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（GLUT 4）从细胞内储库重新定位到质膜，促进葡萄糖摄取到脂肪细胞和肌肉细胞中。易位涉及由三种SNARE- syntaxin 4，SNAP-23和VAMP 2-以及许多调节蛋白（包括Munc 18 c，synip和tomosyn）介导的囊泡融合步骤。虽然SNARE调节蛋白的生理重要性是清楚的，但由于细胞环境的复杂性，它们的分子作用机制和它们之间的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”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，问关键的机制问题GLUT 4囊泡融合，特别是有关调节蛋白如何单独或协同作用，以控制胞吐在分子水平上的问题。这项拟议研究背后的具体假设是，调节蛋白控制SNARE组装周期的不同阶段，并有助于GLUT 4囊泡融合的时空调节。调节蛋白将作为纯重组蛋白或在细胞表面上表达为翻转蛋白添加。每种调节剂的动力学效应可以在将其添加（单独或组合）到SNARE的核心融合机器时进行评估。提出了三个具体目标：1）确定Sec 1/Munc 18（SM）蛋白Munc 18 c对GLUT 4胞吐SNARE的复合物组装和融合动力学的每个阶段的影响; 2）建立胞吐特异性调节剂对SNARE蛋白组装、Munc 18 c-SNARE复合物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。长期目标是一个蛋白质接一个蛋白质地工作，直到我们能够重建GLUT 4胞吐作用的基本特性和微调。胰岛素调节的GLUT 4转运对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。了解SNARE调节器如何工作可能会为治疗干预确定新的靶点。由于GLUT 4转运的许多组分是保守的，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.