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家族包括称为毒蕈碱受体（M1 R-M5 R）的五个成员。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由许多细胞内蛋白控制。该提案集中于理解G蛋白信号传导（RGS）的神经特异性调节剂Gbeta 5-RGS 7复合物对3型毒蕈碱受体（M3 R）的调节。该实验室最近的原始发现表明，Gbeta 5-RGS 7复合物可以通过涉及M3 R和RGS 7之间直接结合的新机制抑制M3 R信号传导。M3 R活化也引起Gbeta 5-RGS 7复合物的亚细胞定位的显著变化。这些现象以M3 R选择性方式发生。此外，对缺乏Gbeta 5的小鼠的研究表明它们与缺乏M3 R的小鼠相似。该项目将测试Gbeta 5-RGS 7复合物通过一种新的机制调节M3 R信号传导和/或将信号从M3 R转移到神经元的细胞内区室的假设。具体目标1将确定参与这种相互作用的M3 R和RGS 7的结构元件。M3 R和RGS 7的显性突变体将用作特定目的2和3中的分子工具。此外，将使用一系列体外试验研究Gbeta 5-RGS 7复合物与M3 R的其他结合配偶体之间的关系。目的2研究M3 R和Gbeta 5-RGS 7从胞质溶胶到内体囊泡的亚细胞再定位。这些实验将使用先进的成像方法，细胞分级分离和生物化学方法来鉴定亚细胞区室，其中M3 R和Gbeta 5-RGS 7在受体激活后重新定位，并寻找这种蛋白质复合物的新结合伴侣。目的3：探讨天然细胞中M3 R：G β 5-RGS 7相互作用的生理意义。实验设计基于发现Gbeta 5敲除小鼠肾上腺素水平增加，这与这些动物的高运动活性和其他变化一致。拟议的研究将集中在分析从Gbeta 5基因敲除小鼠的肾上腺髓质嗜铬细胞的儿茶酚胺分泌。实验还将研究具有改变的Gbeta 5-RGS 7表达的神经内分泌PC 12细胞系，并将利用成像和生物化学方法。这项研究将导致更好地了解神经元毒蕈碱乙酰胆碱受体的调节，并阐明RGS蛋白在信号转导和其他细胞功能的调节中的新作用。 公共卫生相关性：该建议调查调节神经元毒蕈碱乙酰胆碱受体3型功能的分子机制。实验集中在一个新的机制，涉及G蛋白信号，RGS 7的神经元调节。拟议的实验将使用生化分析、先进的成像方法和对转基因小鼠的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了解神经系统的认知、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.