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）：这项研究的长期目标是 阐明乙醇的分子和细胞机制 在中枢神经系统中的营地信号通路上。营地信号 据推测，转导在动物中起重要作用 生理和行为对乙醇的反应以及 人类酗酒。腺苷环酶（AC）的活性，酶，该酶 生成营地，通过药理学相关的浓度增强了 乙醇，以及乙醇对AC活性的增强作用是AC 同工型特异性。这表明在营地生成系统中 由G蛋白偶联受体，异三聚体G蛋白和AC？s组成， AC是乙醇作用的目标。如果是这种情况， 乙醇敏感的AC分子具有一个结构元素，是 乙醇的作用。要检验的假设是乙醇敏感 AC的同工型包含一个负责乙醇影响的域 关于该酶的活性。该结构元素将被称为 “乙醇响应域。” 确定AC的乙醇响应域，嵌合在 将生成对AC的乙醇敏感和对乙醇的不敏感同工型 使用细菌的体内重组系统和位于定位的嵌合体 基于聚合酶链反应的形成。在这些嵌合突变体中， 只有使用与酶活性的酶嵌合 大肠杆菌菌株在其AC基因中有缺陷。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。确定重要的 乙醇对AC活性的影响所必需的氨基酸残基 将在此乙醇响应域中引入突变 通过位置诱变的AC的乙醇敏感同工型，以及 突变体上的乙醇将进行分析。我们将获得的知识是 对于阐明乙醇调节机制的至关重要 AC的活性。将生成和分析的AC的一系列突变体 在拟议的研究中，将是未来研究的宝贵资源 交流分子的结构/功能关系。