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

描述（由申请人提供）：G蛋白信号（RGS）蛋白调节剂通过异三聚体G蛋白作为G蛋白GTPase激活剂下调信号。因此，它们通过血清素、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蛋白的一些最重要的问题仍然没有答案。如何控制RGS蛋白，使它们只能在适当的情况下下调神经信号？RGS蛋白的不同结构域和亚基有什么作用？本研究旨在通过对线虫的两种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R7 RGS蛋白，具有与G蛋白3亚基相似的结构域，并组成性地与称为G25的分化的g2样蛋白结合。秀丽隐杆线虫的遗传实验有力地支持了G25/RGS二聚体的功能与常规G123异源三聚体的23组分相似，从而在特定情况下与G1蛋白组装形成G1/25/RGS“非常规异源三聚体”的假设。非常规的异源三聚体模型可以解释EGL-10和EAT-16 10年的遗传结果，并为理解R7 RGS蛋白存在的全部目的提供了一个框架。本实验将通过三个独立的细胞系实验来检验非常规的异源三聚体模型，分析EGL-10和EAT-16形成的蛋白复合物的性质。第一个目标将使用共免疫沉淀实验来测试秀丽隐杆线虫蛋白裂解物中是否存在非常规异源三聚体，并检查这些蛋白复合物的性质。一种从转基因秀丽隐杆线虫中快速产生生化量裂解物的新方法将用于促进这些实验。第二个目标将使用生化和遗传实验来分析G25的一种不寻常的突变体的影响，这种突变体预计会特别破坏非常规的异源三聚体。第三个目标是产生纯化的重组非常规异三聚体复合物并分析其生化特性。公共卫生相关性：大脑中神经递质（如血清素和多巴胺）对信号的错误调节会导致抑郁症和精神分裂症等疾病。这一建议旨在了解通常控制这些神经递质信号水平的机制。这可能会导致了解人类精神障碍的原因和治疗方法。

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.