DISSECTING G PROTEIN PATHWAYS

剖析 G 蛋白通路

基本信息

批准号：
8076367
负责人：
JOSHUA M KAPLAN
金额：
$ 40.08万
依托单位：
MASSACHUSETTS GENERAL HOSPITAL
依托单位国家：
美国
项目类别：
财政年份：
1997
资助国家：
美国
起止时间：
1997-08-01 至 2014-03-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8076367
关键词：
Address Aldicarb Binding Biochemical Biological Assay C-terminal Cellular biology Clathrin Complex Coupled Data Defect Dense Core Vesicle Dissociation Docking Dynamin Endocytosis Equilibrium Exocytosis Funding GTP-Binding Proteins Genes Genetic Goals Guanine Nucleotides Guanosine Triphosphate Health Mammals Membrane Membrane Proteins Modeling Monomeric GTP-Binding Proteins Mutation N-terminal Neuropeptides Optics Paralysed Pathway interactions Pattern Play Probability Process RNA Interference Recruitment Activity Recycling Regulation Role SH3 Domains SNAP receptor Scaffolding Protein Signal Transduction Specificity Synapses Synaptic Transmission Synaptic Vesicles Synaptic plasticity Testing base design insight interest mutant presynaptic rab3 GTP-Binding Proteins research study synaptojanin vesicle-associated membrane protein vesicular SNARE proteins

项目摘要

DESCRIPTION (provided by applicant): The long term goal of this project is to characterize pre-synaptic mechanisms regulating synaptic transmission, with a focus on G protein pathways. To maintain a stable pattern of synaptic transmission, the rates of SV exocytosis and endocytosis must remain in balance. Thus, G protein pathways that modulate synaptic transmission must produce coordinated changes in SV exocytosis and endocytosis. Here we propose to continue this project by analyzing how exocytosis and endocytosis are regulated. RAB-3 is a small GTPase that undergoes a cycle of association and dissociation with synaptic vesicles (SVs) in a manner that depends upon its bound guanine nucleotide. Prior studies showed that RAB-3 regulates SV docking, and the probability that an SV will fuse following depolarization. Despite its impact on synaptic transmission, relatively little is known about how RAB-3 is regulated. In the last funding period, we identified three new regulators of RAB-3, and showed that RAB-3 promotes both SV and dense core vesicle (DCV) secretion. We also initiated a new project characterizing the mechanism by which Endophilin, a conserved membrane associated protein, promotes SV endocytosis. Based on these preliminary data, we propose three new Aims. First, we will determine the mechanism by which Synaptobrevin and EGL-30 G1q regulate RAB-3. Second, we will screen for new regulators of RAB-3. Third, we will test the importance of Endophilin's two functional domains (the membrane binding N-Bar domain, and the SH3 domain). These experiments will allow us to distinguish between two competing models for Endophilin function. We will also determine how Endophilin is regulated by exocytosis. In summary, these Aims address two interesting aspects of presynaptic cell biology. The ability of synapses to operate over a wide range of signaling rates is dependent upon efficient coordination of the SV cycle of exo- and endocytosis. RAB-3 promotes SV fusion, and its regulation could provide a simple biochemical mechanism for producing synaptic plasticity. The experiments proposed here should give significant new insights into how RAB-3 is regulated, and how its activity is coupled to the SV cycle. Endophilin is required for SV endocytosis, and our preliminary results suggest that its availability at synapses is regulated by the rate of SV exocytosis. Thus, our analysis of Endophilin is likely to provide insights into how these two processes are coordinated. Since RAB-3 and Endophilin play analogous roles in mammalian synaptic transmission, it is likely that our results will also provide new insights into presynaptic mechanisms in mammals. PUBLIC HEALTH RELEVANCE: This proposal describes a coherent set of genetic, biochemical, and biophysical experiments designed to characterize pre-synaptic mechanisms regulating synaptic transmission. In particular, we propose to characterize how two Rab proteins (Rab3 and Rab27) are regulated, and how their activities are coupled to the synaptic vesicle cycle. We also will characterize the mechanism by which Endophilin promotes synaptic vesicle endocytosis.

描述（由申请人提供）：该项目的长期目标是表征调节突触传递的突触前机制，重点关注 G 蛋白途径。为了维持稳定的突触传递模式，SV 胞吐作用和内吞作用的速率必须保持平衡。因此，调节突触传递的 G 蛋白途径必须在 SV 胞吐作用和内吞作用中产生协调变化。在这里，我们建议通过分析胞吐作用和内吞作用的调节方式来继续这个项目。 RAB-3 是一种小型 GTP 酶，它以取决于其结合的鸟嘌呤核苷酸的方式经历与突触小泡 (SV) 的结合和解离循环。先前的研究表明，RAB-3 调节 SV 对接，以及 SV 在去极化后融合的可能性。尽管 RAB-3 对突触传递有影响，但人们对 RAB-3 的调控方式知之甚少。在上一个资助期，我们确定了 RAB-3 的三个新调节因子，并表明 RAB-3 促进 SV 和致密核心囊泡 (DCV) 的分泌。我们还启动了一个新项目，描述内亲蛋白（一种保守的膜相关蛋白）促进 SV 内吞作用的机制。根据这些初步数据，我们提出了三个新目标。首先，我们将确定 Synaptobrevin 和 EGL-30 G1q 调节 RAB-3 的机制。其次，我们将筛选新的RAB-3调节剂。第三，我们将测试 Endophilin 的两个功能域（膜结合 N-Bar 域和 SH3 域）的重要性。这些实验将使我们能够区分内亲蛋白功能的两种竞争模型。我们还将确定内亲蛋白如何通过胞吐作用进行调节。总之，这些目标解决了突触前细胞生物学的两个有趣的方面。突触在广泛的信号传导速率范围内运行的能力取决于外吞作用和内吞作用的 SV 周期的有效协调。 RAB-3促进SV融合，其调节可以为产生突触可塑性提供简单的生化机制。这里提出的实验应该为 RAB-3 的调节方式以及其活性如何与 SV 循环耦合提供重要的新见解。 SV 胞吞作用需要内亲蛋白，我们的初步结果表明，其在突触的可用性受到 SV 胞吐作用速率的调节。因此，我们对内亲蛋白的分析可能会提供有关这两个过程如何协调的见解。由于 RAB-3 和 Endophilin 在哺乳动物突触传递中发挥类似的作用，因此我们的结果很可能也将为哺乳动物的突触前机制提供新的见解。公共健康相关性：该提案描述了一系列连贯的遗传、生物化学和生物物理实验，旨在表征调节突触传递的突触前机制。特别是，我们建议描述两种 Rab 蛋白（Rab3 和 Rab27）的调节方式，以及它们的活性如何与突触小泡周期耦合。我们还将描述内亲蛋白促进突触小泡内吞作用的机制。