Identification of the transmembrane domains important for the heterodimerization of the angiotensin AT1R with other GPCRs

G protein coupled receptors (GPCRs) are a group of membrane proteins that mediate the actions of many actors of cell physiology, including peptide hormones, lipids, ions and even light. While these receptors have been characterized functionally for several decades, the structure of most GPCRs is still unclear. Also, the structural requirements for dimerization of receptor monomers, into either homodimeric or heterodimeric complexes, are also unknown. The study of the importance of homo-oligomer and hetero-oligomer formation is complicated by the predominance of homo-oligomers in most cell culture systems. My laboratory has adapted a technique called bimolecular fluorescence complementation (BiFC), so that it can be used for studies of GPCR dimerization. Using this technique, we have demonstrated the involvement of molecular chaperones in the assembly of GPCR complexes in the endoplasmic reticulum. We propose to examine which receptor domains are involved in the physical interaction between the receptor subunits. Some work has suggested a role for transmembrane domains of GPCRs in homodimer formation, but the role of individual transmembrane domains from individual subunits in initiating dimer formation has not been studied. Specifically, we propose to use the BiFC approach to characterize the interaction interface of various dimers of the Angiotensin II type 1 receptor (AT1R). This receptor has been shown to self-associate into a homodimeric complex, but also forms heterodimer complexes with several other GPCRs including angiotensin II type II (AT2R), the beta2adrenergic (beta2AR), bradykinin B2, cannabinoid CB1 and apelin APJ receptors. Expression of individual transmembrane domains of AT1R will be used to determine if one or more of these domains is involved in dimerization, as reflected by a decrease in fluorescence. NMR studies will be used to examine the structural characteristics of the physical interaction, using wild-type and mutant proteins. We believe our study will clarify the structural constraints for GPCR dimerization and provide insight into physiological and pathological interactions on cellular signaling via these complexes.