Molecular Biology of the Synapse

突触的分子生物学

• 批准号: 6887098
• 负责人: EILEEN M. LAFER
• 金额: $ 33.76万
• 依托单位: UNIVERSITY OF TEXAS HLTH SCIENCE CENTER
• 依托单位国家: 美国
• 财政年份: 1987
• 资助国家: 美国
• 起止时间: 1987-04-01 至 2008-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6887098
• 关键词: Caenorhabditis elegans; binding sites; clathrin; endocytosis; molecular biology; neural transmission; phosphoproteins; protein binding; protein protein interaction; protein structure function; receptor binding; squid; synapses; synaptic vesicles; synaptotagmin; tissue /cell culture

DESCRIPTION (provided by applicant): The long-term goals of this project are to contribute to the determination of the molecular mechanisms which underlie neurotransmission. Synaptic transmission is a cycle of exo- and endocytosis. During the previous project period, we established that the clathrin pathway is essential for synaptic vesicle endocytosis, even during low physiological rates of stimulation. Furthermore, we elucidated a number of the molecular interactions which are required for synaptic vesicle recycling in vivo. In order to take this work to the next level, we believe two types of approaches are required. One is to utilize biophysical methods including X-ray crystallography, surface plasmon resonance, site-directed mutagenesis and solution biochemistry to elucidate mechanisms by which these complex interactions lead to vesicle assembly and uncoating. The other is to utilize electrophysiological methods to determine the temporal order of assembly of this macromolecular complex in living synapses. Therefore the proposal is organized around the following specific aims: Aim 1: Determine the time-course of a core set of protein-protein interactions that underlie neuronal endocytosis in living synapses. Aim 2: Characterize mechanisms of clathrin coated vesicle assembly and its regulation. We will evaluate the hypothesis that the large subunits of the adaptors contain multiple clathrin binding sites, evaluate the controversial hypothesis that the FxDxF and DPF sequence motifs that are involved in direct interactions with AP-2 are also involved in direct interactions with clathrin, determine where on clathrin TD the various different types of clathrin binding sites interact, and evaluate the hypothesis that Eps15 is a ruler that sets vertex-vertex/edge-edge distances in an assembling clathrin lattice. Aim 3: Characterize mechanisms of clathrin coated vesicle uncoating. We will determine how ATP hydrolysis in the ATPase domain of Hsc70 transmits a conformational change to the substrate binding domain which causes it to either bind or release substrate, determine the nature of the interaction between auxilin and Hsc70, elucidate the mechanism by which the J-domain stimulates the ATPase activity of Hsc70, and determine the role of the 'lid' opening and closing in the Hsc70 mechanism. Successful completion of these studies will allow us to achieve a deeper level of understanding of the fundamental processes of endocytosis and neurotransmission.

描述（由申请人提供）： 该项目的长期目标是为确定神经传递的分子机制做出贡献。突触传播是外吞和内吞作用的循环。在上一个项目期间，我们确定网状蛋白途径对于突触囊泡内吞作用至关重要，即使在低生理刺激速率中也是如此。此外，我们阐明了突触囊泡在体内回收所需的许多分子相互作用。为了将这项工作提升到一个新的水平，我们认为需要两种类型的方法。一种是利用生物物理方法，包括X射线晶体学，表面等离子体共振，定位诱变和溶液生物化学来阐明这些复杂相互作用导致囊泡组装和不涂层的机制。另一个是利用电生理方法来确定活体突触中这种大分子复合物的时间顺序。因此，该提案围绕以下特定目的组织：目标1：确定核心蛋白质蛋白相互作用的时间顺序，这些蛋白质蛋白相互作用是神经元内吞作用的蛋白质蛋白质相互作用。 AIM 2：表征网格蛋白涂层囊泡组件的机制及其调节。我们将评估以下假设：适配器的大亚基包含多个网格蛋白结合位点，评估有争议的假说，即FXDXF和DPF序列基序与与AP-2直接相互作用的序列基序也与ap-2直接相互作用涉及直接相互作用，这也参与了与克拉瑟蛋白直接相互作用在组装晶格晶格中设置顶点/边缘边缘距离的标尺。 AIM 3：表征网格蛋白涂层囊泡脱涂的机制。 We will determine how ATP hydrolysis in the ATPase domain of Hsc70 transmits a conformational change to the substrate binding domain which causes it to either bind or release substrate, determine the nature of the interaction between auxilin and Hsc70, elucidate the mechanism by which the J-domain stimulates the ATPase activity of Hsc70, and determine the role of the 'lid' opening and closing in the Hsc70 机制。这些研究的成功完成将使我们能够更深入地了解内吞和神经传递的基本过程。