Molecular Mechanisms of Plexin Signaling in the Heart and Vascular System

描述（由申请人提供）： 该K 02提案的目标是让主要研究者了解心脏发育中血管模式形成的细胞信号传导机制，为他提供超过75%的时间，他将在未来五年内投入研究。神经系统和发育中的心血管系统中丛蛋白信号之间的相似之处最近才变得清晰。丛状蛋白和相关蛋白质参与先天性心脏病（CHD），这仍然是产前和新生儿保健的重大挑战。具体而言，主要研究者将专注于阐明丛蛋白-A1，-B1和-D1与小GTP酶的功能相互作用的相似性和差异。他将尝试识别新的血管特异性结合伴侣。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允许设计多肽或其他试剂，可用于监测和操纵细胞信号传导事件，通过基因治疗早期诊断和影响CHD。小GTdR Rac 1（和同源Rnd 1蛋白）和丛蛋白-B1的胞质区域之间的复合物形成是小GTdR和跨膜受体之间直接相互作用的第一个有记录的案例之一。一系列关于丛蛋白B1的Rac 1结合胞质结构域的生物物理研究由NIGMS R 01资助（GM 073071）。受plexins在心血管发育中作用的新报告的刺激，主要研究者希望将该项目扩展到plexin-A1和-D1受体的结构和功能的表征。K 02项目的具体目标是：1）在丛蛋白-A1和-D1的细胞质区域中的GT3结合和调节结构域的表征，包括通过NMR/X射线进行建模或结构测定。2)通过酵母双杂交筛选和计算建模鉴定其他丛蛋白-B1和-D1胞质结构域结合蛋白。3)通过使用定点诱变和计算建模操纵GT3结合亲和力和特异性。作为K 02项目的一部分，新技术将被引入一个已经跨学科的实验室。主要研究者将接受额外的培训，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.