Regulation Mechanisms for the GTPase activating protein domain of plexins

描述（申请人提供）：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的排斥信号对于发育中的神经元的正确寻路和神经支配至关重要。神经丛蛋白信号还在调节细胞迁移、血管模式和免疫反应中发挥重要作用。神经丛蛋白信号通路的功能障碍与神经系统疾病、癌症和自身免疫性疾病等多种疾病有关，神经丛蛋白已成为治疗这些疾病的新药物靶点。丛蛋白信号传导的关键是它们的细胞内区域，其中包含R-Ras GTPase激活蛋白（GAP）结构域。GAP结构域通过使R-Ras失活来参与丛蛋白介导的轴突引导，从而导致整合素失活和细胞粘附丧失。丛蛋白GAP结构域通常处于失活状态，其激活需要信号蛋白和RhoGTPase （Rac1、RhoD或Rnd1）同时结合到受体的细胞外区域和细胞内RhoGTPase结合域（RBD）。本研究计划的目的是了解丛蛋白GAP结构域的自抑制和激活的分子机制。我们使用x射线晶体学结合生物化学和细胞生物学的方法来研究其机制。我们解出了丛蛋白A3胞内结构域的晶体结构。该结构表明GAP结构域采用非活性构象，并表明RBD和n端片段有助于稳定这种自抑制状态。我们的结构分析也导致了一个假设，即当信号素诱导丛蛋白二聚时，丛蛋白胞内结构域可以形成一个特定的二聚体，并且RhoGTPase与这种二聚体丛蛋白的rbd结合在变构上诱导GAP结构域的构象变化，从而触发其激活。这个建议的中心是测试这个模型。在目标1中，我们将使用生化GAP测定和基于细胞的测定对自抑制机制进行突变分析。我们还将研究丛蛋白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试验和基于细胞的试验来测试涉及二聚化和RhoGTPase结合的激活机制。在Aim 3中，我们将通过确定与RhoGTPases和R-Ras复合物中的丛蛋白胞内结构域的结构来研究GAP结构域的激活机制。这些研究将揭示丛蛋白GAP结构域自抑制和激活的分子基础，并为未来丛蛋白功能障碍相关疾病的药物设计提供新的途径。公共卫生相关性：丛蛋白是细胞表面表达的重要信号蛋白，参与引导神经元轴突到达适当的目标，这对我们体内神经网络的发育至关重要。神经丛蛋白的功能障碍与神经系统疾病、自身免疫性疾病和癌症等多种疾病有关。本研究的目的是阐明对丛蛋白信号功能至关重要的胞内GTPase激活蛋白域的调控机制。

DESCRIPTION (provided by applicant): 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. The goal of this research program is to understand the molecular mechanisms of autoinhibition and activation of the plexin GAP domain. 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. PUBLIC HEALTH RELEVANCE: Plexins are important signaling proteins expressed on the cell surface that participate in guiding the axons of neurons to their proper targets, which is critical for the development of the neural network in our bodies. Malfunction of plexins is implicated in various diseases such as neurological disorders, autoimmune diseases and cancer. The goal of this study is to elucidate the regulation mechanisms for the intracellular GTPase activating protein domain that is essential to the signaling function of plexins.