KRIT1 and Vascular Integrity

项目总结/摘要 内皮细胞（EC）连接调节血管通透性，并在内皮细胞的发育和分化中发挥重要作用。 心血管系统的功能。Rap 1 GTdR的激活稳定了这些连接， 发现KRIT 1是KRIT 1的Rap 1结合蛋白产物，KRIT 1是与大脑海绵状血管相关的基因， 畸形（CCM），可能介导Rap 1 GT3稳定EC-细胞连接。CCM是一种常见的 血管异常影响超过一百万美国人，使他们一生都有中风的风险， 其他神经系统后遗症目前尚无预防CCM发生或临床进展的治疗方法 病变本申请人还发现，与KRIT 1遗传连锁的受体玻璃心（HEG 1）， 与KRIT 1相互作用，整合素胞质结构域也是如此。KRIT 1直接与CCM 2结合，CCM 2是 另一个与CCM相关的基因，他发现EC中KRIT 1或CCM 2的缺失， Rho激酶（ROCK）。因此，他假设Rap 1结合靶向大分子 含有KRIT 1和CCM 2的复合物与EC-细胞连接处结合，在那里它通过与HEG 1结合而被保留 通过抑制RhoA及其效应物Rho激酶， （岩石）。为了测试这些想法，申请人将绘制KRIT 1上的Rap 1结合区域以制备突变体， 破坏Rap 1结合，并检查它们对KRIT 1功能和定位的影响。同样地，他也会发现 Rap 1的开关1区域的突变体，其破坏与KRIT 1的结合并评估其对Rap 1的影响 EC-细胞连接的稳定化。这些信息将用于破译Rap 1在定位中的作用 KRIT 1的功能其次，他将研究KRIT 1与HEG 1的直接相互作用并创建突变体 每一个没有互动的伙伴。然后，他将测试这些突变体在稳定EC细胞中的功能， 连接和RhoA-ROCK活性。第三，申请人将分析RhoA和ROCK的机制 抑制以扩展KRIT 1-CCM 2抑制RhoA效应物ROCK的活性的假设， 从而稳定EC细胞连接。特别是，他将评估KRIT 1的结构特征， 抑制RhoA和ROCK。他发现CCM 2与KRIT 1的相互作用是抑制所必需的。 RhoA/ROCK活性，他将使用KRIT 1突变体，这些突变体被排除在细胞核之外， CCM 2来测试CCM 2通过控制KRIT 1定位于细胞-细胞连接来调节RhoA的想法。 他还将深入了解KRIT 1如何与整合素结合，以检验KRIT 1招募到EC的假设。 连接限制整合素信号，如RhoA激活，破坏细胞-细胞连接。这些研究将 为新发现的调节血管发育的多蛋白复合物提供了基本的见解 和屏障功能，并有可能确定新的治疗目标，在CCM，一个重大的未满足 医疗需求。

Project Summary/Abstract Endothelial cell (EC) junctions regulate vascular permeability and play a central role in the development and function of the cardiovascular system. Activation of Rap1 GTPase stabilizes these junctions and the applicant found that KRIT1, the Rap1-binding protein product of KRIT1, a gene linked with cerebral cavernous malformations (CCM), may mediate Rap1 GTPase stabilization of EC-cell junctions. CCM is a common vascular anomaly affecting more than a million Americans, predisposing them to a lifetime risk of stroke and other neurologic sequelae. There is currently no therapy to prevent the genesis or clinical progression of CCM lesions. The applicant has also found that heart of glass (HEG1), a receptor genetically-linked to KRIT1, interacts with KRIT1, as do integrin cytoplasmic domains. KRIT1 binds directly to CCM2, the product of another gene associated with CCM and he found that loss of either KRIT1 or CCM2 in ECs, leads to increased activation of Rho Kinase (ROCK). Thus, he hypothesizes that Rap1 binding targets a macromolecular complex containing KRIT1 and CCM2 to EC-cell junctions where it is retained by binding to the HEG1 cytoplasmic tail and stabilizes junctional integrity by inhibiting RhoA and its effector, Rho Kinase (ROCK). To test these ideas the applicant will map the Rap1 binding region on KRIT1 to make mutants that disrupt Rap1 binding and examine their effects on KRIT1 function and localization. Similarly, he will identify mutants of the Switch 1 region of Rap1 that disrupt binding to KRIT1 and assess their effects on Rap1 stabilization of EC-cell junctions. This information will be used to decipher the role of Rap1 in the localization and function of KRIT1. Secondly he will examine the direct interaction of KRIT1 with HEG1 and create mutants of each partner that fail to interact. He will then test the function of these mutants in stabilizing EC-cell junctions and RhoA-ROCK activity. Thirdly, the applicant will analyze the mechanism of RhoA and ROCK inhibition to extend the hypothesis that KRIT1-CCM2 suppresses the activity of the RhoA effector, ROCK, thereby stabilizing EC-cell junctions. In particular, he will assess the structural features of KRIT1 that enable it to inhibit RhoA and ROCK. He has found that the interaction of CCM2 with KRIT1 is required for suppression of RhoA/ROCK activity and he will use KRIT1 mutants that are excluded from the nucleus and fail to bind CCM2 to test the idea that CCM2 regulates RhoA by controlling the localization of KRIT1 to cell-cell junctions. He will also use insights into how KRIT1 binds to integrins to test the hypothesis that KRIT1 recruitment to EC junctions limits integrin signals, such as RhoA activation, that disrupt the cell-cell junctions. These studies will provide fundamental insight into a newly discovered multiprotein complex that regulates vascular development and barrier function and have the potential to identify new therapeutic targets in CCM, a significant unmet medical need.