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

RGS 蛋白与 G beta 亚基 G beta 5 的相互作用。

• 批准号: 7886515
• 负责人: Vladlen Z Slepak
• 金额: $ 31.91万
• 依托单位: UNIVERSITY OF MIAMI SCHOOL OF MEDICINE
• 依托单位国家: 美国
• 财政年份: 1999
• 资助国家: 美国
• 起止时间: 1999-08-01 至 2013-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7886515
• 关键词: Acetylcholine; Adrenal Medulla; Adrenergic Receptor; Agonist; Alzheimer' s Disease; Amino Acids; Animals; Arrestins; Binding; Biochemical; Catecholamines; Cell Fractionation; Cell Line; Cell membrane; Cell physiology; Cells; Chromaffin Cells; Cognitive; Complex; Cyclic AMP-Dependent Protein Kinases; Cytosol; Drug Delivery Systems; Elements; Endocytic Vesicle; Enzymes; Epinephrine; Experimental Designs; Family; Figs - dietary; G Protein-Coupled Receptor Kinase Gene; G Protein-Coupled Receptor Signaling; G protein coupled receptor kinase; G-Protein-Coupled Receptors; G-substrate; GTP-Binding Protein Regulators; GTP-Binding Protein beta Subunits; GTP-Binding Proteins; Guanine Nucleotide Exchange Factors; Guanosine Triphosphate; Heterotrimeric GTP-Binding Proteins; Image; Intracellular Membranes; Ion Channel; Knockout Mice; Laboratories; Mammals; Mediating; Mental Depression; Metabolic; Methodology; Methods; Molecular; Motor; Motor Activity; Mus; Muscarinic Acetylcholine Receptor; Nervous System Physiology; Nervous system structure; Neurons; Neurosecretory Systems; Organelles; PC12 Cells; Parkinson Disease; Pathway interactions; Physiological; Physiological Processes; Protein Kinase; Protein Tyrosine Kinase; Proteins; RGS Proteins; Receptor Activation; Receptor Down-Regulation; Recruitment Activity; Regulation; Reporting; Research; Research Personnel; Role; Sensory; Series; Signal Transduction; Site-Directed Mutagenesis; Structure; Testing; Vesicle; adapter protein; base; casein kinase; dimer; imaging modality; in vitro Assay; inorganic phosphate; member; mutant; nervous system disorder; novel; prevent; programs; protein complex; protein function; public health relevance; receptor; receptor binding; receptor function; receptor internalization; research study; scaffold; sodium-hydrogen exchanger regulatory factor; tool

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.

描述（由申请人提供）：G蛋白偶联受体（GPCR）介导了大量的生理过程。介导乙酰胆碱作用的GPCR家族包括五个称为毒蕈碱受体（M1R-M5R）的成员。这些受体与许多神经系统疾病有关，包括抑郁症，阿尔茨海默氏症和帕金森氏病。 GPCR的活性和亚细胞定位受许多细胞内蛋白的控制。该建议集中于通过GBETA5-RGS7复合物的G蛋白信号传导（RGS）的神经特异性调节剂对3型毒蕈碱受体（M3R）的调节。该实验室的最新发现表明，GBETA5-RGS7复合物可以通过涉及M3R和RGS7之间直接结合的新机制抑制M3R信号传导。 M3R激活还会导致GBETA5-RGS7复合物的亚细胞定位发生巨大变化。这些现象以M3R选择方式出现。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。该项目将检验以下假设：GBETA5-RGS7复合物通过新型机制和/或将信号从M3R转移到神经元的细胞内室。具体目标1将确定与此相互作用有关的M3R和RGS7的结构元素。 M3R和RGS7的主要突变体将用作特定目标2和3的分子工具。此外，一系列体外测定将用于研究GBETA5-RGS7复合物与M3R的其他结合伙伴之间的关系。 AIM 2将研究从细胞质到内体囊泡的M3R和GBETA5-RGS7的亚细胞重新定位。该实验将使用先进的成像方法，细胞分馏和生化方法来识别亚细胞隔室，其中M3R和GBETA5-RGS7在受体激活后重新定位并寻找该蛋白质复合物的新型结合伙伴。 AIM 3将探讨天然细胞中M3R：GBETA5-RGS7相互作用的生理意义。实验设计基于以下发现：GBETA5敲除小鼠的肾上腺素水平升高，这与这些动物的高运动活性和其他变化是一致的。拟议的研究将集中于GBETA5基因敲除小鼠的肾上腺髓质细胞对儿茶酚胺分泌的分析。实验还将研究具有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。这项研究将使人们更好地了解神经毒蕈碱乙酰胆碱受体的调节，并阐明RGS蛋白在调节信号转导和其他细胞功能中的新作用。 公共卫生相关性：该提案研究了调节3型神经毒蕈碱乙酰胆碱受体功能的分子机制。实验集中于一种涉及G蛋白信号传导神经元调节剂RGS7的新机制。提出的实验将使用生化分析，高级成像方法和转基因小鼠的研究研究该蛋白质的功能。这项研究将有助于理解神经系统的认知，感觉，运动，代谢和其他功能。