Action of Ethanol on Cyclic AMP Signal Transduction

描述(申请人提供)：这项研究的长期目标是阐明乙醇对中枢神经系统中环磷酸腺苷(CAMP)信号通路影响的分子和细胞机制。腺酰环化酶(AC)是一种产生cAMP的酶，它的活性被药理上相关浓度的乙醇以AC异构体特异性的方式增强。这种选择性表明，在cAMP生成系统中，AC是乙醇作用的主要目标。在这笔赠款的支持下，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的影响非常重要(乙醇响应域)，以及这些区域中可能导致乙醇增强效果的氨基酸残基。我们现在建议继续这个项目，通过检验乙醇通过直接与AC分子在特定结合位点相互作用来增强AC活性的假设(S)。在具体目标1中，利用cAMP积累实验中的一系列突变体AC7，我们将确定乙醇响应域中与乙醇效应有关的关键氨基酸残基，并确定每个残基的哪些物理化学性质是重要的。在具体目标2中，我们将使用细菌表达系统设计和生产重组AC7蛋白。我们将确定AC7催化域的三维结构，包括乙醇响应域，识别与乙醇相互作用的关键氨基酸残基，并利用核磁共振波谱检测乙醇对AC7构象的影响。这两个特定目标中提出的研究将相辅相成，以回答以下问题：1)乙醇反应区域中的哪些氨基酸残基对乙醇的作用重要，这些残基在蛋白质三维结构中的位置是什么？2)与乙醇结合的关键残基是什么，它们的功能贡献是什么？3)乙醇是否改变了乙醇反应区域的结构和动力学？如果是这样的话，哪些氨基酸残基参与了乙醇诱导的构象变化(S)，这些残基的功能贡献是什么？我们将获得的知识对于阐明乙醇调节AC活性的机制至关重要。这项研究将为未来针对AC分子的药物开发提供合理的基础。本研究所采用的方法也可以适用于酒精研究领域中其他重要蛋白质的研究。

DESCRIPTION (provided by applicant): 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.