Molecular Mechanisms of Plexin Signaling in the Heart and Vascular System

描述(由申请人提供)： 这项K02计划的目标是让首席研究人员了解心脏发育过程中血管模式形成的细胞信号机制，为他在未来五年中投入研究的时间提供75%以上的时间。神经丛蛋白信号在神经系统和发育中的心血管系统之间的相似之处直到最近才变得清晰起来。丛状蛋白及其相关蛋白与先天性心脏病(CHD)有关，先天性心脏病对产前和新生儿保健仍是一个重大挑战。具体地说，首席调查员将专注于阐明丛状蛋白-A1、-B1和-D1与小GTP酶功能相互作用的异同。他将尝试确定新的、特定于血管的结合伙伴。长期目标是了解相似的机制如何在不同的细胞和细胞功能中使用，以及信号特异性如何从分子相互作用的细节中产生。这些基础知识最终将使我们能够设计多肽或其他试剂，用于监测和操纵细胞信号事件，通过基因治疗早期诊断和影响CHD。小GTPase rac1(和同源RND1蛋白)与Plexin-B1细胞质区域之间的复合体形成是第一个小GTPase与跨膜受体直接相互作用的案例之一。NIGMS R01拨款(GM073071)资助了一系列关于丛蛋白-B1的rac1结合细胞质结构域的生物物理研究。受到丛状蛋白在心血管发育中作用的新报道的启发，首席研究员希望将该项目扩展到丛状蛋白-A1和-D1受体的结构和功能的表征。K02项目的具体目标是：1)表征Plexin-A1和-D1细胞质区域的GTPase结合和调控结构域，包括通过核磁共振/X射线进行建模或结构确定。2)通过酵母双杂交筛选和计算机模拟，鉴定了其他的丛状蛋白-B1和-D1胞质结构域结合蛋白。3)利用定点突变和计算模型控制GTP酶结合亲和力和特异性。作为K02项目的一部分，新技术将被引入一个已经跨学科的实验室。首席调查员将接受额外的培训，并将扩大与细胞和心血管生物学家的互动。

DESCRIPTION (provided by applicant): The goal of this K02 proposal is to allow the Principal Investigator to understand cellular signaling mechanisms for vascular pattern formation in heart development, providing him with greater than 75% of time that he will devote to research over the next five years. Parallels between plexin signaling in the nervous system and in the developing cardiovascular system have only recently become clear. Plexins and associated proteins are involved in congenital heart defects (CHD), which remains a significant challenge to pre- and neonatal health care. Specifically the Principal Investigator will focus on elucidating similarities and differences in functional interactions of plexins-A1, -B1 and -D1 with small GTPases. He will try to identify novel, vasculature specific binding partners. The long term aim is to understand how similar mechanisms are used in different cells and cellular functions, and how signaling specificity arises from the details of the molecular interactions. This basic knowledge will eventually allow the design polypeptides or other agents that can be used to monitor and manipulate cell signaling events, diagnose and affect CHDs early via gene therapy. The complex formation between the small GTPase Rac1 (and homologous Rnd1 protein) and the cytoplasmic region of plexin-B1 is one of the first documented cases of a direct interaction between a small GTPase and a transmembrane receptor. A range of biophysical studies on the Rac1 binding cytoplasmic domain of plexin-B1 are funded by NIGMS R01 grant (GM073071). Stimulated by the new reports of plexins role in cardiovascular development, the Principal Investigator would like to expand the project to the characterization of the structure and function of plexin-A1 and -D1 receptors. Specific aims of the K02 project are: 1) Characterization of the GTPase binding and regulatory domains in the cytoplasmic regions of plexin-A1 and -D1, including modeling or structure determination by NMR/X-ray. 2) Identification of other plexin-B1 and -D1 cytoplasmic domain binding proteins by yeast two hybrid screening and computational modeling. 3) Manipulation of GTPase binding affinity and specificity by use of site directed mutagenesis and computational modeling. As part of the K02 project new techniques will be introduced into an already interdisciplinary laboratory. The Principal Investigator will undergo additional training and interactions with cellular and cardiovascular biologists will be expanded.