Action of Ethanol on Cyclic AMP Signal Transduction

描述(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 在中枢神经系统的cAMP信号通路上。营地信号 转导被认为在动物身上扮演着重要的角色--S 酒精的生理和行为反应及其发展 人类中的酒精中毒。腺酰环化酶(AC)的活性，该酶 产生cAMP，是由药理上相关的浓度 乙醇对AC活性的增强作用为AC 同型异构体。这表明，在一个营地生成系统中， 由G蛋白偶联受体、异三聚体G蛋白和AC？S组成， AC是乙醇作用的靶标。如果是这样的话，一个 乙醇敏感的AC分子具有作为靶标的结构元件 乙醇的作用。需要检验的假设是，对酒精敏感 AC的异构体含有一个负责乙醇作用的结构域 这种酶的活性。该结构元素将被称为 “乙醇响应域。” 为了鉴定AC的乙醇响应域，嵌合体之间 将产生对乙醇敏感和对乙醇不敏感的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， 只有具有酶活性的嵌合体将通过基因筛选被分离出来 一种AC基因缺陷的大肠杆菌菌株。乙醇对人血清白蛋白的影响 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域。找出重要的 乙醇对AC活性影响所必需的氨基酸残基， 突变将被引入到乙醇反应区域中 乙醇敏感的AC异构体的定点突变及其作用 乙醇对突变体的影响将被分析。我们将获得的知识是 对于阐明乙醇调节细胞周期的机制至关重要。 AC的活动性。将产生和分析的AC突变体系列 建议的研究将是未来研究的宝贵资源 AC分子的结构/功能关系。

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.