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摄取葡萄糖 转运蛋白4(GLUT4)从胞内库到质膜。易位 涉及由三个SNARs介导的囊泡融合步骤--Synaxin 4、SNAP-23和SNAP-23 VAMP2-和一些调节蛋白，包括MunciSc、Synip和Tomosyn。而当 SNARE调节蛋白的生理重要性是明确的，它们的分子机制是 它们之间的动作和功能交互是未知的，因为 蜂窝环境。最近，我们重组了SNARE依赖的GLUT4囊泡融合 脂质体(合成双层)和“翻转”SNARE细胞-细胞(自然膜)融合 系统。在这里，我们建议利用这些独特的发展来询问关键机制 关于GLUT4囊泡融合的问题，特别是关于调节蛋白如何 单独或协同行动，在分子水平上控制胞吐。背后的具体假设 这项拟议的研究是调控蛋白控制圈套组装的不同阶段。 循环并参与GLUT4胞吐的时空变化。监管 蛋白质将作为纯重组蛋白或以翻转蛋白的形式在TH& 细胞表面。当添加调节器(单独)时，可以评估每个调节器的动力学效果 或结合在一起)到圈套的核心融合机器。提出了两个具体目标：1) 确定SNARs和调节因子的协同作用如何控制GLUT4囊泡融合； 2)表征翻转后GLUT4胞吐的融合孔动力学和过渡态 诱捕融合系统。我们的长期TEMN目标是一种又一种蛋白质，直到我们能够 重构GLUT4胞吐的基本性质和微调。胰岛素调节的GLUT4 转运对葡萄糖稳态至关重要，这一过程中的不平衡可能导致2型 糖尿病。了解诱捕调节剂的工作原理可能会确定治疗的新靶点 干预。由于GLUT4运输的许多组件是保守的，我们的工作也可以 其他胞外途径，如血小板和肺上皮细胞分泌。

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.