Global analysis of GPCR dimerization using AdBiFC assay

Abstract

G protein-coupled receptors (GPCRs) are the largest class of cell surface receptors that mediate numerous physiological responses to various extracellular stimuli. GPCRs are believed to exist as monomers, but emerging evidences suggest that GPCRs may form dimers (or higher-order oligomers) leading to an alteration of their signals. In this study, we developed an adenovirus-based bimolecular fluorescence complementation (AdBiFC) assay system, combining a gateway-compatible high-throughput technology and an adenovirus-mediated gene delivery system, to visualize protein-protein interactions in living human cells. Using the AdBiFC assay system, we generated 164 recombinant adenoviruses which can express C-terminally VN- or VC-tagged GPCR receptors, and performed a global mapping of GPCR dimerization. From this large-scale screening, we identified 245 reliable GPCR dimeric forms including 26 homodimerizations. Furthermore, we have developed a tool for β-arrestin recruitment assay based on G protein-coupled receptor activations using BiFC technique which is readily adapted to high-throughput drug screening in living cells. Among GPCR dimeric combinations, we also elucidated that activation of CXCR4 chemokine receptor by CXCL12 elevates intracellular calcium levels via heterodimerization with endothelin receptor B (EDNRB) in COS-7 cells.