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

G蛋白偶联受体(GPCRs)是一组膜蛋白，介导多种细胞生理因子的作用，包括多肽激素、脂质、离子甚至光。虽然这些受体的功能特征已经有几十年了，但大多数GPCRs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体的结构要求也是未知的。在大多数细胞培养体系中，均相低聚物的优势使研究同源低聚物和异源低聚物形成的重要性变得复杂起来。我的实验室已经采用了一种名为双分子荧光互补(BIFC)的技术，因此它可以用于GPCR二聚体的研究。利用这项技术，我们已经证明了分子伴侣参与了内质网中gpr复合体的组装。我们建议研究哪些受体结构域参与受体亚单位之间的物理相互作用。一些工作表明GPCRs的跨膜区在同源二聚体的形成中发挥了作用，但还没有研究单个亚基的跨膜区在启动二聚体形成中的作用。具体地说，我们建议使用BIFC方法来表征血管紧张素II 1型受体(AT1R)的不同二聚体的相互作用界面。该受体不仅与血管紧张素II(AT2R)、β2肾上腺素能(Beta2AR)、缓激肽B2、大麻素CB1和APJ受体等GPCRs形成异二聚体复合体。AT1R的单个跨膜结构域的表达将被用来确定这些结构域中的一个或多个是否参与二聚化，如荧光减弱所反映的。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，我们的研究将澄清GPCR二聚体的结构限制，并通过这些复合体提供对细胞信号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.