EFFECT OF ETHANOL ON G-PROTEINS

描述：（改编自研究者摘要） 乙醇激活几类G蛋白，导致刺激 几种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。在长期的基础上，乙醇诱导深刻的 不同G蛋白表达的改变， 后果乙醇影响G蛋白活性的机制， 表达只是部分理解。最近的研究表明， 乙醇显著干扰动态G蛋白棕榈酰化， 重要的质膜定位和调节的相互作用， α亚单位和效应子这一提议的总体假设是， 乙醇导致的G蛋白棕榈酰化循环紊乱， 其对G蛋白活性和表达的影响。这些问题将 在神经元NG-108细胞和培养的大鼠小脑颗粒细胞中研究 在基础和受体刺激条件下。α的棕榈酰化 将研究功能上受乙醇影响的亚基（Gs，Gi和 Gq）。乙醇抑制的可能位点包括棕榈酰 酰基硫酯酶（脱棕榈酰化）和棕榈酰酰基转移酶 （棕榈酰化）。乙醇对棕榈酰α亚基库大小的影响 将通过HPLC测定。急性乙醇中毒的功能后果 将通过分析α亚单位在细胞中的分布来评估效果。 细胞质和质膜中。突变cDNA方法将用于 评估G蛋白棕榈酰化对效应物刺激的作用 使用完整的细胞和质膜制备物。我们将探讨 假设慢性乙醇治疗减少了特定的 棕榈酰化的G蛋白可用于效应刺激，包括 这是一个似乎与酪氨酸激酶受体相关的特异性池。的 α亚基的池大小将在来自 在体外用乙醇慢性处理神经细胞，并且在获得的细胞中， 从长期喂食乙醇的老鼠身上。最后，慢性酒精的影响 将对各种G蛋白的动态棕榈酰化进行评估 班总之，拟议的研究旨在 乙醇诱导的一个关键信号转导家族的结构改变 分子导致急性和慢性基础上的不同功能作用。

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.