structural and molecular bases of activation mechanisms of the 7TM of mGluRs. It will be largely beneficial for the development of new allosteric modulators with even more precise action, by structure-based drug design, and it will open the way to more direct structural studies like X-ray crystallography.