Interaction of RGS Protein with G beta subunit G beta 5.

描述（由申请人提供）：G蛋白偶联受体（gpcr）介导大量生理过程。介导乙酰胆碱作用的gpcr家族包括五个成员，称为毒蕈碱受体（M1R-M5R）。这些受体与许多神经系统疾病有关，包括抑郁症、阿尔茨海默病和帕金森病。gpcr的活性和亚细胞定位受许多细胞内蛋白的控制。本研究的重点是了解G蛋白信号（RGS）的神经特异性调节因子Gbeta5-RGS7复合物对3型毒蕈碱受体（M3R）的调节。该实验室最近的原始研究表明，Gbeta5-RGS7复合物可以通过M3R和RGS7直接结合的新机制抑制M3R信号传导。M3R激活还会引起Gbeta5-RGS7复合物亚细胞定位的剧烈变化。这些现象以m3r选择性的方式发生。此外，对缺乏Gbeta5的小鼠的研究表明，它们与缺乏M3R的小鼠相似。该项目将测试Gbeta5-RGS7复合物通过一种新机制调节M3R信号和/或将信号从M3R传递到神经元的细胞内隔室的假设。具体目标1将确定参与这种相互作用的M3R和RGS7的结构要素。M3R和RGS7的显性突变体将作为特异性目标2和3的分子工具。此外，我们还将通过一系列体外实验来研究Gbeta5-RGS7复合物与M3R的其他结合伙伴之间的关系。目的2将研究M3R和Gbeta5-RGS7从细胞质到内体囊泡的亚细胞再定位。实验将使用先进的成像方法、细胞分离和生化方法来识别M3R和Gbeta5-RGS7在受体激活后重新定位的亚细胞区室，并寻找该蛋白复合物的新结合伙伴。目的3将探讨M3R:Gbeta5-RGS7相互作用在天然细胞中的生理意义。实验设计是基于Gbeta5基因敲除小鼠肾上腺素水平升高的发现，这与这些动物的高运动活动和其他变化是一致的。本研究将集中分析Gbeta5基因敲除小鼠肾上腺髓质的嗜铬细胞分泌儿茶酚胺的情况。实验还将研究Gbeta5- RGS7表达改变的神经内分泌PC12细胞系，并将利用成像和生化方法。本研究将有助于更好地理解神经元毒蕈碱类乙酰胆碱受体的调控，并阐明RGS蛋白在调控信号转导和其他细胞功能中的新作用。公共卫生相关性：本研究旨在研究调节神经元毒蕈碱乙酰胆碱受体3型功能的分子机制。实验集中在一种涉及G蛋白信号的神经元调节器RGS7的新机制上。拟议的实验将利用生化分析、先进的成像方法和转基因小鼠研究来研究这种蛋白质的功能。这项研究将有助于在分子水平上理解神经系统的认知、感觉、运动、代谢和其他功能。

DESCRIPTION (provided by applicant): G protein coupled receptors (GPCRs) mediate a large number of physiologic processes. A family of GPCRs that mediates the action of acetylcholine includes five members called muscarinic receptors (M1R-M5R). These receptors are implicated in a number of neurological disorders including depression, Alzheimer's and Parkinson's diseases. The activity and subcellular localization of GPCRs are controlled by a number of intracellular proteins. This proposal concentrates on understanding the regulation of type 3 muscarinic receptor (M3R) by a neurospecific regulator of G protein signaling (RGS), the Gbeta5-RGS7 complex. Recent original findings in this laboratory show that Gbeta5-RGS7 complex can inhibit M3R signaling via a novel mechanism that involves direct binding between M3R and RGS7. M3R activation also causes a dramatic change in subcellular localization of the Gbeta5-RGS7 complex. These phenomena occur in an M3R-selective manner. Furthermore, studies of mice lacking Gbeta5 revealed their similarity to mice lacking M3R. This project will test the hypothesis that the Gbeta5-RGS7 complex regulates M3R signaling via a novel mechanism and/or transfers signals from M3R to intracellular compartments in neurons. Specific Aim 1 will determine structural elements of M3R and RGS7 that are involved in this interaction. Dominant mutants of M3R and RGS7 will be used as molecular tools in Specific Aims 2 and 3. In addition, a series of in vitro assays will be used to study the relationship between the Gbeta5-RGS7 complex and other binding partners of M3R. Aim 2 will study subcellular re-localization of M3R and Gbeta5-RGS7 from the cytosol to endosomal vesicles. The experiments will use advanced imaging methodology, cell fractionation and biochemical approaches to identify subcellular compartments where M3R and Gbeta5-RGS7 re-localize upon receptor activation and search for novel binding partners of this protein complex. Aim 3 will explore the physiologic significance of M3R:Gbeta5-RGS7 interaction in native cells. The experimental design is based on the discovery that Gbeta5 knockout mice have an increased level of epinephrine, which is consistent with high locomotor activity and other changes in these animals. The proposed study will concentrate on the analysis of catecholamine secretion by chromaffin cells of the adrenal medulla from Gbeta5 knockout mice. Experiments will also investigate the neuroendocrine PC12 cell lines with altered expression of Gbeta5- RGS7 and will utilize imaging and biochemical methods. This research will result in better understanding of regulation of neuronal muscarinic acetylcholine receptors, and illuminate novel roles of RGS proteins in regulation of signal transduction and other cellular functions. PUBLIC HEALTH RELEVANCE: This proposal investigates molecular mechanisms that regulate the functions of the neuronal muscarinic acetylcholine receptor type 3. The experiments concentrate on a novel mechanism that involves a neuronal regulator of G protein signaling, RGS7. The proposed experiments will study the function of this protein using biochemical analyses, advanced imaging methods and studies of genetically modified mice. This research will contribute to understanding, at the molecular level, of cognitive, sensory, motor, metabolic and other functions of the nervous system.