Signaling and Regulation Mechanisms of Plexin

描述（由申请人提供）：丛蛋白是信号素的细胞表面受体。神经丛蛋白介导的信号素信号对于神经系统和心血管系统的发育以及免疫反应和骨稳态的调节等过程至关重要。神经丛神经的功能障碍与神经系统疾病和癌症有关。了解神经丛的功能将为开发靶向治疗方法来对抗相关疾病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一个GTPase激活蛋白（GAP）结构域，这对功能至关重要。在之前的研究中，我们已经确定了小的GTPase Rap是丛蛋白GAP结构域的真正底物，并确定了GAP结构域如何被信号蛋白诱导的二聚化激活，以及它如何通过非规范催化机制使Rap失活的结构基础。目标。研究丛状蛋白的调控机制，以及丛状蛋白与其几个关键结合伙伴之间的相互调控。研究设计。基于我们的新晶体结构，我们将首先分析抑制二聚体在丛蛋白调控中的作用，这是该领域长期存在的问题。丛蛋白信号不仅需要其RapGAP活性，还需要其在质膜上组装和控制多蛋白信号复合物活性的能力。许多蛋白质与丛蛋白相互作用，但其作用的结构基础在很大程度上是未知的。我们将重点讨论一些重要的结合伙伴，探讨它们如何结合丛蛋白并与丛蛋白相互调节的问题。在目的1中，我们将测试神经丛蛋白调控的抑制二聚体模型。我们确定了PlexinA4的两种晶体结构，它采用一种新的构象，形成紧凑的二聚体，GAP活性位点埋在二聚体界面中。我们认为这种二聚体和先前报道的丛蛋白胞外区的载脂蛋白二聚体结构共同介导了全长丛蛋白在细胞表面的自抑制二聚体状态。将进行基于结构的突变分析来验证这一假设。在Aim 2中，我们将研究RND1/Rac和RhoD对丛蛋白信号传导的相反作用的基础。rhogtpase Rac1和RND1与丛蛋白相互作用，促进丛蛋白结合并被信号蛋白激活。相反，RhoD抑制丛蛋白信号传导，尽管它以相同的方式以相似的亲和力结合丛蛋白。我们的结构分析导致了一个解释这个悖论的假设，这个假设将在本目的中进行测试。在Aim 3中，我们将分析丛蛋白对FARPs的相互作用和调控。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，并对其信号传导起重要作用。我们将研究丛蛋白/FARP复合物的结构，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的自抑制作用。

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.