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.
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.
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