Identification of the direct effector of the major brain G protein, G(alpha)o

项目摘要 我的论文项目旨在阐明大脑中含量最丰富的Gα蛋白亚基的信号机制。 大多数神经递质都能与G蛋白偶联受体结合并激活G蛋白偶联受体(GPCRs)，通过G蛋白偶联受体传递信号 Gαo和Gαo信号的改变被认为与许多神经疾病有关。GPCRs 通过促进国内生产总值与国内生产总值之间的互换来激活Gαo。这会导致Gβγ亚基的解离 来自Gαo并且潜在地允许Gαo和Gβγ两者结合并调节不同的靶分子的行为， 称为效应器。遗传学研究表明，G-αo具有阻止神经递质释放的功能，但 这种情况如何发生的分子细节尚不清楚，主要是因为Gαo结合的效应器(S)和 监管机构仍不清楚。虽然一些领域推测Gαo可能只是用来释放Gβγ 二聚体进行信号传递，对线虫的研究驳斥了这一观点，并提出Gαo必须直接发出信号 通过它自己的效应器。我假设Gαo通过直接结合效应蛋白(S)而发出信号，并识别 分析这些效应器将是理解大脑主要G蛋白发出的信号的关键。我 对小鼠脑中活化和失活的G-αo蛋白复合体进行了免疫纯化 通过质谱学鉴定候选的Gαo效应分子。我已经产生了一大组质量 光谱数据，并已确定相对未研究的Ras GTP酶激活剂Rasa2/3为强 候选人将成为人们期待已久的Gαo效应者。在这项建议中，我将使用体外和体内实验 表征Gαo与Rasa2/3相互作用的方法 我的第一个目标是研究G⍺o和Rasa2/3之间的生化相互作用。 蛋白质。我将提纯Gαo和Rasa2/3以及对照RASA结合蛋白和对照Gαo-GTP结合 蛋白。我将测量Rasa2/3的活性和非活性Gαo的结合亲和力，并确定小的- Rasa2/3、Ca~(2+)和IP3的分子配体改变了这种结合。我将把Rasa2/3的绑定接口映射到Gαo。 我的第二个目标是利用线虫遗传学来分析GAP-1，即接近线虫的功能 哺乳动物Rasa蛋白的同源基因，以确定其在活体中是否以及如何在Gαo信号中发挥作用。我 已经获得了一个零突变体GAP-1，并将进行分析以确定它是否复制了已经 广泛描述的Gαo突变对线虫特定行为的影响。我也会决定 哪些神经元表达GAP-1，并指导我对这些神经元的功能进行分析。我会用双变种人 研究了解Gαo、GAP-1和RAS在体内的功能关系。

Project Summary My thesis project aims to clarify the signaling mechanism of the most abundant Gα protein subunit in the brain, Gαo. Most neurotransmitters can bind to and activate G Protein Coupled Receptors (GPCRs) that signal through Gαo, and alterations in Gαo signaling have been implicated in a number of neurological disorders. GPCRs activate Gαo by promoting exchange of a bound GDP for GTP. This causes the dissociation of the Gβγ subunits from Gαo and potentially allows both Gαo and Gβγ to bind and modulate the behavior different target molecules, known as effectors. Genetic studies show that Gαo functions to prevent the release of neurotransmitters, but the molecular details of how this occurs remains unclear, largely because the effector(s) that Gαo binds to and regulates remain unknown. While some field have speculated that Gαo may simply serve to release the Gβγ dimer to carry out signaling, studies in C. elegans refute this idea and suggest that Gαo must directly signal through its own effectors. I hypothesize that Gαo signals by directly binding effector protein(s) and that identifying and analyzing these effectors will be the key to understanding signaling by the major G protein of the brain. I have employed immunopurification of activated and inactive Gαo protein complexes from mouse brain followed by mass spectrometry to identify candidate Gαo effector molecules. I have already generated a large set of mass spectrometry data and have identified the relatively unstudied Ras GTPase activators Rasa2/3 as strong candidates to be the long-sought Gαo effectors. In this proposal I will use in vitro and in vivo experimental approaches to characterize the interaction between Gαo and Rasa2/3. My first is aim is to characterize the biochemical interactions between G⍺o and Rasa2/3 using purified proteins. I will purify Gαo and Rasa2/3 as well as a control Rasa-binding protein and a control Gαo-GTP binding protein. I will measure the binding affinities of active and inactive Gαo for Rasa2/3 and determine if the small- molecule ligands of Rasa2/3, Ca2+ and IP3, alter this binding. I will map the binding interface of Rasa2/3 for Gαo. My second aim is to use C. elegans genetics to analyze the functions of GAP-1, the close C. elegans ortholog of mammalian Rasa proteins, to determine if and how it functions in Gαo signaling in vivo. I have obtained a null mutant gap-1 and will analyze to determine if it phenocopies aspects of the already extensively-characterized effects of Gαo mutations on specific behaviors in C. elegans. I will also determine which neurons express gap-1 and direct my analysis to functions of those neurons. I will use double-mutant studies to understand the in vivo functional relationship between Gαo, GAP-1, and Ras.