Continued support is requested to analyze mechanisms whereby components of the Cerebral Cavernous Malformations (CCM) complex contribute to cardiovascular development. Work in the previous funding period established that Heart of Glass (HEG1), a transmembrane protein essential for vertebrate cardiovascular development, anchors a Rap effector, KRIT1, and thus the CCM complex to endothelial cell (EC) cell junctions. Our analysis of the HEG1 interactome revealed a novel interaction of the HEG1 cytoplasmic domain with RASIP1, another Rap effector that plays a critical role in vascular development and integrity. We thus propose that RASIP1 binding to Rap1 regulates its interaction with HEG1 to localize RASIP1 to assembling EC junctions thus mediating some, but not all, of the RASIP1 functions involved in vascular development. To test these ideas we propose to ask if binding of RASIP1 to HEG1 mediates RASIP1 localization to EC junctions and its capacity to suppress RhoA/ROCK activity to maintain EC barrier function. We will minimize the regions of HEG1 and RASIP1 that interact and then test, using purified recombinant fragments, whether the interaction is direct. Functions will be probed by examining the effect of mutants that perturb the interaction on the capacity of RASIP1 to localize to EC junctions, to support endothelial monolayer integrity, to inhibit RhoA/ROCK activity, and to support cardiovascular development in zebrafish. We will also test the hypothesis that Rap1 binding to RASIP1 regulates its interaction with HEG1. Molecular modeling will predict mutations in the RA domain of RASIP1 that block Rap1 binding. The effect of these mutations on the interaction of RASIP1 with HEG1 in cells and RASIP1 junctional localization, regulation of vascular integrity, and cardiovascular development will be assessed. Preliminary data suggest that RASIP1 is auto-inhibited; by mapping self-interacting sites in conjunction with analysis of Rap1 binding we will examine potential mechanisms whereby Rap1 might alleviate such auto inhibition. Thirdly, we will assess whether there are HEG1-independent functions of RASIP1. In addition to suppressing RhoA/ROCK signaling, RASIP1 supports integrin activation and the activities of CDC42 and Rac1. Using the information gleaned in Aims 1 and 2, we will test whether RASIP1 mutants that fail to bind to HEG1 or Rap1 can support these functions. Conversely we will examine the effect of loss of HEG1 on these functions and assess the capacity of HEG1 deficient in RASIP1 binding to support them. Finally, we will test the idea that RASIP1 and KRIT1 might regulate each other's interactions with HEG1. These studies will serve to elucidate the mechanisms whereby HEG1, RASIP1, and KRIT1 play a critical role in cardiovascular development.
This application proposes to analyze the mechanism by which RASIP1, a protein that is essential for normal vascular development and integrity, is recruited to endothelial cell cell junctions and performs its functions in regulating the signaling of small GTPases. The work will provide new insights into the mechanisms of vascular development with the potential to identify therapeutic targets in angiogenesis and vascular malformations.
| Lopez-Ramirez, Miguel Alejandro; Fonseca, Gregory; Zeineddine, Hussein A et al. (2017) Thrombospondin1 (TSP1) replacement prevents cerebral cavernous malformations. J Exp Med 214:3331-3346 |
| Gingras, Alexandre R; Puzon-McLaughlin, Wilma; Bobkov, Andrey A et al. (2016) Structural Basis of Dimeric Rasip1 RA Domain Recognition of the Ras Subfamily of GTP-Binding Proteins. Structure 24:2152-2162 |
| de Kreuk, Bart-Jan; Gingras, Alexandre R; Knight, James Dr et al. (2016) Heart of glass anchors Rasip1 at endothelial cell-cell junctions to support vascular integrity. Elife 5:e11394 |
| Lagarrigue, Frederic; Kim, Chungho; Ginsberg, Mark H (2016) The Rap1-RIAM-talin axis of integrin activation and blood cell function. Blood 128:479-87 |
| Zhang, Ping; Ye, Feng; Bastidas, Adam C et al. (2015) An Isoform-Specific Myristylation Switch Targets Type II PKA Holoenzymes to Membranes. Structure 23:1563-1572 |
| Ablack, Jailal N G; Metz, Patrick J; Chang, John T et al. (2015) Ubiquitylation of CD98 limits cell proliferation and clonal expansion. J Cell Sci 128:4273-8 |
| Lagarrigue, Frederic; Vikas Anekal, Praju; Lee, Ho-Sup et al. (2015) A RIAM/lamellipodin-talin-integrin complex forms the tip of sticky fingers that guide cell migration. Nat Commun 6:8492 |
| Yago, Tadayuki; Petrich, Brian G; Zhang, Nan et al. (2015) Blocking neutrophil integrin activation prevents ischemia-reperfusion injury. J Exp Med 212:1267-81 |
| Estrach, Soline; Lee, Sin-Ae; Boulter, Etienne et al. (2014) CD98hc (SLC3A2) loss protects against ras-driven tumorigenesis by modulating integrin-mediated mechanotransduction. Cancer Res 74:6878-89 |
| Ye, Feng; Lagarrigue, Frederic; Ginsberg, Mark H (2014) SnapShot: talin and the modular nature of the integrin adhesome. Cell 156:1340-1340.e1 |
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