EFFECT OF ETHANOL ON G-PROTEINS

乙醇对 G 蛋白的影响

• 批准号: 6371829
• 负责人: Raphael Rubin
• 金额: $ 24.84万
• 依托单位: THOMAS JEFFERSON UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2000
• 资助国家: 美国
• 起止时间: 2000-09-19 至 2005-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6371829
• 关键词: G protein; biological signal transduction; cell line; drug adverse effect; ethanol; fatty acylation; high performance liquid chromatography; palmitates; protein structure function; tissue /cell culture

DESCRIPTION: (Adapted from the Investigator's Abstract) Ethanol activates several classes of G-proteins leading to the stimulation of several intracellular signal transduction pathways, including adenylyl cyclase, phospholipase C and ion channels. On a chronic basis, ethanol induces profound alterations in the expression of diverse G-proteins with associated functional consequences. The mechanisms by which ethanol affects G-protein activity and expression is only partially understood. Recent studies have demonstrated that ethanol markedly interferes with dynamic G-protein palmitoylation, which is important for plasma membrane localization and modulates the interaction of alpha subunits with effectors. The overall hypothesis of this proposal is that the derangement of the G-protein palmitoylation cycle by ethanol contributes to its effects on G-protein activity and expression. These issues will be investigated in neuronal NG-108 cells and cultured rat cerebellar granule cells under basal and receptor-stimulated conditions. Palmitoylation of the alpha subunits that are functionally affected by ethanol will be studied (Gs, Gi and Gq). Possible sites of ethanol inhibition to be studied included palmitoyl acylthioesterase (depalmitoylation) and palmitoyl acyltransferase (palmitoylation). The effect of ethanol on palmitoyl alpha subunit pool sizes will be determined by HPLC. The functional consequences of the acute ethanol effect will be assessed by analyzing the distribution of alpha subunits in the cytosol and in the plasma membranes. A mutational cDNA approach will be used to assess the role of G-protein palmitoylation on the stimulation of effectors using intact cells and plasma membrane preparations. We will explore the hypothesis that chronic ethanol treatment reduces the specific pool of palmitoylated G-protein available for effector stimulation, including a specific pool that appears to be associated with tyrosine kinase receptors. The pool sizes of alpha subunits will be measured in membranes and cytosol from neural cells treated chronically with ethanol in vitro, and in cells obtained from rats fed ethanol chronically. Finally, the effect of chronic ethanol treatment on dynamic palmitoylation will be assessed for various G-protein classes. In summary, the proposed studies are designed to relate ethanol-induced structural alterations in a family of key signal transduction molecules leading to diverse functional effects on acute and chronic bases.

描述：（根据调查员的摘要进行了改编） 乙醇激活几类G蛋白，导致刺激 几个细胞内信号转导途径，包括腺苷酸环化酶， 磷脂酶C和离子通道。在慢性的基础上，乙醇诱导了深刻的 与相关功能的不同G蛋白表达的改变 结果。乙醇影响G蛋白活性的机制和 表达仅被部分理解。最近的研究表明 乙醇明显干扰了动态的G蛋白棕榈酰化，这是 对于质膜定位至关重要，并调节相互作用 带有效应子的α亚基。该提议的总体假设是 乙醇对G蛋白棕榈酰化周期的危险有助于 它对G蛋白活性和表达的影响。这些问题将是 在神经元NG-108细胞和培养的大鼠小脑颗粒细胞中进行了研究 在基础和受体刺激的条件下。阿尔法的棕榈酰化 将研究受乙醇功能影响的亚基（GS，GI和 GQ）。可能研究的乙醇抑制部位包括棕榈酰 酰基三酰酯酶（去氨木酰化）和棕榈酰酰基转移酶 （棕榈酰化）。乙醇对棕榈酰α亚基池尺寸的影响 将由HPLC确定。急性乙醇的功能后果 将通过分析α亚基的分布来评估效果 细胞质和质膜中。突变cDNA方法将用于 评估G蛋白棕榈酰化在刺激效应子中的作用 使用完整的细胞和质膜制剂。我们将探索 假设慢性乙醇治疗减少了特定库 棕榈酰化的G蛋白可用于效应子刺激，包括A 似乎与酪氨酸激酶受体有关的特定池。这 将在膜和细胞质中测量α亚基的池尺寸 在体外用乙醇慢性处理的神经细胞，并在获得的细胞中 从长期喂养乙醇的大鼠。最后，慢性乙醇的作用 将评估各种G蛋白的动态棕榈酰化治疗 课程。总而言之，拟议的研究旨在与 关键信号转导家族的乙醇诱导的结构改变 分子导致对急性和慢性碱基的功能作用。