G Protein Signaling in the C. elegans Nervous System

线虫神经系统中的 G 蛋白信号传导

• 批准号: 6430665
• 负责人: MICHAEL R KOELLE
• 金额: $ 30.43万
• 依托单位: YALE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1997
• 资助国家: 美国
• 起止时间: 1997-12-08 至 2005-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6430665
• 关键词: Caenorhabditis elegans; G protein; G protein coupled receptor kinase; biological signal transduction; diacylglycerols; enzyme activity; gene expression; gene mutation; genetic mapping; genetically modified animals; in situ hybridization; molecular cloning; molecular genetics; nervous system; neurons; neurotransmitters; northern blottings; ovulation; polymerase chain reaction; protein protein interaction; protein signal sequence; protein structure function; transcription factor; western blottings

DESCRIPTION (provided by applicant): The long-term goal of this proposal is to understand how neurotransmitters signal through G protein coupled receptors to modulate the activities of neurons. Egg-laying behavior in C. elegans is regulated by neurotransmitter signaling through the G proteins Ga0 and Ga. To identify novel G protein signaling components and understand their mechanisms of action, we: isolate and analyze mutations that disrupt C. elegans egg-laying behavior; 2) clone the genes identified by these mutations; 3) purify the signaling proteins encoded by the cloned genes; and 4) study the properties and interactions of the purified proteins. The signaling proteins analyzed so far have close homologs expressed in the mammalian brain, suggesting that C. elegans is a useful model for understanding neurotransmission through G proteins in humans. The potential of our approach is illustrated by the fact that we have already used it to discover a novel class of regulators of G protein signaling (RGS proteins) that inhibit signaling by acting as G protein GTPase activators. The first major aim of this proposal is to exploit C. elegans genetics to identify and analyze more G protein signaling pathway components. We recently isolated mutations in at least three novel signaling genes. We will clone and molecularly analyze these genes. We also recently cloned an additional genetically identified signaling gene, egl-47, that encodes two putative G protein-coupled receptors, and propose further analysis of their functions. As an additional genetic project, we will knock out the genes encoding most or all of the 13 RGS proteins of C. elegans and use these mutants to define the in vivo functions of RGS proteins. The second major aim of this proposal is to carry out biochemical studies of the signaling proteins already identified. First, we will carry out enzymological studies of diacylglycerol kinase-1, which genetic and biochemical experiments show is a direct effector of Ga0. We will characterize the activities of the purified enzyme and its activation by purified Galphao. Second, we will study the in vitro activities of the RGS proteins EGL-lO and EAT-16. They contain a G gamma-like domain via which they complex with the GB5-like subunit GPB-2 to regulate Galphao and Galphaq. These proteins may form a novel type of G protein heterotrimer containing a Ga subunit, the GB protein GPB-2, and an RGS protein rather than a conventional gamma subunit. We will examine formation of complexes between the RGS, GB and Ga subunits in vitro and determine which regions of the RGS proteins direct them to act specifically on their genetically identified Ga targets.

描述（由申请人提供）：本提案的长期目标是 了解神经递质如何通过G蛋白偶联受体发出信号， 调节神经元的活动。产卵行为在C.埃莱甘斯群岛 通过G蛋白Ga 0和Ga的神经递质信号传导调节。到 识别新的G蛋白信号传导成分并了解其机制 我们的行动是：分离和分析破坏C的突变。秀丽隐翅虫产卵 行为; 2）克隆由这些突变鉴定的基因; 3）纯化 由克隆的基因编码的信号蛋白;和4）研究性质， 纯化蛋白质的相互作用。到目前为止分析的信号蛋白 在哺乳动物脑中有相似的同源物表达，表明C. elegans是一个有用的模型，了解神经传递通过G 人体内的蛋白质我们的方法的潜力是由以下事实说明的： 我们已经用它发现了一类新的G 通过G蛋白抑制信号传导的蛋白质信号传导（RGS蛋白） GT3激活剂。 该方案的第一个主要目的是利用C。elegans遗传学 鉴定和分析更多G蛋白信号通路组分。我们最近 在至少三个新的信号传导基因中的孤立突变。我们将克隆， 对这些基因进行分子分析我们最近还克隆了一个 一种基因鉴定的信号基因egl-47，编码两个假定的G 蛋白偶联受体，并提出进一步分析其功能。作为 一个额外的基因工程，我们将敲除编码大部分或全部 C.并利用这些突变体来定义 RGS蛋白的体内功能。 这项建议的第二个主要目的是进行生物化学研究， 已经识别的信号蛋白。首先，我们将开展 二酰基甘油激酶-1的酶学研究， 实验表明，Ga 0.我们将描述 纯化的酶的活性及其被纯化的Galphao激活。 其次，我们将研究RGS蛋白EGL-10和EGL-14的体外活性。 EAT-16它们含有一个G γ样结构域，通过该结构域，它们与 GB 5样亚基GPB-2调节Galphao和Galphaq。这些蛋白质可以形成 一种新型的含有Ga亚基的G蛋白异源三聚体，GB蛋白 GPB-2和RGS蛋白而不是常规的γ亚基。我们将 检查体外RGS、GB和Ga亚基之间的复合物形成， 确定RGS蛋白的哪些区域指导它们特异性地作用于 他们的基因识别Ga目标。