A Novel Mechanism of Ras Regulation Necessary for Normal Vascular Development

The Ras-Mitogen-activated protein kinase (MAPK) signaling pathway is a critical regulator of blood vascular development and function. Dysregulated Ras-MAPK signaling in blood vascular endothelial cells (EC) leads to different types of vascular malformations, including arteriovenous malformations in which arteries connect di- rectly with veins. Ras is a small GTP-binding protein that cycles between inactive GDP-bound and active GTP- bound states in response to growth factor stimulation. Ras cycling is mediated by Ras guanine nucleotide exchange factors that exchange Ras-bound GDP for GTP, and Ras GTPase-activating proteins (RasGAPs) that promote Ras hydrolysis of GTP to GDP. RASA1 is one member of a family of RasGAPs that is essential for normal Ras cycling in the vasculature, and loss of function of RASA1 in EC results in AVM. In addition to its catalytically active GAP domain, RASA1 contains several modular binding domains, including a protein kinase C2 homology domain. Missense mutations in the C2 domain have been identified in families with a type of brain AVM known as Vein of Galen AVM (VGAM), which is the most common pediatric cerebrovascular mal- formation that carries the risk of hemorrhage, stroke, epilepsy, and other complications. The role of the C2 domain in RASA1 function has hitherto been unclear. However, our preliminary data, which comprises in vitro enzymatic assays, crystal structure determination, structure-based conservation assessment, and mutational analyses, indicate that the C2 domain directly contacts Ras and promotes GAP domain-mediated Ras cycling. We generated a VGAM-associated RASA1 C2 domain mutation in mice and found that homozygous mutant mice show similar embryonic vascular defects to those observed in RASA1 null mice. Together, our preliminary findings lead us to our central hypothesis that the RASA1 C2 domain is required for normal regulation of Ras, which is necessary to prevent vascular malformations such as VGAM. In this new MPI grant, we will address this hypothesis in three specific aims that combine highly complementary and synergistic expertise in the King and Boggon laboratories. In the first aim, we will employ enzymatic analyses to understand the mechanism by which the C2 domain promotes RASA1 catalytic activity against different Ras isoforms. In the second aim, we will further interrogate RASA1 mutant mouse models to provide additional support for our proposed mechanism in vivo, including its role in protection from the development of AVM. In the third aim, we will conduct RASA1- Ras mutant complementation studies in vitro and in vivo and will perform structural analyses of a RASA1-Ras complex to provide a detailed understanding of how RASA1 C2 domain interaction with Ras promotes normal vascular development. Our proposed studies are highly innovative and significant in that they will explore a heretofore unappreciated mechanism involved in Ras cycling that is relevant to an understating of the patho- genesis and potential treatment of vascular malformations such as VGAM.