DISSECTING G PROTEIN PATHWAYS

剖析 G 蛋白通路

• 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
• 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

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播的突触前机制，重点是G蛋白途径。为了保持突触传播的稳定模式，必须保持平衡的SV外胞菌病和内吞作用的速率。因此，调节突触传播的G蛋白途径必须在SV胞吞和内吞作用中产生协调的变化。在这里，我们建议通过分析如何调节胞吐和内吞作用来继续该项目。 RAB-3是一个小的GTPase，它以取决于其结合的鸟嘌呤核苷酸的方式经历与突触囊泡（SV）的缔合和解离循环。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。尽管它对突触传播的影响，但对RAB-3的调节方式知之甚少。在最后一个资金期间，我们确定了RAB-3的三个新调节剂，并表明RAB-3促进了SV和密集的核心囊泡（DCV）分泌。我们还启动了一个新项目，该项目表征了这种机制，该机制是一种保守的膜相关蛋白来促进SV内吞作用。基于这些初步数据，我们提出了三个新目标。 首先，我们将确定突触和EGL-30 G1Q调节RAB-3的机制。其次，我们将筛选RAB-3的新调节器。第三，我们将测试内菲尔蛋白两个功能域（膜结合N-BAR结构域和SH3域）的重要性。这些实验将使我们能够区分两个竞争模型的内po蛋白功能。我们还将确定如何受到胞吐作用调节。 总而言之，这些目的涉及突触前细胞生物学的两个有趣方面。突触在广泛的信号传导速率上运行的能力取决于胞吞和内吞作用的SV周期的有效协调。 RAB-3促进SV融合，其调节可以提供一种简单的生化机制来产生突触可塑性。此处提出的实验应对RAB-3的调节方式以及其活性如何与SV周期耦合，给出重大的新见解。内吞和胞吞作用是必需的，我们的初步结果表明其突触的可用性受SV胞吐作用率调节。因此，我们对内po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和内菲尔在哺乳动物的突触传播中起着相似的作用，因此我们的结果可能还会为哺乳动物的突触前机制提供新的见解。 公共卫生相关性：该提案描述了一组连贯的遗传，生化和生物物理实验，旨在表征调节突触传播的突触前机制。特别是，我们建议表征如何调节两种RAB蛋白（RAB3和RAB27），以及它们的活性如何与突触囊泡周期耦合。我们还将表征内菲尔蛋白促进突触囊泡内吞作用的机制。