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: 6438493
• 负责人: Masami Yoshimura
• 金额: $ 22.65万
• 依托单位: UNIVERSITY OF COLORADO DENVER
• 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/6438493
• 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 阐明乙醇影响的分子和细胞机制 中枢神经系统中 cAMP 信号通路的作用。环腺苷酸信号 转导被认为在动物体内发挥着重要作用 对乙醇的生理和行为反应以及 人类酗酒。腺苷酸环化酶 (AC) 的活性，该酶 产生cAMP，通过药理学相关浓度增强 乙醇，乙醇对AC活性的增强作用为AC 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 蛋白和 AC? 组成， AC是乙醇的作用目标。如果是这种情况，则 乙醇敏感的 AC 分子具有一个结构元件，该元件是 乙醇的作用。要检验的假设是乙醇敏感 AC 的亚型包含一个负责乙醇作用的结构域 关于这种酶的活性。该结构元素将被称为 “乙醇响应结构域。” 为了鉴定 AC 的乙醇响应结构域，嵌合体 将产生乙醇敏感和乙醇不敏感的AC亚型 使用细菌体内重组系统和定点嵌合体 基于聚合酶链式反应的形成。在这些嵌合突变体中， 使用基因筛选仅分离具有酶活性的嵌合体 AC 基因有缺陷的大肠杆菌菌株。乙醇对的影响 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结构域。识别重要的 乙醇影响AC活性所必需的氨基酸残基， 突变将被引入到该乙醇响应结构域中 通过定点诱变获得乙醇敏感的 AC 亚型，以及 将分析突变体上的乙醇。我们将获得的知识是 对于阐明乙醇调节的机制至关重要 AC 的活性。将生成和分析的一系列 AC 突变体 拟议的研究将成为未来研究的宝贵资源 AC分子的结构/功能关系。