Transmembrane signaling mechanisms of plexin

丛状蛋白是单通道跨膜受体，其作为用于引导 信号蛋白分子。一些脑信号蛋白也需要神经纤毛蛋白辅助受体来结合和激活 丛蛋白。脑信号蛋白/丛蛋白/神经毡蛋白介导的信号传导对于许多过程是必需的，包括 神经系统和心血管系统的发育。这种信号通路的故障是 与神经系统疾病和癌症等疾病有关。更好地理解机制 这一途径的研究将为开发针对这些疾病的靶向治疗和改善 损伤后神经元再生。丛蛋白和神经毡蛋白都是大蛋白，并且使用它们的N-末端， 膜远端结构域结合semaphorin。脑信号蛋白是二聚体分子，并通过以下方式激活丛蛋白： 诱导活性二聚体的形成。一个主要的机械问题是， 丛蛋白和神经毡蛋白的近端和跨膜区在N-末端偶联脑信号蛋白结合 这些结构域与丛蛋白胞质结构域的激活有关，丛蛋白胞质结构域将信号传递到下游途径。 有证据表明，膜近端和跨膜区发挥积极作用， 丛蛋白激活。另一个悬而未决的问题是， 丛蛋白有助于信号传导。拟议的研究将集中在解决这些机制 问题.在目的1中，我们将分析丛蛋白的跨膜区介导的相互作用是如何进行的。 调节丛蛋白活性二聚体的形成。我们将设计plexin结构， 跨膜区和胞质区，但不是N-末端自身抑制结构域。这些构建体 预期在没有脑信号蛋白结合的情况下自发形成活性二聚体。我们将确定 通过X射线晶体学或冷冻电子显微镜观察这种活性二聚体的结构 跨膜区如何相互作用并促进丛蛋白活性二聚体的形成。在目标2中， 我们将利用低温电子显微镜对神经丛蛋白和神经纤毛蛋白与脑信号蛋白复合物的全长结构进行研究 显微镜这些结构将提供整个受体/配体复合物的直接视图，并揭示如何 近膜区和跨膜区在N-末端偶联脑信号蛋白的结合 域到胞质域。在目标3中，我们将研究丛蛋白的相互作用和调节， 调节蛋白这些蛋白质中的一些可能仅在脂质的情况下发挥其调节功能。 双层，我们将通过使用在脂质盘中重构的丛蛋白来研究。结构研究将是 通过体外生物物理学和基于细胞的功能测定进行关联。此外，还开发了新的方法， plexin系统将用于研究其他跨膜信号蛋白，以获得一个通用的 了解跨膜信号传导的原理。

Plexins are single-pass transmembrane receptors that serve as the primary receptors for the guidance molecules semaphorins. Some semaphorins also require the neuropilin co-receptor for binding and activating plexin. Semaphorin/plexin/neuropilin-mediated signaling is essential for many processes, including the development of the nervous system and the cardiovascular system. Malfunction of this signal pathway is associated with diseases such as neurological disorder and cancer. A better understanding of the mechanisms of this pathway will provide a foundation for developing targeted therapies for these diseases and improving neuronal regeneration after injury. Plexin and neuropilin are both large proteins, and use their N-terminal membrane distal domains to bind semaphorin. Semaphorin are dimeric molecules, and activate plexin by inducing the formation of the active dimer. One major remaining mechanistic question is how the membrane proximal and transmembrane regions in plexin and neuropilin couple the semaphorin binding at the N-terminal domains to the activation of the plexin cytoplasmic domain, which relays the signal to downstream pathways. There is evidence suggesting that the membrane proximal and transmembrane regions play active roles in plexin activation. Another outstanding question concerns how many of the newly identified binding partners of plexin contribute to the signaling. The proposed research will be focused on addressing these mechanistic questions. In Aim 1, we will analyze how the interactions mediated by the transmembrane region of plexin regulate the formation of the plexin active dimer. We will design plexin constructs that contain the transmembrane region and cytoplasmic region but not the N-terminal autoinhibitory domains. These constructs are expected to form the active dimer spontaneously in the absence of semaphorin binding. We will determine the structure of this active dimer through either X-ray crystallography or cryo-electron microscopy to visualize how the transmembrane region interacts and promotes the formation of the plexin active dimer. In Aim 2, we will pursue the structure of full-length plexin and neuropilin in complex with semaphorin by using cryo-electron microscopy. These structures will provide a direct view of the entire receptor/ligand complex, and reveal how the membrane proximal and transmembrane regions couple the binding of semaphorin at the N-terminal domains to the cytoplasmic domains. In Aim 3, we will investigate the interactions and regulation of plexin by regulatory proteins. Some of these proteins may only exert their regulatory function in the context of the lipid bilayer, which we will investigate by using plexin reconstituted in lipid discs. The structural studies will be correlated by in vitro biophysical and cell-based functional assays. In addition, new approaches developed for the plexin system will be used to study other transmembrane signaling proteins in order to gain a general understanding of the principles of transmembrane signaling.