ARF PROTEINS IN CELLULAR REGULATION OF PHOSPHOLIPASE D

描述(摘自申请人的摘要) ARF小GTP结合蛋白家族的成员发挥着重要的作用 在膜交通调节中的作用，最近已经被证明 激活细胞功能的重要调节物--细胞磷脂酶D 以及由不同类型的细胞外控制的增殖 信号。然而，细胞外信号对细胞状态的影响 ARF蛋白的激活还没有被研究。因此，虽然这些影响 ARF对磷脂酶D激活的强烈提示在 这些蛋白质，细胞外信号对其活性的调节 还有待于证明。《校长》的最新工作 研究人员的实验室表明，ARF的激活可以通过 细胞外信号。用过表达野生型的细胞作为模型细胞 人胰岛素受体，我们已经证明了纯化后的激活 胰岛素可增强质膜介导的牛ARF。细胞 过度表达突变的受体不会表现出这种行为。同样， 一种破坏细胞内膜的类异戊二烯Brefeldin A 并且其效果与抑制ARF激活有关， 已被证明可以阻断胰岛素诱导的受体内化和 磷脂酶D的激活。可能的详细调查 胰岛素可能诱导ARF激活的机制显示 胰岛素受体和ARF与固定化胰岛素琼脂糖凝胶共沉淀 带有特定的抗胰岛素受体抗体。这种共沉淀是 被鸟嘌呤核苷酸抑制，其方式使人想起相互作用 异源三聚体G蛋白及其特定受体。有建议说 继续这些研究以确定ARF相互作用的性质 与胰岛素受体信号系统有关的蛋白质。建议进行的研究 将侧重于以下几个方面：a)描述 ARF与胰岛素受体相互作用的体外研究 激活和结合重建分析是最近在这方面开发的 实验室；b)分析受体和ARF之间的相互作用 使用酵母双杂交系统；c)建立重组试验，以 体外研究生长因子对ARF的激活作用；d)培养 应用无细胞重组实验研究ARF蛋白在胰岛素抵抗中的作用 和生长因子介导的磷脂酶D的激活；以及e)测试 ARF蛋白在受体介导的PLD激活中的作用 激活的和阴性的ARF突变体在体外和在表达 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.