Biochemical and genetic analysis of Regulator of G protein Signaling (RGS) protei

G 蛋白信号转导调节器 (RGS) 蛋白的生化和遗传分析

• 批准号: 7529991
• 负责人: MICHAEL R KOELLE
• 金额: $ 18.62万
• 依托单位: YALE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2008
• 资助国家: 美国
• 起止时间: 2008-07-01 至 2010-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7529991
• 关键词: Binding; Biochemical; Biochemical Genetics; Brain; Caenorhabditis elegans; Caenorhabditis elegans Proteins; Co-Immunoprecipitations; Complex; Data; Disease; Dopamine; Epitopes; G-Protein-Coupled Receptors; GTP Binding; GTP Phosphohydrolase Activators; GTP-Binding Protein Regulators; GTP-Binding Proteins; Genetic; Heterotrimeric GTP-Binding Proteins; Homologous Gene; Human; In Vitro; Lead; Mental Depression; Mental Health; Mental disorders; Methods; Modeling; Mutation; Nervous system structure; Neurotransmitters; Nucleotides; Phenotype; Property; Proteins; Proteus; Public Health; Purpose; RGS Proteins; Recombinants; Schizophrenia; Series; Serotonin; Signal Transduction; Site; Structural Models; Structure; Testing; Thinking; Transgenic Organisms; Work; base; design; dimer; genetic analysis; in vivo; innovation; mutant; neurotransmission; novel; novel strategies; null mutation; receptor; relating to nervous system; research study

DESCRIPTION (provided by applicant): Regulator of G protein Signaling (RGS) proteins downregulate signaling through heterotrimeric G proteins by acting as G protein GTPase activators. They thus regulate signaling by serotonin, dopamine, and other neurotransmitters critical to human mental health. Some of the most important questions about RGS proteins remain unanswered. How are RGS proteins controlled so they can downregulate neural signaling only under appropriate circumstances? What do the various domains and subunits of RGS proteins do? This proposal seeks to answer these questions by carrying out a detailed analysis of two C. elegans RGS proteins, EGL-10 and EAT-16. These are so-called R7 RGS proteins, which have a domain similar to G protein 3 subunits and are constitutively bound to a divergent G2-like protein called G25. Genetic experiments in C. elegans strongly support the hypothesis that G25/RGS dimers function like the 23 component of conventional G123 heterotrimers, and thus assemble under certain circumstances with G1 proteins to form G1/25/RGS "unconventional heterotrimers". The unconventional heterotrimer model can explain 10 years of genetic results with EGL-10 and EAT-16 and provides a framework for understanding the whole purpose for which R7 RGS proteins exist. The proposed experiments will test the unconventional heterotrimer model using three independent lines experiments to analyze properties of the protein complexes formed by EGL-10 and EAT-16. The first aim will use co-immunoprecipitation experiments to test whether unconventional heterotrimers exist in C. elegans protein lysates, and to examine the properties of these protein complexes. A novel approach for rapidly generating biochemical quantities of lysates from transgenic C. elegans will be used to facilitate these experiments. The second aim will use both biochemical and genetic experiments to analyze the effects of an unusual mutant of G25 predicted to specifically disrupt unconventional heterotrimers. The third aim seeks to generate purified recombinant unconventional heterotrimer complexes and to analyze their biochemical properties. PUBLIC HEALTH RELEVANCE: Misregulation of signaling in the brain by neurotransmitters such as serotonin and dopamine leads to disorders such as depression and schizophrenia. This proposal seeks to understand the mechanisms that normally control levels of signaling by these neurotransmitters. This may lead to understanding the causes of human mental disorders and to therapies for their treatment.

描述（由申请人提供）：G蛋白信号传导调节剂（RGS）蛋白通过充当G蛋白GT3激活剂下调通过异源三聚体G蛋白的信号传导。因此，它们调节血清素、多巴胺和其他对人类心理健康至关重要的神经递质的信号传导。关于RGS蛋白的一些最重要的问题仍然没有答案。RGS蛋白是如何控制的，只有在适当的情况下才能下调神经信号？RGS蛋白的不同结构域和亚基有什么作用？本文试图通过对两个C.线虫RGS蛋白，EGL-10和EAT-16。这些是所谓的R7 RGS蛋白，其具有类似于G蛋白3亚基的结构域，并且组成性地结合到称为G25的趋异G2样蛋白。C.秀丽线虫强烈支持这样的假设：G25/RGS二聚体的功能与传统G123异源三聚体的23组分相似，因此在某些情况下与G1蛋白组装形成G1/25/RGS“非常规异源三聚体”。非传统的异源三聚体模型可以解释EGL-10和EAT-16的10年遗传结果，并为理解R7 RGS蛋白存在的整个目的提供了一个框架。所提出的实验将使用三个独立的线实验来测试非常规异源三聚体模型，以分析由EGL-10和EAT-16形成的蛋白质复合物的性质。第一个目标将使用免疫共沉淀实验来测试C. elegans蛋白裂解物，并检查这些蛋白复合物的性质。一种从转基因C.线虫将被用来促进这些实验。第二个目标将使用生化和遗传实验来分析G25的一种不寻常突变体的影响，这种突变体预计会特异性地破坏非常规的异源三聚体。第三个目的是寻求产生纯化的重组非常规异源三聚体复合物，并分析其生化特性。公共卫生相关性：神经递质如5-羟色胺和多巴胺对大脑信号的错误调节会导致抑郁症和精神分裂症等疾病。这项提案旨在了解通常控制这些神经递质信号水平的机制。这可能会导致理解人类精神障碍的原因和治疗方法。