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.

项目总结