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

G蛋白偶联受体（GPCR）是一组介导细胞生理学的许多作用者的作用的膜蛋白，包括肽激素、脂质、离子和甚至光。虽然这些受体已经在功能上表征了几十年，但大多数GPCR的结构仍然不清楚。此外，受体单体二聚化成同二聚体或异二聚体复合物的结构要求也是未知的。同源寡聚体和异源寡聚体形成的重要性的研究是复杂的同源寡聚体在大多数细胞培养系统中的优势。我的实验室采用了一种称为双分子荧光互补（BiFC）的技术，因此它可以用于GPCR二聚体的研究。使用这种技术，我们已经证明了参与GPCR复合物在内质网中的组装分子伴侣。我们建议检查哪些受体结构域参与受体亚基之间的物理相互作用。一些工作已经表明GPCR的跨膜结构域在同二聚体形成中的作用，但是来自单个亚基的单个跨膜结构域在引发二聚体形成中的作用尚未被研究。具体而言，我们建议使用BiFC方法来表征血管紧张素II 1型受体（AT1R）的各种二聚体的相互作用界面。该受体已被证明自缔合成同源二聚体复合物，但也与几种其他GPCR形成异源二聚体复合物，包括血管紧张素II II II型（AT2R）、β 2肾上腺素能（β 2AR）、缓激肽B2、大麻素CB 1和apelin APJ受体。AT1R的单个跨膜结构域的表达将用于确定这些结构域中的一个或多个是否参与二聚化，如荧光减少所反映的。NMR研究将用于检查物理相互作用的结构特征，使用野生型和突变蛋白质。我们相信我们的研究将澄清GPCR二聚化的结构限制，并通过这些复合物提供对细胞信号传导的生理和病理相互作用的见解。

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.