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

G蛋白偶联受体（gpcr）是一组膜蛋白，介导多种细胞生理因子的作用，包括肽激素、脂质、离子甚至光。虽然这些受体在功能上已经被表征了几十年，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的结构要求也是未知的。由于在大多数细胞培养系统中均寡聚物占主导地位，对同聚物和异聚物形成重要性的研究变得复杂。我的实验室采用了一种叫做双分子荧光互补（BiFC）的技术，这样它就可以用于GPCR二聚化的研究。使用这种技术，我们已经证明了分子伴侣参与内质网中GPCR复合物的组装。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。一些研究表明gpcr的跨膜结构域在同型二聚体的形成中起作用，但是来自单个亚基的单个跨膜结构域在启动二聚体形成中的作用尚未得到研究。具体来说，我们建议使用bbic方法来表征血管紧张素II型1受体（AT1R）的各种二聚体的相互作用界面。该受体已被证明可自结合成同二聚体复合物，但也可与其他几种gpcr形成异二聚体复合物，包括血管紧张素II II型（AT2R）、β 2肾上腺素能受体（β 2 ar）、缓激肽B2、大麻素CB1和apelin APJ受体。单个AT1R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那样。核磁共振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。我们相信我们的研究将阐明GPCR二聚化的结构限制，并提供通过这些复合物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.