Novel AT1a Receptor Interaction Patners

描述(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)包含七个疏水跨膜区域。因此，它们的胞内结构域由三个环和一个羧基末端组成。众所周知，细胞内结构域，特别是第三个细胞内环(I3环)氨基和羧基末端附近的短片段对于G蛋白的相互作用和激活是至关重要的。最近的研究表明，G蛋白以外的一些蛋白质，即所谓的受体相互作用伙伴(RIPs)，可以与某些GPCRs的羧基末端和I3环相互作用。虽然只对一些GPCRs的RIPs进行了详细的研究，但仔细的研究可能会为GPCRs的胞内结构域的作用提供重要的线索。我的实验室的长期目标是确定血管紧张素II 1型受体(AT1R)的新信号机制，并最终开发针对该受体及其相互作用蛋白之间的界面的新疗法。这项K奖提案的短期目标是定义与AT1R细胞内结构域相关的新的信号蛋白复合体，并探索它们的功能意义。最初的工作将集中在鉴定大鼠主动脉血管平滑肌细胞(RASM)中与AT1aR的羧基末端(AT1aR-CT)和/或I3环(AT1aR-i3L)相互作用的新RIPs。我们的策略将使用谷胱甘肽S转移酶(GST)融合蛋白、“下拉”分析和质谱仪。我们将带着两个具体目标来实现我们的短期目标。寻找RASM中与AT1aR-CT和/或AT1aR-i3L.2相互作用的新型受体相互作用伙伴(RIPs)。确定AT1aR-CT和/或AT1aR-i3L与新型RIPs相互作用的结构域这个项目将为我提供一个机会，将我以前研究GPCR蛋白质相互作用的经验与使用质谱学方法研究GPCRs的新机会结合起来。由于蛋白质组的重要性正在显现，这个项目的成功完成将为我开发一个独立和自给自足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.