Cell-Surface Protein–Protein Interaction Analysis with Time-Resolved FRET and Snap-Tag Technologies: Application to G Protein-Coupled Receptor Oligomerization
G protein-coupled receptors (GPCRs) are key players in cell–cell communication, the dysregulation of which has often deleterious effects leading to pathologies such as psychiatric and neurological diseases. Consequently, GPCRs represent excellent drug targets, and as such are the object of intense research in drug discovery for therapeutic application. Recently, the GPCR field has been revolutionized by the demonstration that GPCRs are part of large protein complexes that control their pharmacology, activity, and signaling. Moreover, in these complexes, one GPCR can either associate with itself, forming homodimers or homooligomers, or with other receptor types, forming heterodimeric or heterooligomeric receptor entities that display new receptor features. These features include alterations in ligand cooperativity and selectivity, the activation of novel signaling pathways, and novel processes of desensitization. Thus, it has become necessary to identify GPCR-associated protein complexes of interest at the cell surface, and to determine the state of oligomerization of these receptors and their interactions with their partner proteins. This is essential to understand the function of GPCRs in their native environment, as well as ways to either modulate or control receptor activity with appropriate pharmacological tools, and to develop new therapeutic strategies. This requires the development of technologies to precisely address protein–protein interactions between oligomers at the cell surface. In collaboration with Cisbio Bioassay, we have developed such a technology, which combines TR-FRET detection with a new labeling method called SnapTag. This technology has allowed us to address the oligomeric state of many GPCRs.
Key wordsFluorescence resonance energy transfer G protein-coupled receptor Dimerization SnapTag GABAB receptor
We thank Eric Trinquet and his team at Cisbio Bioassay, for the technological and scientific collaboration, the fluorophores, and the development of new fluorescent tools dedicated to TR-FRET analysis. We thank K. Johnsson (Ecole Polytechnique Fédérale de Lausanne) for his support to this project, and for providing some Snap-tag tools. The work described was made possible thanks to the screening facilities (Plate-forme de Pharmacologie Criblage Interactome) of the Institut Fédératif de Recherche 3. This work has been supported by CNRS, INSERM, Cisbio Bioassay, and by Grants from the French Ministry of Research, Action Concertée Incitative “Biologie Cellulaire Moléculaire et Structurale” (ACI-BCM 328), the Agence Nationale de la Recherche (ANR-05-PRIB-02502, ANR-BLAN06-3_135092 and ANR-05-NEUR-035) and by an unrestricted grant from Senomyx.
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