Regulation of GLUT4 Exocytosis

描述(由申请人提供)： 胰岛素通过将葡萄糖转运蛋白4(GLUT4)从细胞内库重新定位到质膜上，促进脂肪细胞和肌肉细胞对葡萄糖的摄取。这种转位涉及一个囊泡融合步骤，该步骤由三个SNARs--Synaxin 4、SNAP-23和VAMP2-以及包括Munc18c、Synip和Tomosyn在内的一系列调节蛋白介导。虽然SNARE调节蛋白的生理重要性是明确的，但由于细胞环境的复杂性，它们的分子作用机制和功能相互作用尚不清楚。最近，我们在脂质体(合成双层)和翻转的SNARE细胞(天然膜)融合系统中重建了SNARE介导的融合。在这里，我建议利用这些独特的发展来提出关于GLUT4囊泡融合的关键机制问题，特别是关于调节蛋白如何在分子水平上单独或协同作用来控制胞吐的问题。这项研究背后的具体假设是，调控蛋白控制着SNARE组装周期的不同阶段，并有助于GLUT4囊泡融合的时间和空间调节。调控蛋白要么以纯重组蛋白的形式添加，要么以翻转蛋白的形式在细胞表面表达。当将每个调节器(单独或组合)添加到诱捕器的核心融合机械中时，可以评估每个调节器的动力学效应。提出了三个具体的目标：1)确定Sec1/Munc18(SM)蛋白Munc18c对GLUT4胞外SNARE复合体组装和融合动力学的各个阶段的影响；2)建立胞吐特异性调节因子对SNAREpin组装、Munc18c-SNARE复合体形成和融合动力学的影响；3)表征SNARE融合途径中的融合孔动力学和过渡态。我们的长期目标是一个蛋白质一个蛋白质地工作，直到我们能够重建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，这一过程中的失衡可能导致2型糖尿病。了解诱捕调节器是如何工作的，可能会确定治疗干预的新靶点。由于GLUT4转运的许多成分是保守的，我们的工作也可以揭示其他胞外途径，如血小板和肺上皮分泌。

DESCRIPTION (provided by applicant): 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 Munc18c, synip and tomosyn. While the physiological importance of the SNARE regulatory proteins are 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 SNARE-mediated fusion in both liposome (synthetic bilayers) and "flipped" SNARE cell (native membranes) fusion systems. Here I 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 regulation of GLUT4 vesicle fusion. Regulatory proteins will be added either as pure recombinant proteins or expressed as flipped proteins on the cell surface. Kinetic effects of each regulator can be assessed when it is added (alone or in combination) to the core fusion machinery of SNAREs. Three specific aims are proposed: 1) Define the effects of the Sec1/Munc18 (SM) protein Munc18c on each stage of complex assembly and fusion kinetics of GLUT4 exocytic SNAREs; 2) Establish the effects of exocytosis-specific regulators on SNAREpin assembly, Munc18c-SNARE complex formation and fusion kinetics; 3) Characterize the fusion pore dynamics and transition states in the SNARE fusion pathway. The long-term 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 the SNARE regulators work will likely identify novel targets for therapeutic intervention. Since many components of GLUT4 transport are conserved, our work can also shed light upon other exocytic pathways such as platelet and lung epithelial secretion.