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最丰富的Gα蛋白亚基的信号转导机制， 去吧。大多数神经递质可以结合并激活G蛋白偶联受体（GPCR）， Gαo和Gαo信号的改变与许多神经系统疾病有关。GPCRs 通过促进结合GDP与GTP的交换来激活Gαo。这导致Gβγ亚基的解离 并可能允许Gαo和Gβγ结合并调节不同靶分子的行为， 称为效应器。遗传学研究表明，Gαo的功能是阻止神经递质的释放，但 这是如何发生的分子细节尚不清楚，主要是因为Gαo结合的效应物， 规则仍然未知。虽然有些领域推测Gαo可能只是为了释放Gβγ 二聚体进行信号传导，在C. elegans反驳了这一观点，认为Gαo必须直接发出信号， 通过自己的效应器。我假设Gαo通过直接结合效应蛋白来传递信号， 分析这些效应器将是理解大脑主要G蛋白信号传递的关键。我 采用免疫纯化的活化和失活Gαo蛋白复合物从小鼠脑， 通过质谱鉴定候选Gαo效应分子。我已经生成了大量的 光谱数据，并已确定相对未经研究的Ras GTdR激活剂Rasa 2/3为强 候选人是长期寻找的Gαo效应子。在这个建议中，我将使用体外和体内实验 方法来表征Gαo和Rasa 2/3之间的相互作用。 我的第一个目的是使用纯化的GSTO和Rasa 2/3之间的生物化学相互作用来表征 proteins.我将纯化Gαo和Rasa 2/3以及对照Rasa结合蛋白和对照Gαo-GTP结合蛋白， 蛋白我将测量活性和非活性Gαo对Rasa 2/3的结合亲和力，并确定小- Rasa 2/3、Ca 2+和IP 3的分子配体改变这种结合。我将为Gαo映射Rasa 2/3的绑定界面。 我的第二个目标是使用C。GAP-1基因的功能分析，并与C. elegans 哺乳动物Rasa蛋白的直系同源物，以确定其是否以及如何在体内Gαo信号传导中起作用。我 我已经获得了一个空突变gap-1，并将进行分析，以确定它是否与已经存在的 Gαo突变对C.优雅我也会决定 哪些神经元表达gap-1并引导我分析这些神经元的功能。我会用双变异体 研究以了解Gαo、GAP-1和Ras之间的体内功能关系。

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.