Regulation Mechanisms for the GTPase activating protein domain of plexins

丛蛋白 GTPase 激活蛋白结构域的调控机制

• 批准号: 8310038
• 负责人: Xuewu Zhang
• 金额: $ 30.78万
• 依托单位: UT SOUTHWESTERN MEDICAL CENTER
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-09-30 至 2014-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8310038
• 关键词: Activation Analysis; Adopted; Autoimmune Diseases; Axon; Binding; Biochemical; Biological; Biological Assay; Biological Neural Networks; Blood Vessels; Cell Adhesion; Cell surface; Cells; Cellular Morphology; Complex; Development; Dimerization; Disease; Drug Delivery Systems; Drug Design; Family; Family member; Future; GTP Binding; GTPase-Activating Proteins; Goals; Guanosine Triphosphate Phosphohydrolases; Immune response; In Vitro; Individual; Integrins; Ligand Binding; Malignant Neoplasms; Mediating; Modeling; Molecular; Molecular Conformation; Mutation; N-terminal; Neurons; Pattern; Play; Regulation; Research; Research Design; Role; Route; Semaphorins; Signal Pathway; Signal Transduction; Signaling Protein; Structure; Subcellular structure; Tertiary Protein Structure; Testing; X-Ray Crystallography; abstracting; axon growth; axon guidance; base; cell motility; dimer; extracellular; member; mutant; nerve supply; nervous system disorder; plexin; programs; protein activation; ras GTPase-Activating Proteins; receptor; response; rho

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.

项目概要/摘要 背景。丛蛋白是信号蛋白轴突引导分子的跨膜受体。 来自信号蛋白结合丛蛋白的排斥信号对于正确的寻路和神经支配至关重要 发育中的神经元。 Plexin 信号在调节细胞迁移、血管模式方面也发挥着重要作用 和免疫反应。丛蛋白信号通路的功能障碍与多种疾病有关，例如 随着神经系统疾病、癌症和自身免疫性疾病，丛蛋白已成为新的药物靶点 对于这些疾病。丛蛋白的信号转导至关重要的是其细胞内区域，其中包含 R-Ras GTP 酶激活蛋白 (GAP) 结构域。 GAP 结构域有助于丛蛋白介导的轴突引导 使 R-Ras 失活，从而导致整合素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信号蛋白和 RhoGTPase (Rac1、RhoD 或 Rnd1) 的胞外区和胞内 RhoGTPase 结合域 (RBD) 分别为受体。 目标。该研究计划的目标是了解其分子机制 丛蛋白 GAP 结构域的自抑制和激活。 研究设计。我们将 X 射线晶体学与生化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。该结构表明 GAP 结构域采用非活性构象，并表明 RBD 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 还导致了一个假设，即当丛蛋白被诱导时，丛蛋白胞内结构域可以形成特定的二聚体 通过信号蛋白二聚化，并且 RhoGTPase 与该二聚体丛蛋白的 RBD 的结合以变构方式诱导 GAP 结构域的构象变化触发其激活。该提案围绕 测试这个模型。 在目标 1 中，我们将使用生化 GAP 对自抑制机制进行突变分析 测定和基于细胞的测定。我们还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 RhoGTPase 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。 丛蛋白胞内结构域与 RhoGTPases 和 R-Ras 形成复合物。 这些研究将共同​​揭示丛蛋白自身抑制和激活的分子基础 GAP 结构域，并为未来针对丛蛋白功能障碍相关疾病的药物设计提供新途径。