Regulation of GLUT4 Exocytosis

胰岛素通过重新定位葡萄糖促进脂肪细胞和肌肉细胞对葡萄糖的摄取 在一些实施方案中，GLUT 4是从细胞内储库到质膜的转运蛋白4（GLUT 4）。易位 涉及由三种SNARE-突触融合蛋白4、SNAP-23和 VAMP 2-和许多调节蛋白，包括MunciSc，synip和tomosyn。而 SNARE调节蛋白的生理重要性是清楚的，它们的分子机制是 它们之间的作用和功能相互作用是不知道的，由于复杂性， 细胞环境最近，我们重建了SNARE依赖的GLUT 4囊泡融合， 脂质体（合成双层）和“翻转”SNARE细胞-细胞（天然膜）融合 系统.在这里，我们建议利用这些独特的发展， 关于GLUT 4囊泡融合的问题，特别是关于调控蛋白如何 单独或协同作用，在分子水平上控制胞吐作用。背后的具体假设 这项拟议的研究是，调节蛋白控制SNARE组装的不同阶段， 循环，并有助于GLUT 4胞吐的时空调控。监管 蛋白质将作为纯重组蛋白或作为翻转蛋白表达加入， 细胞表面当添加调节剂（单独）时，可以评估每种调节剂的动力学效应 或组合）到SNARE的核心聚变机器。提出了两个具体目标： 定义SNARE和调节因子的协同作用如何控制GLUT 4囊泡融合; 2)表征“翻转”中GLUT 4胞吐的融合孔动力学和过渡态 SNARE融合系统。我们的长期目标是一个蛋白质一个蛋白质地提高， 重建GLUT 4胞吐的基本性质和微调。胰岛素调节GLUT 4 转运对于葡萄糖稳态至关重要，这一过程的失衡可能导致2型糖尿病。 糖尿病了解SNARE调节剂如何工作可以确定治疗的新靶点 干预由于GLUT 4转运的许多组分是保守的，我们的工作也可以被用来研究GLUT 4转运。 对其他胞吐途径如血小板和肺上皮分泌的影响。

Insulin facilitates glucose uptake into adipocytes and muscle cells by relocating glucose transporter 4 {GLUT4) from intracellular reservoirs to the plasma membrane. The translocation involves a vesicle fusion step that is mediated by three SNAREs - syntaxin 4, SNAP-23 and VAMP2 - and a number of regulatory proteins including MunciSc, synip and tomosyn. While the physiological importance of the SNARE regulatory proteins is clear, their molecular mechanisms of action and functional interactions among themselves are not known due to the complexity of the cellular environment. Recently, we reconstituted the SNARE-dependent GLUT4 vesicle fusion in both liposome (synthetic bilayers) and "flipped" SNARE cell-cell (native membranes) fusion systems. Here we propose to capitalize on these unique developments to ask key mechanistic questions about GLUT4 vesicle fusion, especially questions concerning how regulatory proteins act, alone or in concert, to control exocytosis at the molecular level. The specific hypothesis behind this proposed research is that regulatory proteins control different stages of the SNARE assembly cycle and contribute to the temporal and spatial Vegulation of GLUT4 exocytosis. Regulatory proteins will be added either as pure recombinant proteins or expressed as flipped proteins on th& cell surface. Kinetic effects of each regulator can be assessed when the regulator is added (alone or in combination) to the core fusion machinery of SNAREs. Two specific aims are proposed: 1) Define how the concerted action of SNAREs and regulatory factors controls GLUT4 vesicle fusion; 2) Characterize the fusion pore dynamics and transition state of GLUT4 exocytosis in the "flipped" SNARE fusion system. Our long-temn goal is to work our way up, protein by protein, until we can reconstitute the basic properties and fine-tuning of GLUT4 exocytosis. Insulin-regulated GLUT4 transport is crucial for glucose homeostasis, and imbalances in this process may lead to type 2 diabetes. Knowledge of how SNARE regulators work may Identify novel targets for therapeutic intervention. Since many components of GLUT4 transport are conserved, our work can also dhed light upon other exocytic pathways such as platelet and lung epithelial secretion.