ARF PROTEINS IN INSULIN SIGNALLING

ARF家族成员的小GTP结合蛋白发挥作用 在膜交通调节中的重要作用，最近已经 被证明能激活细胞磷脂酶D，这是一种重要的调节剂 由多种类型控制的细胞功能和增殖 细胞外信号。然而，细胞外信号的影响 关于ARF蛋白激活状态的研究尚未见报道。因此， 尽管ARF对磷脂酶D的激活有强烈的影响 暗示了这些蛋白质的信号作用，对它们的调节 细胞外信号的活性仍有待证实。非常近 PI实验室的研究表明，ARF的激活可以 受到细胞外信号的刺激。使用作为模型单元的 过度表达野生型人类胰岛素受体，我们已经能够证明 质膜对纯化的牛ARF的激活作用 胰岛素的作用显著增强。过度表达的细胞 突变的受体不会表现出这种行为。同样，类异戊二烯 Brefeldin A，一种扰乱细胞内膜运输和 其作用与抑制ARF激活有关，一直以来 显示能阻断胰岛素受体内化和胰岛素- 磷脂酶D的激活。详细调查 胰岛素可能诱导ARF激活的可能机制被揭示 胰岛素受体和ARF在固定化条件下共沉淀 胰岛素琼脂糖凝胶或特异性抗胰岛素受体抗体。这 鸟嘌呤核苷酸在一定程度上抑制共沉淀 让人想起异源三聚体G蛋白与它们的 特定的受体。建议继续进行这些研究，以确定 ARF蛋白与胰岛素受体相互作用的本质 信令系统。拟议的研究将集中于以下几个方面 各方面：a)区域合作框架与 胰岛素受体在体外ARF激活和结合重组中的应用 本实验室新近开发的化验方法；b)分析 利用酵母双杂交技术研究受体与ARF的相互作用 系统；c)建立一种无细胞重组试验，以研究 ARF蛋白在胰岛素介导的磷脂酶D激活中的作用 (PLD)；d)开发ARF和PLD激活的多肽抑制剂 以所选序列为基础的胰岛素处理膜 次区域论坛大家庭成员的区域；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 使用过度表达野生型和突变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.