Structure and function of plexin - co-receptor interactions

修改后的项目摘要/摘要 神经丛蛋白是一种跨膜受体，在血管和神经元的发育、疾病和再生中做出细胞指导决定。靶向丛状蛋白及其辅助受体，特别是神经纤毛蛋白，是治疗老年性黄斑变性(AMD)和糖尿病视网膜病变等视网膜疾病的一条新途径，但丛状蛋白激活的机制细节尚不清楚。我们将描述丛蛋白和膜脂之间的相互作用。丛状蛋白是一种独特的跨膜受体，其胞内区域直接与膜锚定的小GTP酶相互作用。这些RAS和RhoFamily GTP酶反过来又与视网膜中的细胞迁移有关。目标1的初步结果表明，蛋白质-膜相互作用在丛状蛋白的信号机制中起着关键作用，例如，使GTP酶在系统中的不同功能角色合理化。我们的工作将是第一次在分子水平上研究丛蛋白结构域与脂质双层的相互作用。这包括在AIM1中的Plexin-B1跨膜区，它被预测为三种螺旋间接触状态。总之，这些结果支持一个多态模型，该模型结合了以前和新的结果，但也指出了蛋白质和脂质双层之间关键的新相互作用。目的2研究丛状蛋白辅受体、神经纤毛素和信号素跨膜区的结构和功能。我们假设，这种相互作用建立了与配体和辅助受体的交叉信号。为了解决这一假设，我们将使用活细胞，使用时间分辨荧光光谱和功能实验来检测受体的同源和异源齐聚。与核磁共振和分子动力学一起，这些互补的工具将在不同的复杂程度上解决假说，范围从领域水平到细胞内行为。 神经丛蛋白的功能是通过与辅助受体的相互作用来调节的，例如神经毛膜蛋白，它也是单程跨膜(TM)蛋白。TM区最近被认为是设计用来干扰受体信号转导的多肽的有效靶点。在目标2中，我们将描述丛状蛋白TM与神经粘连蛋白TM结构域的相互作用的结构和功能。我们的数据表明，丛状蛋白与其某些信号素配体之间的TM相互作用也可以在顺式蛋白中发生。这种基于TM的调节将是丛状蛋白领域的一种新机制，也是一种可以靶向抑制视网膜受体的机制。具体地说，TM区域是AMD和糖尿病视网膜病变等眼病抗血管生成治疗的靶点，扩大了神经毛膜肽作为抗肿瘤药物的成功使用。

Modified Project Summary/Abstract Plexins are transmembrane receptors that make cell guidance decisions in vascular and neuronal development, disease, and regeneration. Targeting plexins and their co-receptors, especially neuropilin, is a novel route to treating retinal diseases such as age-related macular degeneration (AMD) and diabetic retinopathy, but the mechanistic details of plexin activation are poorly understood. We will characterize the interactions between plexin and membrane lipids. Plexins are unique transmembrane receptors in that their intracellular regions interact directly with membrane-anchored small GTPases. These Ras and Rhofamily GTPases, in turn, are associated with cell migration in the retina. The preliminary results in aim 1 indicate that protein-membrane interactions play a key role in the signaling mechanisms of plexins, for example, rationalizing the different functional roles of the GTPases in the system. Our work will be the first to study the interactions of plexin domains with the lipid bilayer at the molecular level. This includes, in aim1, the plexin-B1 transmembrane region which has been predicted to assume three states of inter helix contacts. Altogether the results support a multi-state model which incorporates previous and the new results, but also points to critical new interactions between the proteins and the lipid bilayer. Aim 2 seeks to resolve the structure and function of the transmembrane regions of plexin coreceptors, neuropilin and semaphorins. We hypothesize that such interactions set up cross signaling with ligands and co-receptors. In order to address this hypothesis, we will examine receptor homo- and hetero-oligomerization using time resolved fluorescence spectroscopy and functional experiments, both using live cells. Together with NMR and molecular dynamics, these complementary tools will address the hypothesis at different levels of complexity, ranging from domain level to in-cell behavior. Plexin function is modulated by interaction with co-receptors, such as neuropilins which are also single pass transmembrane (TM) proteins. TM regions are recently recognized as an effective target for peptides designed to disrupt receptor signaling. In aim 2, we will characterize the structure and function of plexin TM interactions with the TM domains of neuropilins. Our data suggest that TM interactions between plexins and certain of their semaphorin ligands can also occur in cis. Such TM based regulation would be a novel mechanism in the plexin field, and one that can be targeted to inhibit the receptors in the retina. Specifically, expanding the successful use of neuropilin peptides as anti-tumor agents, TM regions are a target for anti-angiogenesis therapy in eye disease such as AMD and diabetic retinopathy.