The role of the Exocyst complex in insulin regulated glucose transport

描述（由申请方提供）：胰岛素对葡萄糖转运的调节代表葡萄糖利用和储存的限速步骤，已知代表胰岛素抵抗患者的原发性病变。胞吐复合物首先在酵母中被鉴定为靶向胞吐的束缚位点，后来在我们的实验室中发现在葡萄糖转运的调节中发挥作用，将Glut 4囊泡靶向脂肪细胞中的对接和融合位点。这个建议的总体目标是探索外囊组装的分子细节及其在胰岛素刺激的葡萄糖转运中的作用。我们的初步数据表明，与最初的设想相反，外囊可能组装在两个亚复合物中，一个目标（t-）复合物聚集在质膜上，一个囊泡（v-）复合物在Glut 4囊泡上组装。我们假设每个子复合物的组装是由不同的G蛋白激活不同的过程控制，然后统一之前囊泡对接。我们将通过解剖脂肪细胞的分子组成来探讨这一假说。我们将首先研究t-外囊的组装，表征这些蛋白质在体外和细胞中的分子相互作用，检查组装的顺序和脂筏作为组装位点的作用。然后，我们将表征的v-外囊上组装的Glut 4囊泡，评估该复合物的形成的调节。最后，我们将探讨G蛋白RalA在这一过程中的作用，研究它是如何被胰岛素激活的，以及它的分子效应器，介导它的能力，统一整个复杂的囊泡贩运到质膜。我们预计，这些数据将提供一个更清晰的画面的调节Glut 4贩运，对接和融合。

DESCRIPTION (provided by applicant): The regulation of glucose transport by insulin represents the rate limiting step in glucose utilization and storage, and is known to represent a primary lesion in patients with insulin resistance. The exocyst complex was first identified in yeast as a tethering site for targeted exocytosis, and later found in our laboratory to play a role in the regulation of glucose transport, targeting Glut4 vesicles to sites of docking and fusion in fat cells. The overall goal of this proposal is to explore the molecular details of exocyst assembly and its role in insulin-stimulated glucose transport. Our preliminary data suggest that, contrary to what was originally conceived, the exocyst may be assembled in two subcomplexes, a target (t-) complex that comes together at the plasma membrane, and a vesicular (v-) complex that assembles at Glut4 vesicles. We hypothesize that the assembly of each sub-complex is controlled by distinct G proteins activated by a different process, and then unified prior to vesicle docking. We will explore this hypothesis by dissecting the molecular components in adipocytes. We will first study the assembly of the t-exocyst, characterizing the molecular interactions of these proteins in vitro and in cells, examine the order of assembly and the role of lipid rafts as an assembly site. We will then characterize the v-exocyst that assembles on Glut4 vesicles, evaluating the regulation of the formation of this complex. Finally, we will explore the role of the G-protein RalA in this process, examining how it is activated by insulin, and its molecular effectors that mediate its ability to unify the entire complex upon vesicle trafficking to the plasma membrane. We anticipate that these data will present a clearer picture of the regulation of Glut4 trafficking, docking and fusion.