Analysis of GPCR dimerization using acceptor photobleaching resonance energy transfer techniques.
The ability of GPCRs to assemble into multimeric complexes is one of the most recently studied and discussed topics for many reasons, including the possibility that GPCR assemblies show a distinct pharmacological profile offering an innovative avenue for the drug synthesis. In addition, the possible differential coupling of monomeric versus multimeric GPCRs to G proteins and other downstream partners, as well as the signaling, the regulation through desensitization and internalization, and the subcellular localization can well represent additional factors that contribute to GPCR-mediated physiopathological states. The standard biochemical techniques used to identify GPCR interactions, such as coimmunoprecipitation, have obvious limitations owing to the use of nonphysiological buffers and detergents that disrupt the natural cell environment and biological interactions and preclude the analysis of subcellular localization and compartmentalization. In the past decade, new biophysical proximity assays based on the resonance energy transfer (RET) between two chromophores allow the study of dimerization in intact living cells, thus proving more information on GPCR physiological roles. In this chapter, we detail the application of two RET techniques based on fluorescence (FRET) and bioluminescence (BRET) to the study of GPCR dimerization and describe the results that can be obtained.
CNR Institute of Neuroscience, University of Milan, Milan, Italy.
Marta
M
Busnelli
Mario
M
Mauri
Marco
M
Parenti
Bice
B
Chini
D000818:Animals; D031541:Fluorescence Resonance Energy Transfer / Q000379:methods; D006801:Humans; D008163:Luminescent Measurements / Q000379:methods*; D038761:Photobleaching; D055503:Protein Multimerization; D043562:Receptors, G-Protein-Coupled / Q000032:analysis* / Q000235:genetics / Q000378:metabolism*; D018045:Receptors, Oxytocin / Q000032:analysis / Q000235:genetics / Q000378:metabolism; D017482:Receptors, Thromboxane / Q000032:analysis / Q000235:genetics / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000032:analysis / Q000235:genetics / Q000378:metabolism
D043562:Receptors, G-Protein-Coupled; D018045:Receptors, Oxytocin; D017482:Receptors, Thromboxane; D011993:Recombinant Fusion Proteins