STUDIES ON THE GENERATION OF INSULIN MEDIATORS

这项提案的主要目标是对 胰岛素介体生成机制的研究。初步工作显示 胰岛素刺激锚定在细胞内的蛋白质释放 磷脂酰肌醇-葡聚糖(PI-葡聚糖)膜 时间进程与推定的 胰岛素调节剂。此外，我们已经证明，胰岛素 刺激诱导这种介体在大脑中迅速出现 胞外培养液。基于这些和其他实验，使用 特定的蛋白酶和细胞磷脂酶C阻滞剂，它是 假设至少有一些胰岛素作用的介体 来源于PI-葡聚糖锚定的膜蛋白。一个 特定的机理，根据这两个水解步在 膜表面是产生介体所必需的 建议。该机制还假设存在一个 介体转运/摄取系统。本项目致力于 从实验上看，这一模型的关键方面。这些实验 旨在证明胰岛素促进卵裂素的切割 PI-葡聚糖锚定某些膜蛋白的研究 无论这种卵裂是否直接与 产生胰岛素介体，并鉴定至少部分 可能的蛋白质前体。此外，还建议 利用新陈代谢研究卵裂的具体机制 贴标签技术。最后，在一些非常有希望的基础上 初步数据显示，建议利用收盘优势 该变异体的PI-葡聚糖锚定的结构相似性 锥虫表面抗原与PI-葡聚糖锚定的关系 哺乳动物的蛋白质，以探索参与 胰岛素刺激这些蛋白质的释放和假说 介体摄取或转运的机制。

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.