KRIT1 and Vascular Integrity

描述（由申请人提供）：内皮细胞（EC）连接调节血管通透性，在心血管系统的发育和功能中发挥核心作用。Rap1 GTPase的激活稳定了这些连接，申请人发现，与脑海绵状畸形（CCM）相关的基因KRIT1的Rap1结合蛋白产物KRIT1可能介导Rap1 GTPase稳定ec细胞连接。CCM是一种常见的血管异常，影响着超过一百万的美国人，使他们终生有中风和其他神经系统后遗症的风险。目前还没有治疗方法来预防CCM病变的发生或临床进展。申请人还发现，玻璃心脏（HEG1），一种与KRIT1遗传相关的受体，与KRIT1相互作用，整合素细胞质结构域也是如此。KRIT1直接与CCM2结合，CCM2是另一个与CCM相关的基因的产物，他发现ECs中KRIT1或CCM2的缺失都会导致Rho激酶（ROCK）的激活增加。因此，他假设Rap1结合将含有KRIT1和CCM2的大分子复合物靶向到ec细胞连接处，并通过与HEG1细胞质尾部结合而保留，并通过抑制RhoA及其效应物Rho激酶（ROCK）来稳定连接处的完整性。为了验证这些想法，申请人将在KRIT1上绘制Rap1结合区域，以制造破坏Rap1结合的突变体，并检查它们对KRIT1功能和定位的影响。同样，他将识别Rap1的开关1区域的突变体，破坏与KRIT1的结合，并评估它们对Rap1稳定ec细胞连接的影响。这些信息将用于破译Rap1在KRIT1定位和功能中的作用。其次，他将研究KRIT1与HEG1的直接相互作用，并创建无法相互作用的每个伙伴的突变体。然后，他将测试这些突变体在稳定ec细胞连接和RhoA-ROCK活性方面的功能。第三，申请人将分析RhoA和ROCK抑制的机制，以扩展KRIT1-CCM2抑制RhoA效应物ROCK的活性，从而稳定ec细胞连接的假设。特别是，他将评估KRIT1的结构特征，使其能够抑制RhoA和ROCK。他已经发现，抑制RhoA/ROCK活性需要CCM2与KRIT1的相互作用，他将使用被排除在细胞核外且无法结合CCM2的KRIT1突变体来验证CCM2通过控制KRIT1在细胞-细胞连接中的定位来调节RhoA的观点。他还将利用对KRIT1如何与整合素结合的深入研究来验证这样一个假设，即KRIT1在EC连接上的募集限制了整合素信号，如RhoA激活，从而破坏细胞-细胞连接。这些研究将为新发现的调节血管发育和屏障功能的多蛋白复合物提供基础见解，并有可能确定CCM的新治疗靶点，这是一个重大的未满足的医疗需求。

DESCRIPTION (provided by applicant): 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. PUBLIC HEALTH RELEVANCE: Vascular leak plays a major role in a wide variety of diseases and there is presently no therapy to reduce the development of cerebral cavernous malformations (CCM), nor the significant associated morbidity, despite the fact that more than one million Americans bear these lesions. Our studies have already implicated the RhoA/ROCK pathway in this disease; the existence of relatively well-tolerated drugs that can modulate this pathway, has suggested new directions for therapy of vascular leak and CCM. These studies will provide fundamental insight into the CCM multi-protein complex that contains three proteins whose genes are linked to CCM; these insights may lead to identification of additional therapeutic targets for these diseases.