ARF PROTEINS IN CELLULAR REGULATION OF PHOSPHOLIPASE D

描述（摘自申请人摘要） ARF家族的小GTP结合蛋白的成员发挥重要作用， 在调节膜运输中的作用，最近已被证明， 激活细胞磷脂酶D，一种重要的细胞功能调节剂 以及由各种类型的细胞外 信号. 然而，细胞外信号对细胞状态的影响， 尚未研究ARF蛋白的活化。 因此，虽然影响 ARF对磷脂酶D激活的影响强烈表明ARF对磷脂酶D的信号作用。 这些蛋白质，通过细胞外信号调节其活性， 还有待证明。 最近的工作在校长 研究者的实验室已经表明，ARF激活可以刺激 细胞外信号 使用过表达野生型的细胞作为模型 人胰岛素受体，我们已经表明，激活纯化的 牛ARF通过质膜被胰岛素增强。 的细胞 过表达突变的受体不显示这种行为。 同样， 类异戊二烯Brefeldin A，一种破坏细胞内膜的试剂 其作用与抑制ARF激活有关， 已显示阻断胰岛素诱导的受体内化， 磷脂酶D的活化。详细调查了 胰岛素可能诱导ARF激活的机制表明， 胰岛素受体和ARF与固定化胰岛素-琼脂糖共沉淀或 有特异性抗胰岛素受体抗体 该共沉淀是 抑制鸟嘌呤核苷酸的方式让人想起的相互作用 异源三聚体G蛋白与其特异性受体的结合。 拟 继续这些研究，以确定ARF相互作用的性质， 胰岛素受体信号系统的蛋白质。 拟议的研究 将侧重于以下几个方面： ARF与胰岛素受体的相互作用 激活和结合重建分析最近在这方面发展起来， 实验室; B）受体和ARF之间的相互作用的分析 使用酵母双杂交系统; c）开发重建测定， 体外研究生长因子激活ARF; d）开发一种 无细胞重建试验，研究ARF蛋白在胰岛素- 和生长因子介导的磷脂酶D的活化;和 ARF蛋白在受体介导的PLD激活中的作用 在体外和表达ARF的细胞中， 突变的ARF蛋白。

DESCRIPTION (Taken from the applicant's Abstract) The members of the ARF family of small GTP-binding proteins play important roles in the regulation of membrane traffic and recently have been shown to activate cellular phospholipase D, an important modulator of cell function and proliferation that is controlled by various types of extracellular signals. However, the effects of extracellular signals on the state of activation of ARF proteins has not been studied. Thus, although the effects of ARF on phospholipase D activation strongly suggest a signalling role for these proteins, the regulation of their activity by extracellular signals remains to be demonstrated. Very recent work in the Principal Investigator's laboratory has shown that ARF activation can be stimulated by extracellular signals. Using as a model cells that overexpress wild type human insulin receptors, we have shown that the activation of purified bovine ARF by plasma membranes is enhanced by insulin. Cells that overexpress mutated receptors do not show this behavior. Likewise, the isoprenoid Brefeldin A, an agent that disrupts intracellular membrane traffic and whose effects are linked to the inhibition of ARF activation, has been shown to block insulin-induced receptor internalization and activation of phospholipase D. A detailed investigation of the possible mechanisms by which insulin might induce ARF activation revealed that the insulin receptor and ARF co-precipitate with immobilized insulin-agarose or with specific anti-insulin receptor antibodies. This co-precipitation was inhibited by guanine nucleotides in a manner reminiscent of the interactions of heterotrimeric G proteins with their specific receptors. It is proposed to continue these studies to determine the nature of the interactions of ARF proteins with the insulin receptor signalling system. The proposed studies will focus on the following aspects: a) characterization of the interactions between ARF and the insulin receptor using in vitro ARF activation and binding reconstitution assays recently developed in this laboratory; b) analysis of the interactions between the receptor and ARF using a yeast two-hybrid system; c) development of a reconstitution assay to study ARF activation by growth factors in vitro; d) development of a cell-free reconstitution assay to study the role of ARF proteins in insulin- and growth factor-mediated activation of phospholipase D; and e) test the role of ARF proteins in receptor-mediated PLD activation using dominant activated and negative ARF mutants in vitro and in cells that express the mutated ARF protein.