Innovation for the study of cell surface receptosomes: application to the GABAB receptor – InnovGABAB

Hormone and neurotransmitter receptors have long been considered to diffuse freely in the plasma membrane. Data accumulated these last few years revealed that these receptor are associated with their effectors and to other signaling proteins. These protein complexes called receptosomes play critical roles in signaling. For example, G protein-coupled receptors appear to be constitutively associated with their targeted G-proteins as well as their primary effectors, such as ionic channels or enzymes producing second messengers. This is the case for the GABAB receptor, a GPCR for the main inhibitory neurotransmitter GABA that activates different signaling cascades depending on the receptosome in which it is associated. In pre-synaptic elements, it inhibits neurotransmitter release by controlling at least the opening of Ca-channels to which it is physically associated. In contrast, in the post-synaptic side, it regulates other effectors such as other receptors (including the metabotropic glutamate receptor 1) and K-channels. However, very few tools can be used to study these signalization complexes, and most importantly the functional relevance of such interactions, their stability, their dynamics, etc' Our project has two main goals: 1- Technologic: we will develop new methods to analyze protein-protein interaction by combining specific labeling of proteins using snap-tags or ACP-tags, and fluorophores compatible with time-resolved FRET (TR-FRET). We recently demonstrated the powerfulness of such an approach compare to FRET or BRET measurements. We will develop technologies to validate proximity between proteins in native systems, using antibodies or ligands labeled with TR-FRET compatible fluorophores. 2- Scientific: these tools will be used to study the interaction between the different partners of the GABAB receptosomes, the functional relevance of these interactions, their stability and their dynamics. To that aim we have access to several protein partners identified using a proteomic approach from immunoprecipitate from brains of wild-type and mutant mice. We will confirm these interactions and will analyze their functional relevance. The GABAB receptor is a promising target for the treatment of various diseases (anxiety, absene epilepsy, spasticity, '), as well illustrated by Lioresal® that is being used for the treatment of multiple sclerosis patients. However, the absence of GABAB receptor subtypes, and its expression in most brain regions, make difficult the development of drugs targeting this receptor without side effects. By better defining the functioning of GABAB receptosomes, we hope to provide novel perspective for the development of more selective drugs. In addition to providing new information on the functioning of GABAB receptosomes, this study will provide a general framework and tools to the study of other signaling complexes. Our work will provide tools for the development of new screening methodologies, more precise, more specific, better defined, in association with a company leader in this field.