Régulation du fonctionnement des RCPG : hétéro-oligomérisation et organisation dynamique des récepteurs dans la membrane – GPCR D-I-F

In recent years, the general mechanism of G protein-coupled receptor (GPCR) signaling has been largely revisited, in part because it was shown that a given receptor could form homo- or heteromers with other GPCRs. In addition, the notion that GPCRs must function in the context of a specific multiprotein complex or 'signalosome' has emerged and was confirmed by the observation that GPCRs diffusion can be restricted to microdomains of the plasma membrane. Although the main GPCR biochemical processes have been individually identified, the molecular structures, the dynamics of each partner, and the successive protein'protein interactions governing these multi-molecular events remain to be established. The proposed partnership, which relies on pluridisciplinary and recognized expertise of biologists, biophysicists, chemists and physicists, has already produced valuable results in the GPCR field and now wishes to extend further our understanding of these new concepts by developing and applying new methodologies. We will focus on the interaction between mu opioid (MOP) and Neuropeptide FF (NPFF2) receptors. The NPFF system is part of an "opioid-modulating system" that regulates opioid responses in order to maintain homeostasis. The mechanism underlying the modulation of opioid response by NPFF is likely to involve a functional interaction between receptors in the same cell, rather than a regulation due to neuronal circuitry. Indeed, exposure of isolated neurons or model human neuroblastoma SH-SY5Y cells to NPFF agonists reduces the inhibitory effect of MOP agonists on voltage-gated Ca2+ channels. By using this cellular model we have already demonstrated by fluorescent techniques that exposure to NPFF agonist, not only promoted the association of NPFF receptor to MOP receptor as observed by Fluorescence Resonance Energy Transfer, but also modified the lateral mobility of MOP receptor in the cell membrane as observed by vrFRAP (Fluorescence Recovery After Photobleaching at variable radii). We now need to understand how this interaction between receptors in the membrane results in an inhibition of MOP receptor signaling. The specific aims of the project are: 1)The detailed vrFRAP analysis of the regulation of MOP and NPFF2 receptors (dynamic behavior and its regulation by agonists and antagonists, reciprocity and selectivity of the interaction). 2)The characterization of the molecular mechanism involved in the modification of membrane compartmentalization of the MOP receptor induced by activation of the NPFF2 receptor. 3) The analysis of the relationship between the change in lateral mobility of MOP receptors and their reduced ability to regulate calcium channels. We will thus study whether interfering with receptor diffusion modifies receptor signaling. 4) The demonstration of these mechanisms in single neurons in which the anti-opioid effect of NPFF has already been described. To achieve these goals, several new tools and technical improvements will be developed: 1) a dual-color FRAP apparatus that will enable us to follow the diffusion of NPFF2 and MOP receptors simultaneously, 2) geneticallyengineered receptors, in particular receptors fused to split GFP to follow specifically the diffusion of receptor dimers, and 3) new fluorescently labeled ligands to mark up endogenous untagged receptors and bivalent ligands with improved affinity for receptor heteromers to define the link between diffusion and heteromerization. The present multidisciplinary project will provide technological advances in laser FRAP microscopy and fluorescent tool design. It will also extend our understanding of the mechanisms of GPCR cross-talk, with elaboration of models of the dynamic organization of GPCR signaling complexes. In the case of MOP/NPFF receptor interactions, identification of potential targets and ligands to manipulate specifically transmembrane signaling of the complex will provide leads for the improvement of opiate therapeutic use.