Regulation Mechanisms for the GTPase activating protein domain of plexins

项目摘要/摘要 背景资料。神经丛蛋白是信号素轴突引导分子的跨膜受体。 来自信号素结合丛的排斥信号对于正确的路径发现和神经支配是至关重要的 发育中的神经元。丛状蛋白信号在调节细胞迁移、血管构型等方面也发挥着重要作用 和免疫反应。丛状蛋白信号通路的故障与多种疾病有关，如 作为神经疾病、癌症和自身免疫性疾病，以及网络蛋白已成为新的药物靶点 治疗这些疾病。丛蛋白信号转导的关键是它们的胞内区，其中包含一个R-RAS GTP酶激活蛋白(GAP)结构域。GAP结构域通过以下途径参与丛连蛋白介导的轴突引导 R-RAS失活，导致整合素失活和细胞黏附丧失。丛蛋白GAP结构域是 通常保持不活跃，它的激活需要信号素和RhoGTP酶的同时结合 (rac1、Rhod或RND1)的胞外区和胞内RhoGTP酶结合域(RBD)。 分别是受体。 目标。这项研究的目标是了解人类免疫缺陷的分子机制。 丛状间隙结构域的自身抑制和激活。 研究设计。我们将X射线结晶学与生化和细胞生物学相结合。 研究这些机制的方法。我们已经解决了丛状蛋白胞内结构域的晶体结构 A3.结构表明，GAP结构域采用非活性构象，表明RBD 而N-末端片段有助于稳定这种自抑制状态。我们对结构的分析 也导致了一种假设，即当网织蛋白被诱导为 通过信号素进行二聚，并将RhoGTP酶与该二聚体网络蛋白的RBDS结合变构诱导 GAP结构域中的构象变化触发其激活。这项提议的中心是 测试这个模型。 在目标1中，我们将使用生化间隙对自抑制机制进行突变分析。 化验和细胞化验。我们还将研究Plexin家族其他成员的晶体结构。 在目标2中，我们将使用相同的GAP分析和基于细胞的分析来测试激活机制 涉及二聚化和RhoGTP酶结合。 在目标3中，我们将通过确定GAP结构域的结构来研究GAP结构域的激活机制 RhoGTP酶与R-RAS的复合体中的网织蛋白胞内结构域。 这些研究将揭示神经丛蛋白自身抑制和激活的分子基础 GAP结构域，并为未来与丛蛋白功能障碍相关的疾病的药物设计提供了新的途径。

Project Summary/Abstract Background. Plexins are transmembrane receptors for the semaphorin axon guidance molecules. Repulsive signals from semaphorin-bound plexins are critical for proper pathfinding and innervation of developing neurons. Plexin signals also play important roles in regulating cell migration, vascular patterning and immune responses. Malfunction of the plexin signaling pathways is implicated in a variety of diseases such as neurological disorders, cancer and autoimmune diseases, and plexins have emerged as new drug targets for these diseases. Essential to the signaling of plexins is their intracellular regions, which contain a R-Ras GTPase activating protein (GAP) domain. The GAP domain contributes to plexin-mediated axon guidance by inactivating R-Ras, which leads to inactivation of integrin and loss of cell adhesion. The plexin GAP domain is normally kept inactive, and its activation requires simultaneous binding of semaphorin and a RhoGTPase (Rac1, RhoD or Rnd1) to the extracellular region and the intracellular RhoGTPase binding domain (RBD) of the receptor, respectively. Objectives. The goal of this research program is to understand the molecular mechanisms of autoinhibition and activation of the plexin GAP domain. Research Design. We use X-ray crystallography in combination with biochemical and cell biological approaches to study the mechanisms. We have solved the crystal structure of the intracellular domain of plexin A3. The structure shows that the GAP domain adopts an inactive conformation, and suggests that the RBD and a N-terminal segment contribute to stabilization of this autoinhibited state. Our analyses of the structures also led to a hypothesis that the plexin intracellular domain can form a specific dimer when plexin is induced to dimerize by semaphorin, and binding of a RhoGTPase to the RBDs of this dimeric plexin allosterically induces a conformational change in the GAP domain which triggers its activation. This proposal is centered around testing this model. In Aim 1 we will perform mutational analyses of the autoinhibition mechanism using a biochemical GAP assay and a cell-based assay. We will also pursue crystal structures of other plexin family members. In Aim 2 we will use the same GAP assay and cell-based assay to test the activation mechanism involving both dimerization and RhoGTPase binding. In Aim 3 we will study the activation mechanism for the GAP domain by determining structures of the plexin intracellular domains in complex with RhoGTPases and R-Ras. These studies together will reveal the molecular basis for the autoinhibition and activation of the plexin GAP domain, and provide new routes to future drug design for diseases associated with plexin malfunction.