Action of ethanol on cyclic AMP signal transduction

乙醇对环磷酸腺苷信号转导的作用

• 批准号: 7125896
• 负责人: Masami Yoshimura
• 金额: $ 22.05万
• 依托单位: LOUISIANA STATE UNIV A&M COL BATON ROUGE
• 依托单位国家: 美国
• 财政年份: 2002
• 资助国家: 美国
• 起止时间: 2002-03-01 至 2007-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7125896
• 关键词: Escherichia coli; adenylate cyclase; biological signal transduction; cell line; cyclic AMP; enzyme activity; ethanol; isozymes; polymerase chain reaction; protein isoforms; recombinant DNA; site directed mutagenesis; tissue mosaicism; transfection

DESCRIPTION (provided by applicant): The long-term goal of this research is to elucidate the molecular and cellular mechanisms underlying effects of ethanol on the cAMP signaling pathway in the central nervous system. cAMP signal transduction has been postulated to play an important role in an animal?s physiological and behavioral responses to ethanol and the development of alcoholism in humans. The activity of adenylyl cyclase (AC), the enzyme which generates cAMP, is enhanced by pharmacologically relevant concentrations of ethanol, and the enhancing effect of ethanol on AC activity is AC isoform-specific. This indicates that within a cAMP generating system, which consists of G protein-coupled receptors, heterotrimeric G proteins, and AC?s, AC is the target of action of ethanol. If this is the case, an ethanol-sensitive AC molecule has a structural element that is a target of action of ethanol. The hypothesis to be tested is that ethanol-sensitive isoforms of AC contain a domain that is responsible for the effect of ethanol on the activity of this enzyme. This structural element will be termed an "ethanol-responsive domain." To identify the ethanol-responsive domain of AC, chimeras between ethanol-sensitive and ethanol-insensitive isoforms of AC will be generated using a bacterial in vivo recombination system and also site-directed chimera formation based on polymerase chain reaction. Out of these chimeric mutants, only enzymatically active chimera will be isolated using genetic screening with an Escherichia coli strain defective in its AC gene. The effect of ethanol on the activity of a series of the chimeric AC mutants will be examined in mammalian cells to locate the ethanol-responsive domain. To identify important amino acid residues necessary for the effect of ethanol on the activity of AC, mutations will be introduced in this ethanol-responsive domain of the ethanol-sensitive isoform of AC by site-directed mutagenesis, and the effect of ethanol on the mutants will be analyzed. The knowledge we will obtain is crucial for elucidation of the mechanism by which ethanol modulates the activity of AC. The series of mutants of AC that will be generated and analyzed in the proposed study will be a valuable resource for future studies of the structure/function relationships of AC molecules.

描述（由申请人提供）：本研究的长期目标是 阐明乙醇作用的分子和细胞机制 对中枢神经系统cAMP信号通路的影响。cAMP信号 转导被假定在动物中起重要作用？S 对乙醇的生理和行为反应以及 人类的酒精中毒腺苷酸环化酶（AC）的活性， 产生cAMP，并通过与cAMP浓度相关的 乙醇对活性炭活性的增强作用最大， 异构体特异性。这表明在cAMP生成系统中， 由G蛋白偶联受体，异源三聚体G蛋白，AC？s， AC是乙醇的作用对象。如果是这种情况， 乙醇敏感性AC分子具有作为靶点的结构元件， 乙醇的作用。待检验的假设是乙醇敏感的 AC的同种型含有负责乙醇作用的结构域 这种酶的活性。该结构元件将被称为 “乙醇响应域。" 为了鉴定AC的乙醇响应结构域， 将产生AC的乙醇敏感型和乙醇不敏感型亚型 使用细菌体内重组系统以及定点嵌合体 基于聚合酶链反应的形成。在这些嵌合突变体中， 只有酶活性嵌合体将使用遗传筛选分离， 一种AC基因缺陷的大肠杆菌菌株。乙醇对 一系列嵌合AC突变体的活性将在 哺乳动物细胞定位乙醇响应域。识别重要 乙醇对AC活性的影响所必需的氨基酸残基， 突变将被引入到这个乙醇响应域的 通过定点诱变获得AC的乙醇敏感性同种型，以及 将分析乙醇对突变体的影响。我们将获得的知识是 这对于阐明乙醇调节 AC的活动。将产生和分析的AC的一系列突变体 在拟议的研究将是一个宝贵的资源，为未来的研究， AC分子的结构/功能关系。