KRIT1 and Vascular Integrity

项目摘要/摘要 血管内皮细胞(EC)连接调节血管通透性，在血管的发育和发育中发挥核心作用。 心血管系统的功能。RAP1 GTP酶的激活稳定了这些连接和申请者 发现与脑海绵体相关的基因KRIT1的Rap1结合蛋白产物KRIT1 畸形(CCM)，可能介导RAP1 GTP酶稳定的EC细胞连接。CCM是一种常见的 血管异常影响着100多万美国人，使他们有终身中风和 其他神经系统后遗症。目前尚无治疗方法可阻止CCM的发生或临床进展。 损伤。申请人还发现玻璃之心(HEG1)，一种与KRIT1基因连锁的受体， 与KRIT1相互作用，整合素胞质结构域也是如此。KRIT1直接与CCM2结合，CCM2是 另一个与CCM相关的基因，他发现在ECs中KRIT1或CCM2的丢失会导致 Rho Kinase(ROCK)的激活。因此，他假设RAP1结合的目标是一个大分子 含有KRIT1和CCM2与EC细胞连接的复合体，其中它通过与HEG1结合而保留 胞质尾巴通过抑制RhoA及其效应物Rho Kinase来稳定连接完整性 (摇滚)。为了测试这些想法，申请人将在KRIT1上绘制RAP1结合区的图谱，以制造 干扰RAP1结合并检测它们对KRIT1功能和定位的影响。同样，他也会认为 破坏与KRIT1结合的RAP1开关1区突变体及其对RAP1的影响 EC-细胞连接的稳定性。这些信息将用于破译Rap1在本地化中的作用 和KRIT1的功能。其次，他将研究KRIT1与HEG1的直接相互作用，并创建突变体 无法互动的每个合作伙伴的信息。然后，他将测试这些突变体在稳定EC细胞方面的功能 结点和RhoA-Rock活动。第三，申请人将分析RhoA和ROCK的机理 抑制扩展KRIT1-CCM2抑制RhoA效应器，ROCK， 从而稳定EC-细胞连接。特别是，他将评估KRIT1的结构特征，使其能够 它能抑制RhoA和ROCK。他发现CCM2与KRIT1的相互作用是抑制所必需的 RhoA/ROCK活性，他将使用KRIT1突变体，这些突变体被排除在细胞核之外，无法结合 CCM2测试CCM2通过控制KRIT1对细胞-细胞连接的定位来调节RhoA的想法。 他还将利用对KRIT1如何与整合素结合的洞察来检验KRIT1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 提供对新发现的调节血管发育的多蛋白复合体的基本见解 和屏障功能，并有可能在CCM中发现新的治疗靶点，这是一个重大的未实现的问题 医疗需要。

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.