Regulation Mechanisms for the GTPase activating protein domain of plexins

描述(申请人提供)：神经丛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发育中神经元的正确寻路和神经支配是至关重要的。丛状蛋白信号在调节细胞迁移、血管构型和免疫反应方面也发挥着重要作用。丛状蛋白信号通路的功能障碍与多种疾病有关，如神经系统疾病、癌症和自身免疫性疾病，丛状蛋白已成为治疗这些疾病的新药靶点。丛蛋白信号转导所必需的是其胞内区域，该区域含有R-RAS GTP酶激活蛋白(GAP)结构域。GAP结构域通过使R-RAS失活，从而导致整合素失活和细胞黏附丧失，从而参与网络蛋白介导的轴突引导。丛状GAP结构域通常保持不活跃，它的激活需要信号素和RhoGTP酶(rac1、Rhod或RND1)分别与受体的胞外区和胞内RhoGTPase结合域(RBD)同时结合。本研究的目的是了解丛状蛋白间隙结构域自身抑制和激活的分子机制。我们使用X射线结晶学结合生化和细胞生物学的方法来研究这种机制。我们已经解决了丛状蛋白A3胞内结构域的晶体结构。结构表明GAP结构域采用非活性构象，表明RBD和N-末端片段有助于这种自抑制状态的稳定。我们对结构的分析也导致了一个假设，即当信号素诱导Plexin二聚化时，Plexin胞内域可以形成一个特定的二聚体，并且RhoGTP酶与这个二聚体Plexin的RBDS结合后，变构地诱导GAP结构域的构象变化，从而触发其激活。该提案以测试该模型为中心。在目标1中，我们将使用生化缺口分析和基于细胞的分析对自身抑制机制进行突变分析。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涉及二聚化和RhoGTP酶结合的激活机制。在目标3中，我们将通过测定RhoGTP酶和R-RAS复合体中网织蛋白胞内结构域的结构来研究GAP结构域的激活机制。这些研究将揭示丛状蛋白GAP结构域自身抑制和激活的分子基础，并为未来与丛状蛋白功能障碍相关的疾病的药物设计提供新的途径。与公共健康相关：丛状蛋白是表达在细胞表面的重要信号蛋白，参与引导神经元轴突到达合适的目标，这对我们体内神经网络的发展至关重要。神经丛功能障碍与多种疾病有关，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GTP酶激活蛋白结构域的调节机制，该结构域对丛状蛋白的信号功能至关重要。

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.