MECHANISM OF ACTION OF DYNAMIN

DYNAMIN 的作用机制

• 批准号: 2444492
• 负责人: Richard Bert Vallee
• 金额: $ 21.82万
• 依托单位: UNIV OF MASSACHUSETTS MED SCH WORCESTER
• 依托单位国家: 美国
• 财政年份: 1979
• 资助国家: 美国
• 起止时间: 1979-07-01 至 1999-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2444492
• 关键词: clathrin; endocytosis; enzyme mechanism; gene mutation; genetic mapping; genetic techniques; guanine nucleotides; guanosinetriphosphatases; immunoprecipitation; intermolecular interaction; laboratory mouse; laboratory rat; molecular cloning; yeasts

Dynamin is a 100 kD GTPase identified in this laboratory which is involved in the initial stages of endocytosis. Mutations in dynamin and shibire,its Drosophila homologue, block the formation of coated and non-coated vesicles at the plasma membrane and the internalization of cell surface ligands and receptors. Mutations in shibire also block the reformation of synaptic vesicles at neuromuscular junctions and interneuronal synapses once the vesicles have discharged their contents. Dynamin has been found to interact with a number of macromolecular factors, most recently the SH3 domains of proteins involved in signal transduction, but which of these factors are involved in dynamin function in the cell remains uncertain. The long-term objective of this project is to understand the mechanism of action of dynamin, and, in particular, the steps in its GTPase cycle. To address this problem, we will take advantage of our extensive mutational analysis of the protein, and we will use a variety of molecular genetic, biochemical, ultrastructural and genetic approaches. We will seek to identify novel dynamin-interacting proteins and to characterize known dynamin interactions further. We will determine the dependence of dynamin interactions on the state of the guanine nucleotide in the dynamin active site, to help define the functional cycle of the protein. We will determine whether dynamin self-associates in the cell, and whether self- association activates the dynamin GTPase. We will characterize the association of dynamin with coated pits and other endocytic precursor structures by ultrastructural and biochemical means. Finally, we will attempt to identify a true dynamin homologue in yeast. An understanding of the dynamin functional cycle has a number of medical implications. This knowledge may ultimately lead to methods to control the entry of viruses, LDL, and other potentially deleterious agents into the cell. it may also provide a means to control the lifetime of activated growth factor receptors on the cell surface, and, therefore, a means to control normal and abnormal cell growth. Finally, because temperature-sensitive shibire mutations mimic certain conditional paralytic conditions of humans, an understanding of how dynamin functions could be of value in understanding and controlling these conditions.

发动蛋白是本实验室鉴定的一种100 kD的GT酶， 在胞吞作用的初始阶段。发动蛋白和Shibire的突变， 果蝇同源物，阻止被膜和非被膜的形成 质膜上的小泡和细胞表面的内化 配体和受体。Shibire的突变也阻碍了 神经肌肉接头和神经元间突触处的突触囊泡 一旦囊泡排出了它们的内容物。发动蛋白已被发现 与许多大分子因子相互作用，最近的SH3 参与信号转导的蛋白质结构域，但这些结构域中 参与细胞中发动蛋白功能的因素仍然不确定。 本项目的长期目标是了解 发动蛋白的作用，特别是其GT3循环中的步骤。到 解决这个问题，我们将利用我们广泛的突变， 分析蛋白质，我们将使用各种分子遗传学， 生物化学、超微结构和遗传学方法。我们将寻求 鉴定新动力蛋白相互作用蛋白并表征已知的 dynamin互动进一步。我们将确定发动蛋白 在发动蛋白活性中鸟嘌呤核苷酸状态的相互作用 位点，以帮助定义蛋白质的功能周期。我们将 确定发动蛋白是否在细胞中自缔合，以及是否自缔合。 结合激活发动蛋白GT3。我们将描述 发动蛋白与被膜小凹和其他内吞前体的结合 通过超微结构和生物化学手段来观察结构。最后我们将 试图在酵母中鉴定一种真正的发动蛋白同源物。了解 发动蛋白功能循环具有许多医学意义。这 这些知识可能最终导致控制病毒进入的方法， 低密度脂蛋白和其他潜在的有害物质进入细胞。它也可以 提供了一种控制活化生长因子寿命的方法 细胞表面的受体，因此，一种控制正常细胞的方法， 和异常细胞生长。最后，因为温度敏感的shibire 突变模仿人类的某些条件性麻痹状况， 了解发动蛋白的功能对于理解 控制这些条件。