Regulation Mechanisms for the GTPase activating protein domain of plexins

描述（由申请人提供）：丛蛋白是脑信号蛋白轴突导向分子的跨膜受体。来自信号蛋白结合丛蛋白的排斥信号对于发育中的神经元的正确寻路和神经支配至关重要。丛状蛋白信号在调节细胞迁移、血管形成和免疫应答中也起重要作用。丛蛋白信号通路的功能障碍与多种疾病如神经系统疾病、癌症和自身免疫性疾病有关，并且丛蛋白已成为这些疾病的新药物靶标。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 GT3激活蛋白（GAP）结构域。差距域通过使R-Ras失活而有助于丛蛋白介导的轴突引导，这导致整联蛋白失活和细胞粘附丧失。丛蛋白GAP结构域通常保持无活性，并且其激活需要同时将脑信号蛋白和RhoGT 3（Rac 1、RhoD或Rnd 1）分别结合到受体的细胞外区域和细胞内RhoGT 3结合结构域（RBD）。本研究计划的目标是了解丛蛋白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方法来研究机制。我们已经解决了plexin A3的胞内结构域的晶体结构。结构表明差距域采用非活性构象，并表明RBD和N-末端片段有助于稳定这种自抑制状态。我们的结构分析还导致了一个假设，即丛蛋白的细胞内结构域可以形成一个特定的二聚体时，丛蛋白诱导二聚体的脑信号蛋白，并结合RhoGTdR的这种二聚体丛蛋白的RBD变构诱导差距域的构象变化，触发其激活。本提案围绕测试该模型展开。在目标1中，我们将使用生物化学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涉及二聚化和RhoGT 3结合的活化机制。在目的3中，我们将通过确定与RhoGTPases和R-Ras复合的丛蛋白胞内结构域的结构来研究差距结构域的激活机制。这些研究将揭示丛蛋白GAP结构域的自抑制和激活的分子基础，并为未来与丛蛋白功能障碍相关的疾病的药物设计提供新的途径。公共卫生相关性：丛蛋白是表达在细胞表面的重要信号蛋白，参与引导神经元的轴突到达其适当的目标，这对我们体内神经网络的发育至关重要。丛蛋白的功能障碍涉及各种疾病，如神经系统疾病、自身免疫性疾病和癌症。本研究的目的是阐明细胞内GT3激活蛋白结构域的调控机制，该结构域对丛蛋白的信号传导功能至关重要。

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.