Action of Ethanol on Cyclic AMP Signal Transduction

这项研究的长期目标是阐明 乙醇对环腺苷影响的分子和细胞机制 中枢神经系统中的一磷酸(CAMP)信号通路。腺苷酰的活性 环化酶(AC)，产生cAMP的酶，通过药理上的相关作用而增强 乙醇浓度以AC异构体特有的方式。这种选择性表明，在 CAMP生成系统，AC是乙醇作用的主要靶点。在此之前的支持下 Grant，我们已经确定了7型AC(AC7)的三个离散区域，这些区域对乙醇对 其活性(乙醇响应区)，以及这些区域内的氨基酸残基 可能对乙醇的增强作用负责。我们现在提议继续这个项目 通过检验乙醇通过直接与AC分子相互作用来增强AC活性的假设 特异性结合位点(S)。 在特定的目标1中，使用一系列突变型AC7在cAMP积累试验中，我们将鉴定 乙醇反应区域中的关键氨基酸残基是导致 乙醇，并确定每个残留物的哪些物理化学性质是重要的。以特定的目标 2、我们将利用细菌表达系统设计和生产重组AC7蛋白。我们会 确定AC7包括乙醇在内的催化域的三维结构 响应区域，确定与乙醇相互作用中涉及的关键氨基酸残基，以及 用核磁共振波谱研究乙醇对AC7构象的影响。建议进行的研究 在这两个具体目标中将相辅相成地回答以下问题：1)哪一种氨基 乙醇响应区中的酸残基对乙醇的作用很重要，那么 这些残基在蛋白质三维结构中的位置？2)关键是什么 残基参与与乙醇的结合，它们的功能贡献是什么？3)乙醇 改变乙醇响应域的结构和动力学？如果是这样，哪种氨基酸 残基参与乙醇诱导的构象变化(S)，其功能是什么？ 这些残留物的贡献呢？我们将获得的知识对于阐明这种机制是至关重要的 乙醇通过它来调节AC的活性。拟议的研究将为以下方面提供合理的基础 针对AC分子的未来药物开发。这项研究采用的方法也可以是 适应酒精研究领域中其他重要蛋白质的研究。

The long-term goal of this research is to elucidate the molecular and cellular mechanisms underlying the effects of ethanol on the cyclic adenosine monophosphate (cAMP) signaling pathway in the central nervous system. The activity of adenylyl cyclase (AC), the enzyme that generates cAMP, is enhanced by pharmacologically relevant concentrations of ethanol in an AC isoform-specific manner. This selectivity indicates that within a cAMP generating system, AC is a primary target of ethanol's action. With previous support from this grant, we have identified three discrete regions of type7 AC (AC7) important for the effect of ethanol on its activity (ethanol responsive domains), as well as the amino acid residues within these regions that are potentially responsible for the enhancing effect of ethanol. We now propose to continue this project by testing the hypothesis that ethanol enhances AC activity by directly interacting with AC molecules at specific binding site(s). In Specific Aim 1, using a series of mutant AC7s in the cAMP accumulation assay, we will identify crucial amino acid residues in the ethanol responsive domains that are responsible for the effect of ethanol and determine which physicochemical properties of each residue are important. In Specific Aim 2, we will design and produce recombinant AC7 proteins using a bacterial expression system. We will determine the three dimensional structure of the catalytic domains of AC7 including the ethanol responsive domains, identify key amino acid residues involved in the interaction with ethanol, and examine the effect of ethanol on the conformation of AC7 using NMR spectroscopy. Studies proposed in the two Specific Aims will complement each other to answer the following questions: 1) Which amino acid residues in the ethanol responsive domains are important for ethanol's effect, and what are the locations of these residues in the three dimensional structure of the protein? 2) What are the key residues involved in binding to ethanol, and what are their functional contributions? 3) Does ethanol change the structure and dynamics of the ethanol responsive domains? If so, which amino acid residues are involved in ethanol-induced conformational change(s), and what is the functional contribution of those residues? The knowledge we will obtain is crucial for elucidating the mechanism by which ethanol modulates the activity of AC. The proposed research will provide a rational basis for future drug development targeting AC molecules. The approach employed in this research can also be adapted to the study of other proteins important in the alcohol research field.