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: 7647054
• 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 2011-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7647054
• 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 Health; Mental disorders; Methods; Modeling; Mutation; Nervous system structure; Neurotransmitters; Nucleotides; Phenotype; Property; Proteins; Proteus; RGS Proteins; Recombinants; Schizophrenia; Series; Serotonin; Signal Transduction; Site; Structural Models; Structure; Testing; Transgenic Organisms; Work; base; depression; design; dimer; genetic analysis; in vivo; innovation; mutant; neurotransmission; novel; novel strategies; null mutation; protein complex; public health relevance; 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(申请人提供)：G蛋白信号转导调节(RGS)蛋白通过异源三聚体G蛋白作为G蛋白GTP酶激活剂下调信号。因此，它们通过5-羟色胺、多巴胺和其他对人类精神健康至关重要的神经递质来调节信号。关于RGS蛋白质的一些最重要的问题仍然没有答案。RGS蛋白是如何控制的，以便它们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单位起什么作用？这项提案试图通过对线虫的两种RGS蛋白EGL-10和EAT-16进行详细分析来回答这些问题。这些是所谓的R7RGS蛋白，它们的结构域类似于G蛋白3亚单位，并结构性地与一种名为G25的发散的类似G2的蛋白结合。线虫的遗传学实验有力地支持了这一假说，即G25/RGS二聚体的功能类似于常规G123异源三聚体的23个组分，从而在某些情况下与G1蛋白组装形成G1/25/RGS“非常规异源三聚体”。非传统的杂三聚体模型可以解释10年来EGL-10和EAT-16的遗传结果，并为理解R7 RGS蛋白存在的全部目的提供了一个框架。建议的实验将使用三个独立的线实验来测试非传统的杂三聚体模型，以分析由EGL-10和EAT-16形成的蛋白质复合体的性质。第一个目标将使用免疫共沉淀实验来测试线虫蛋白裂解物中是否存在非传统的杂三聚体，并检查这些蛋白质复合体的性质。一种从转基因线虫中快速产生生化量裂解产物的新方法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的一个不寻常的突变的影响，预测该突变将专门破坏非传统的异源三聚体。第三个目标是制备纯化的重组非常规杂三聚体复合体，并分析它们的生化性质。与公共健康相关：5-羟色胺和多巴胺等神经递质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。这项提议试图了解通常通过这些神经递质控制信号水平的机制。这可能会导致理解人类精神障碍的原因，并找到治疗方法。