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

鉴定主要脑 G 蛋白 G(α)o 的直接效应子

• 批准号: 10535634
• 负责人: Halie Adesin Sonnenschein
• 金额: $ 4.68万
• 依托单位: YALE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-07-01 至 2024-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10535634
• 关键词: Affect; Affinity; Amino Acids; Animals; Basic Science; Behavior; Binding; Binding Proteins; Biochemical; Biological Assay; Biological Process; Brain; C2 Domain; Caenorhabditis elegans; Clustered Regularly Interspaced Short Palindromic Repeats; Data; Defect; Developmental Delay Disorders; Disease; Dissociation; Dyskinetic syndrome; Engineering; Family; G Protein-Coupled Receptor Signaling; G-Protein-Coupled Receptors; GNAO1 encephalopathy; GTP Phosphohydrolase Activators; GTP-Binding Protein alpha Subunits, Gs; GTP-Binding Proteins; Genes; Genetic; Genetic study; Green Fluorescent Proteins; Guanosine Triphosphate; Guanosine Triphosphate Phosphohydrolases; In Vitro; Inositol Phosphates; Ligands; Locomotion; Maps; Mass Spectrum Analysis; Measures; Mediating; Molecular; Mus; Mutation; Neurons; Neurotransmitters; Orthologous Gene; PH Domain; Phenocopy; Physiological; Plasma; Point Mutation; Protein Subunits; Proteins; Recombinant Proteins; Recombinants; Reporter; Seizures; Signal Transduction; Surface; Testing; Transgenes; Work; dimer; drug of abuse; egg; experimental study; gain of function mutation; in vivo; mutant; nervous system disorder; neuromechanism; neurotransmitter release; null mutation; prevent; protein complex; small molecule

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.

项目概要 我的论文项目旨在阐明大脑中最丰富的 Gα 蛋白亚基的信号传导机制， 大多数神经递质可以结合并激活通过 G 蛋白偶联受体 (GPCR) 发出信号。 Gαo 和 Gαo 信号传导的改变与许多神经系统疾病有关。 通过促进结合的 GDP 与 GTP 的交换来激活 Gαo，这会导致 Gβγ 亚基的解离。 来自 Gαo 并可能允许 Gαo 和 Gβγ 结合并调节不同目标分子的行为， 遗传学研究表明，Gαo 具有阻止神经递质释放的作用，但 这种现象如何发生的分子细节仍不清楚，很大程度上是因为 Gαo 结合的效应器和 虽然一些领域推测 Gαo 可能只是释放 Gβγ。 二聚体来进行信号传导，线虫研究驳斥了这一观点，并表明 Gαo 必须直接发出信号 我通过它自己的效应子来对抗 Gαo 信号，通过直接结合效应蛋白并识别。 分析这些效应器将是理解大脑 I 主要 G 蛋白信号传导的关键。 采用免疫纯化小鼠大脑中活化和失活的 Gαo 蛋白复合物 通过质谱法来识别候选 Gαo 效应分子 我已经生成了大量质量。 光谱数据，并已确定相对未经研究的 Ras GTPase 激活剂 Rasa2/3 具有很强的 在本提案中，我将使用体外和体内实验作为长期寻找的 Gαo 效应器的候选者。 表征 Gαo 和 Rasa2/3 之间相互作用的方法。 我的第一个目标是使用纯化的方法来表征 G⍺o 和 Rasa2/3 之间的生化相互作用 我将纯化 Gαo 和 Rasa2/3 以及对照 Rasa 结合蛋白和对照 Gαo-GTP 结合蛋白。 我将测量活性和非活性 Gαo 对 Rasa2/3 的结合亲和力，并确定小- Rasa2/3、Ca2+ 和 IP3 的分子配体改变了这种结合，我将绘制 Rasa2/3 与 Gαo 的结合界面。 我的第二个目标是利用线虫遗传学来分析 GAP-1（近缘线虫）的功能 哺乳动物 Rasa 蛋白的直向同源物，以确定它是否以及如何在体内 Gαo 信号传导中发挥作用 I。 已经获得了无效突变gap-1，并将进行分析以确定它是否与已经存在的突变体的表型相同 我还将确定 Gαo 突变对线虫特定行为的一般影响。 哪些神经元表达间隙-1 并指导我对这些神经元功能的分析，我将使用双突变体。 研究了解 Gαo、GAP-1 和 Ras 之间的体内功能关系。