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: 10651659
• 负责人: Halie Adesin Sonnenschein
• 金额: $ 3.26万
• 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/10651659
• 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; Experimental Genetics; 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; candidate identification; dimer; domain mapping; drug of abuse; egg; experimental study; gain of function mutation; in vivo; mutant; nervous system disorder; neuromechanism; neurotransmitter release; null mutation; prevent; protein complex; protein purification; ras GTPase-Activating Proteins; small molecule; transmission process

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蛋白亚基的信号传导机制， GαO。大多数神经递质都可以与G蛋白偶联受体（GPCR）结合并激活通过 在许多神经系统疾病中，GαO和GαO信号的改变已被暗示。 GPCR 通过促进GDP的GDP交换来激活GαO。这导致Gβγ亚基的解离 从GαO和可能允许GαO和Gβγ的行为不同的靶标分子，也可以调节行为， 称为效果。遗传研究表明，GαO的功能可防止神经递质的释放，但是 这种情况如何发生的分子细节尚不清楚，主要是因为GαO与和 监管仍然未知。虽然某些领域推测GαO可以简单地释放Gβγ 二聚体进行信号传导，在秀丽隐杆线虫中进行研究反驳这一想法，并建议GαO必须直接发出信号 通过其自身的效果。我假设通过直接结合效应蛋白的Gα信号，并识别 分析这些效果将是理解大脑主要G蛋白信号传导的关键。我 跟随小鼠脑的激活和非活性GαO蛋白复合物的免疫性 通过质谱法确定候选GαO效应分子。我已经产生了大量的质量 光谱数据数据并确定了相对未研究的RAS GTPase激活剂RASA2/3 候选人是长期追求的GαO效应。在此提案中，我将在体外和体内实验 表征GαO与RASA2/3之间相互作用的方法。 我的第一个目的是使用纯化来表征G⍺O和RASA2/3之间的生化相互作用 蛋白质。我将纯化GαO和RASA2/3以及对照RASA结合蛋白和对照GαO-GTP结合 蛋白质。我将测量RASA2/3的活性GαO和非活性GαO的结合亲和力，并确定是否较小 RASA2/3，Ca2+和IP3的分子配体改变了这种结合。我将为GαO绘制RASA2/3的结合界面。 我的第二个目的是使用秀丽隐杆线虫遗传学分析GAP-1的功能，即秀丽隐杆线虫。 哺乳动物RASA蛋白的直系同源物，以确定其在体内GαO信号传导中的功能以及如何在体内发挥作用。 已经获得了零突变体GAP-1，并将分析以确定它是否已经 GαO突变对秀丽隐杆线虫特定行为的广泛特征化作用。我还将确定 神经元表达GAP-1并将我的分析转向这些神经元的功能。我将使用双人突动剂 了解GαO，GAP-1和RAS之间的体内功能关系的研究。