ARF PROTEINS IN INSULIN SIGNALLING

小GTP结合蛋白的ARF家族成员发挥着 在膜运输的调节中起重要作用， 被证明可以激活细胞磷脂酶D，一种重要的调节剂 细胞的功能和增殖是由不同类型的 细胞外信号。然而，细胞外信号的作用 对ARF蛋白激活状态的影响尚未研究。因此，在本发明中， 尽管ARF对磷脂酶D的激活有强烈的影响， 表明这些蛋白质的信号作用，它们的调节， 细胞外信号的活性仍有待证实。非常近期 PI实验室的工作表明，ARF激活可以 受到细胞外信号的刺激作为一个模型细胞， 过表达野生型人胰岛素受体，我们已经能够证明， 纯化的牛ARF被质膜激活， 胰岛素的作用显著增强。过度表达的细胞 突变的受体不显示这种行为。同样， 布雷菲德菌素A，一种破坏细胞内膜运输的药物， 其作用与抑制ARF激活有关， 显示阻断胰岛素指示的受体内化和胰岛素- 表明磷脂酶D的活化。详细调查了 胰岛素可能诱导ARF激活的可能机制揭示了 胰岛素受体和ARF共沉淀， 胰岛素-琼脂糖或特异性抗胰岛素受体抗体。这 共沉淀被鸟嘌呤核苷酸抑制， 让人想起异源三聚体G蛋白与它们的 特定受体建议继续进行这些研究， ARF蛋白与胰岛素受体相互作用的性质 信号系统拟议的研究将集中于以下方面 （a）东盟区域论坛与联合国 利用体外ARF活化和结合重建的胰岛素受体 B）分析在该实验室中最近开发的测定; 应用酵母双杂交技术研究受体与ARF的相互作用 系统; c）开发无细胞重建测定以研究 ARF蛋白在胰岛素介导的磷脂酶D活化中的作用 (PLD)d）通过以下方法开发ARF和PLD活化的肽抑制剂： 胰岛素处理的膜，其使用所选择的 ARF家族成员的区域;以及e）影响分析 胰岛素对受体内化、葡萄糖摄取和PLD激活的影响 使用过表达野生型和突变的ARF基因的细胞。

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 PI'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 been able to show that the activation of purified bovine ARF by plasma membranes is significantly enhanced by the effects of 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-indiced receptor internalization and insulin- indiced 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 cell-free reconstitution assay to study the role of ARF proteins in insulin-mediated activation of phospholipase D (PLD); d) development of peptide inhibitors of ARF and PLD activation by insulin-treated membranes using as a basis the sequence of selected regions of the members of the ARF family; and e) analysis of the effects of insulin on receptor internalization, glucose uptake and PLD activation using cells that overexpress wild type and mutated ARF genes.