EFFECT OF ETHANOL ON G-PROTEINS

描述：(改编自《调查者摘要》) 乙醇激活几类G-蛋白，从而刺激 几个细胞内信号转导通路，包括腺苷环化酶， 磷脂酶C和离子通道。在慢性基础上，乙醇会导致深刻的 与功能相关的多种G蛋白表达的变化 后果。乙醇影响G蛋白活性和G蛋白活性的机制 人们对这一表达只有部分理解。最近的研究表明， 乙醇显著干扰动态G蛋白棕榈酰化，这是 重要的质膜定位和调节相互作用 带有效应器的Alpha子单元。这项提议的总体假设是 乙醇对G蛋白棕榈酰化循环的干扰 它对G蛋白活性和表达的影响。这些问题将是 神经元NG-108细胞和培养的大鼠小脑颗粒细胞的研究 在基础和受体刺激条件下。α-棕榈酰化 将研究受乙醇影响的亚基(Gs、Gi和 GQ)。可能的乙醇抑制部位包括棕榈酰基。 酰基硫酯酶(脱氨基)和棕榈酰转移酶 (棕榈酰化)。乙醇对棕榈酰α亚基池大小的影响 将通过高效液相色谱来测定。急性乙醇的功能后果 将通过分析阿尔法亚基在体内的分布来评估效果 胞浆和质膜中。突变的cdna方法将用于 评估G蛋白棕榈酰化在刺激效应器中的作用 使用完整的细胞和质膜制剂。我们将探索 假设慢性乙醇治疗会减少特异性库 可用于效应器刺激的棕榈酰化G蛋白，包括 似乎与酪氨酸激酶受体相关的特异池。这个 α亚基的池大小将在膜和胞浆中测量，从 乙醇处理的神经细胞在体外和在获得的细胞中 来自长期喂食乙醇的老鼠。最后，慢性酒精的影响 动态棕榈酰化治疗将评估各种G蛋白 上课。总而言之，拟议的研究旨在与 乙醇诱导的一个关键信号转导家族的结构改变 对急性和慢性碱基产生不同功能影响的分子。

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.