Regulation Mechanisms for the GTPase activating protein domain of plexins

项目总结/摘要 背景丛状蛋白是脑信号蛋白轴突导向分子的跨膜受体。 来自脑信号蛋白结合丛蛋白的排斥信号对于神经元的正确寻路和神经支配是至关重要的。 发育中的神经元丛蛋白信号在调节细胞迁移、血管形成等方面也发挥重要作用 和免疫反应。丛蛋白信号通路的功能障碍与多种疾病有关， 随着神经系统疾病、癌症和自身免疫性疾病的出现，丛蛋白已成为新的药物靶点， 对于这些疾病。对于丛蛋白的信号传导至关重要的是它们的细胞内区域，其含有R-Ras GTP酶激活蛋白（GAP）结构域。差距域有助于丛蛋白介导的轴突导向， R-Ras失活，导致整联蛋白失活和细胞粘附丧失。丛蛋白GAP结构域是 通常保持非活性，其激活需要同时结合脑信号蛋白和RhoGT 3 (Rac1、RhoD或Rnd 1）与细胞外区域和细胞内RhoGT 3结合结构域（RBD）的结合。 受体，分别。 目标.这项研究计划的目标是了解 丛蛋白GAP结构域的自身抑制和激活。 研究设计。我们将X射线晶体学与生物化学和细胞生物学相结合， 研究机制的方法。我们已经解决了丛蛋白胞内结构域的晶体结构 A3.结构表明差距域采用无活性构象，并表明RBD 和N-末端片段有助于这种自抑制状态的稳定。我们的结构分析 这也导致了一种假设，即当丛蛋白被诱导时，丛蛋白细胞内结构域可以形成特异性二聚体， 二聚化，并且RhoGT受体与这种二聚丛蛋白的RBD的结合变构诱导 差距域中触发其激活的构象变化。该提案围绕 测试这个模型。 在目标1中，我们将使用生物化学GAP对自抑制机制进行突变分析。 测定和基于细胞的测定。我们也将研究其他丛蛋白家族成员的晶体结构。 在目标2中，我们将使用相同的GAP测定和基于细胞的测定来测试激活机制 涉及二聚化和RhoGT结合。 在目的3中，我们将通过确定GAP结构域的结构来研究GAP结构域的活化机制。 与RhoGTP酶和R-Ras复合的丛蛋白胞内结构域。 这些研究将揭示丛蛋白自抑制和激活的分子基础 GAP结构域，并提供了新的途径，未来的药物设计与丛蛋白功能障碍的疾病。

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.