Novel AT1a Receptor Interaction Patners

描述（由申请人提供）：异源三聚体鸟嘌呤核苷酸结合蛋白（G蛋白）偶联受体（GPCR）含有7个疏水跨膜区。因此，它们的胞内结构域由三个环和一个羧基末端组成。已知胞内结构域，特别是第三胞内环（i3环）的氨基和羧基末端附近的短片段对于与G蛋白的相互作用和G蛋白的活化是至关重要的。最近的证据表明，一些蛋白质以外的G-蛋白，所谓的受体相互作用伴侣（RIP），可以与羧基末端和一些GPCR的i3环相互作用。虽然只有一小部分GPCR的RIPs被详细研究，但仔细的研究可能会为GPCR的胞内结构域的作用提供重要线索。我实验室的长期目标是确定血管紧张素II亚型1受体（AT 1 R）的新信号传导机制，并最终开发针对受体及其相互作用蛋白之间界面的新疗法。这个K奖提案的短期目标是定义与AT 1 R胞内结构域相关的新型信号蛋白复合物，并探索其功能意义。初步工作将集中在识别大鼠主动脉血管平滑肌细胞（RASM）中的新型RIPs，其与AT 1aR的羧基端（AT 1aR-CT）和/或i3环（AT 1aR-i3 L）相互作用。我们的策略将使用谷胱甘肽S-转移酶（GST）融合蛋白，“下拉”分析和质谱。我们将通过两个具体目标来实现我们的短期目标。目的1.鉴定RASM中与AT 1aR-CT和/或AT 1aR-i3 L相互作用的新型受体相互作用伴侣（RIPs）。为了确定与新型RIP相互作用的AT 1aR-CT和/或AT 1aR-i3 L的结构域，该项目将为我提供机会，将我以前研究GPCR蛋白质-蛋白质相互作用的经验与使用质谱方法研究GPCR的新机会相结合。由于蛋白质组的重要性日益显现，成功完成这个项目将为我发展一个独立和自我维持的研究计划提供坚实的基础。

DESCRIPTION (provided by applicant):The heterotrimeric guanine nucleotide binding protein (G-protein) coupled receptors (GPCR) contain seven hydrophobic membrane-spanning regions. Thus, their intracellular domains consist of three loops and one carboxyl terminus. It is known that the intracellular domains, especially the short fragments near the amino- and carboxyl termini of the third intracellular loop (i3 loop) are critical for interaction with and activate of G-proteins. Recent evidence indicates that some proteins other than G-proteins, so called receptor interaction partners (RIPs), can interact with the carboxyl termini and the i3 loops of some GPCRs. Although only a minute percentage of the RIPs for some GPCRs have been examined in detail, careful studies might yield important clues to the roles of intracellular domains of GPCRs. The long-term objective of my laboratory is to identify novel signaling mechanisms of the angiotensin II subtype 1 receptor (AT1R), and eventually to develop novel therapeutics that target the interface between the receptor and its interacting proteins. The short-term objective of this K award proposal is to define novel signaling protein complexes that are associated with the AT1R intracellular domains, and to explore their functional significance. The initial work will focus on the identification of novel RIPs in rat aortic vascular smooth muscle cells (RASM), which interact with the carboxyl terminus (AT1aR-CT) and/or i3 loop (AT1aR-i3L) of the AT1aR. Our strategy will use glutathione S-transferase (GST) fusion proteins, "pull-down" assays and mass spectrometry. We will approach our short-term goals with two specific aims.1. To identify novel receptor interaction partners (RIPs) in RASM, which interact with AT1aR-CT and/or AT1aR-i3L.2. To identify the domains of AT1aR-CT and/or AT1aR-i3L that interact with the novel RIPsThis project will provide me with the opportunity to blend my previous experience in studying GPCR protein-protein interactions with new opportunities to use mass spectrometric methods to study GPCRs. Because of the emerging importance of the proteome, successful completion of this project will provide me with a solid foundation for developing an independent and self-sustaining research program.