Signaling and Regulation Mechanisms of Plexin

描述(申请人提供)：丛状蛋白是信号素的细胞表面受体。丛蛋白介导的信号素信号对神经系统和心血管系统的发育、免疫反应和骨平衡的调节等过程是必不可少的。神经丛功能障碍与神经紊乱和癌症有关。了解丛状蛋白的功能将为开发抗击相关疾病的靶向疗法和改善损伤后的神经元再生铺平道路。丛状蛋白胞内区含有GTP酶激活蛋白(GAP)结构域，是发挥功能所必需的。在过去的一段时间里，我们已经确定小GTP酶Rap是Plexin GAP结构域的真实底物，并确定了GAP结构域如何被信号素诱导的二聚化激活以及它如何通过非规范的催化机制失活Rap。目标。研究网络蛋白的其他层次的调节机制，以及网络蛋白与其几个关键结合伙伴之间的相互调节。研究设计。基于我们的一个新的晶体结构，我们将首先分析抑制性二聚体在丛连蛋白调节中的作用，这是该领域一个长期存在的问题。丛蛋白信号不仅需要它的RapGAP活性，还需要它在质膜上组装和控制多蛋白信号复合体的活性。许多蛋白质与丛状蛋白相互作用，但其作用的结构基础在很大程度上是未知的。我们将集中于一些重要的结合伙伴，解决他们如何结合丛状蛋白以及与丛状蛋白相互调节的问题。在目标1中，我们将测试抑制二聚体模型在丛状蛋白调节中的作用。我们确定了PlexinA4的两种晶体结构，它采用了一种新的构象，形成了致密的二聚体，GAP活性中心埋在二聚体的界面上。我们认为，该二聚体和先前报道的丛状蛋白胞外区的脱辅基二聚体结构共同介导了细胞表面全长丛状蛋白的自抑制二聚体状态。将进行基于结构的突变分析来检验这一假设。在目标2中，我们将研究RND1/RAC和Rhod对丛蛋白信号转导的相反作用的基础。RhoGTP酶rac1和RND1与丛蛋白相互作用，并通过信号素促进其结合和激活。相反，Rhod抑制丛状蛋白信号转导，尽管它以相同的方式以相似的亲和力结合丛状蛋白。我们的结构分析导致了一个解释这一悖论的假设，这一假设将在这一目标中得到检验。在目标3中，我们将分析Plexin对FARPs的相互作用和调节。FARP1和FARP2是两个相关的鸟嘌呤核苷酸交换因子(GEF)，已被证明直接与丛蛋白相互作用，并在其信号转导中发挥重要作用。我们将探索Plexin/FARP复合体的结构，以阐明它们相互作用的基础，并分析这种相互作用如何帮助释放FARP的自身抑制。

DESCRIPTION (provided by applicant): Plexins are the cell surface receptors of semaphorins. Plexin-mediated semaphorin signaling is essential for processes such as the development of the nervous system and the cardiovascular system and regulation of immune responses and bone homeostasis. Malfunction of plexins has been associated with neurological disorder and cancer. Understanding how plexins function will pave the way for developing targeted therapeutics for fighting the associated diseases and improving neuronal regeneration after injury. The plexin intracellular region contains a GTPase Activating Protein (GAP) domain that is essential for function. In the previous period, we have identified the small GTPase Rap as the authentic substrate for the plexin GAP domain, and have determined the structural basis for how the GAP domain is activated by semaphorin-induced dimerization and how it inactivates Rap through a non-canonical catalytic mechanism. Objectives. To study additional layers of regulation mechanisms of plexins, and mutual regulation between plexins and several of their key binding partners. Research Design. Based on a new crystal structure of ours, we will first analyze the role of the inhibitory dimer in plexin regulation, a long-standing question in the fiel. Plexin signaling requires not only its RapGAP activity, but also its ability to assemble and contro the activity of a multi-protein signaling complex at the plasma membrane. Many proteins interact with plexins, but the structural basis of their actions is largely unknown. We will focus on some of the essential binding partners, address the questions how they bind plexin and exert mutual regulation with plexins. In Aim 1 we will test an inhibitory dimer model in plexin regulation. We have determined two crystal structures of PlexinA4, which adopts a new conformation and forms a compact dimer with the GAP active site buried in the dimer interface. We propose that this dimer and the apo dimer structure of the plexin extracellular region reported previously together mediate the autoinhibited dimeric state of full-length plexin on the cell surface. Structure-based mutational analyses will be performed to test this hypothesis. In Aim 2 we will study the basis for the opposite effects of RND1/Rac and RhoD on plexin signaling. The RhoGTPases Rac1 and RND1 interact with plexin and facilitate its binding and activation by semaphorin. In contrast, RhoD inhibits plexin signaling, although it binds plexin in the same mode with similar affinity. Our structure analyses led to a hypothesis that explains this paradox, which will be tested in this aim. In Aim 3 we will analyze the interaction and regulation of FARPs by plexin. FARP1 and FARP2 are two related guanine nucleotide exchange factors (GEFs) that have been shown to interact directly with plexin and make essential contributing to its signaling. We will pursue a structure of the plexin/FARP complex to elucidate the basis for their interaction, and analyze how this interaction helps release the autoinhibition of FARPs.