GENERATION OF INSULIN MEDIATORS

该提案的主要目标是描述 胰岛素介体产生的机制。 初步工作表明， 胰岛素刺激蛋白质的释放， 磷脂酰肌醇-聚糖（PI-聚糖）膜， 时间进程，这是一致的产生一个假定的 胰岛素介体。 此外，我们已经证明，胰岛素 刺激诱导这种介质在细胞中的快速出现， 细胞外介质 基于这些和其他实验， 特异性蛋白酶和细胞磷脂酶C阻断剂， 假设至少有一些胰岛素作用的介质 来源于PI-聚糖锚定的膜蛋白。 一 根据这两个水解步骤的具体机制， 膜表面是产生介质所必需的， 提出了 该机制还假设存在一个 介质转运/摄取系统。 该项目涉及 在实验上验证了这个模型的重要性。 实验 目的是证明胰岛素促进切割的 某些膜蛋白的PI-聚糖锚，以研究 无论这种分裂是否直接与 产生胰岛素介质，并确定至少一些 可能的蛋白质前体。 还拟议 研究使用代谢的特定机制的分裂 标签技术。 最后，根据一些非常有希望的 初步数据，建议利用接近 变体的PI-聚糖锚的结构相似性 锥虫的表面抗原与PI-聚糖锚 哺乳动物的蛋白质，以探测参与的酶， 胰岛素刺激这些蛋白质的释放， 介质摄取或运输的机制。

The main goal of this proposal is the characterization of the mechanism of insulin mediator generation. Preliminary work shows that insulin stimulates the release of proteins anchored to the membrane by phosphatidylinositol-glycan (PI-glycan) following a time course which is consistent with the generation of a putative insulin mediator. Furthermore, we have shown that insulin stimulation induces a rapid appearance of this mediator in the extracellular medium. Based on these and other experiments using specific protease and cellular phospholipase C blockers, it is hypothesized that at least some of the mediators of insulin action originate from the PI-glycan anchored membrane proteins. A specific mechanism according to which two hydrolytic steps at the membrane surface are required for the generation of mediators is proposed. This mechanism also hypothesizes the existence of a mediator transport/uptake system. This project addresses experimentally the critical aspects of this model. The experiments are aimed to demonstrate that insulin promotes the cleavage of the PI-glycan anchor of certain membrane proteins, to investigate whether or not this cleavage is directly associated to the generation of insulin mediators and to identify at least some of the possible protein precursors. It is also proposed to investigate the specific mechanisms of cleavage using metabolic labeling techniques. Finally, on the basis of some very promising preliminary data, it is proposed to take advantage of the close structural similarities of the PI-glycan anchor of the variant surface antigens of trypanosomes with the PI-glycan anchor of mammalian proteins in order to probe the enzymes involved in the insulin-stimulated release of these proteins and the hypothesized mechanisms of mediator uptake or transport.