Assemblage oligomérique des récepteurs couplés aux protéines G avec les RAMPs

G protein coupled receptors (GPCRs) constitute the largest family of membrane receptors involved in signal transduction. Traditionally, signal transduction by GPCRs involves the activation of a hetero-trimeric G protein which will then modulate the activity of several intracellular effectors. We can now appreciate the fact that in addition to their interaction with G proteins, GPCRs also associate with several other proteins, in order to allow proper signal transduction. In particular, the discovery of a family of proteins called receptor activity-modifying proteins (RAMPs) has challenged the traditional views of signal transduction by some GPCRs. In the case of the calcitonin-like receptor (CLR), the association with RAMPs allows the proper cell surface targeting of the receptor in addition to modulate it’s pharmacological properties. Co-expression of CLR with RAMP1 leads to a calcitonin gene-related peptide (CGRP) receptor, whereas CLR association with RAMP2 or RAMP3 promotes the formation of an adrenomedullin receptor. In addition to their interaction with transmembrane accessory proteins such as RAMPs, GPCRs can also interact with other receptors to form receptors oligomers. In this thesis, we were interested in the interactions between GPCRs and RAMPs, and particularly, in the link between these GPCR/RAMP interactions and the assembly of receptor oligomers, using CGRP1 receptor as a model. We first confirmed the interaction between CLR and RAMP1 in living cells. We showed that this CLR/RAMP1 complex activates G proteins and recruits the signalling protein -arrestin upon CGRP stimulation. Next, we demonstrated that even if the CLR requires hetero-oligomeric assembly with RAMPs in order to be active, this receptor can still interact with other GPCRs. In addition to CLR homo-oligomers, we observed that RAMPs can also self-associate to form oligomeric complexes which can involve different subtypes (RAMP1/RAMP2 and RAMP1/RAMP3). This observation of the presence of CLR and RAMP1 homo-oligomers raised the question of the stoiechiometry of interaction of the CLR/RAMP1 complex. In order to establish the molecular composition of the CGRP1 receptor in vivo, we developed a novel approach allowing the detection of the interaction between three proteins in living cells. This method called BRET/BiFC is based on the bioluminescence resonance energy transfer between a luminescent energy donor, Renilla luciferase, and a fluorescent energy acceptor, the yellow fluorescent protein (YFP), reconstituted after the re-association of its two fragments. Using this approach, we showed that the CGRP1 receptor consist of a homo-oligomer of CLR interacting with a monomer of RAMP1. By demonstrating the asymmetrical organization of the CGRP1 receptor complex using a novel biophysical approach, we believe that the results presented herein have contributed to increase our knowledge of the mechanisms of function of the large family of GPCRs and will be useful for the pursuit of research on protein complexes involved in signalling pathways.

Keyphrases: BRET/BiFC, RAMPs, CGRP, G protein coupled receptors, Receptor activity-modifying proteins, Calcitonin-like receptor, Calcitonin gene-related peptide, Oligomerization, Bioluminescence resonance energy transfer, Bimolecular fluorescence complementation