G Protein Signaling in the C. elegans Nervous System

描述(申请人提供)：这项建议的长期目标是 了解神经递质如何通过G蛋白偶联受体传递信号 调节神经元的活动。线虫的产卵行为是 受神经递质信号通过G蛋白GA0和GA调节。至 鉴定新的G蛋白信号成分并了解其作用机制 行动方面，我们：分离和分析扰乱线虫产卵的突变 行为；2)克隆由这些突变识别的基因；3)纯化 由克隆的基因编码的信号蛋白；以及4)研究它们的性质和 纯化蛋白的相互作用。到目前为止分析过的信号蛋白 具有在哺乳动物脑中表达的密切同源基因，这表明C. 线虫是了解G的神经传递的有用模型 人体内的蛋白质。我们的方法的潜力通过以下事实来说明 我们已经用它发现了G的一类新的调节器 蛋白信号转导(RGS蛋白)通过充当G蛋白来抑制信号转导 GTP酶激活剂。 这项提议的第一个主要目标是利用线虫的遗传学来 鉴定和分析更多的G蛋白信号通路组件。我们最近 分离出至少三个新信号基因的突变。我们将克隆和 对这些基因进行分子分析。我们最近还克隆了一个额外的 基因识别的信号基因EGL-47，编码两个推定的G 蛋白质偶联受体，并提出对其功能的进一步分析。AS 一个额外的基因项目，我们将敲除大部分或全部编码基因 线虫的13种RGS蛋白，并利用这些突变体来定义 RGS蛋白的活体功能。 这项建议的第二个主要目的是进行生物化学研究 已经确定的信号蛋白。第一，我们将开展 二酰甘油激酶-1的遗传和生化的酶学研究 实验表明，它是GA0的直接影响因素。我们将描述 纯化的酶的活性及其被纯化的Galpao激活。 第二，我们将研究RGS蛋白EGL-LO和RGS蛋白的体外活性 吃-16。它们含有一个类似G的结构域，它们通过这个结构域与 Gb5样亚单位gpb-2调节Galpao和Galphaq。这些蛋白质可能会形成 一种含有GA亚基的新型G蛋白杂三聚体--GB蛋白 GPB-2和RGS蛋白，而不是传统的伽马亚基。我们会 研究RGS、Gb和Ga亚基在体外和体外形成的复合体 确定RGS蛋白的哪些区域指示它们特定地作用于 他们的基因识别的GA目标。

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.