Hemorrhagic stroke accounts for ~13% of all stroke cases. One of the main underlying cases of this type of stroke is the rupture of intracranial aneurysm (IA), with consequent subarachnoid hemorrhage and critically reduced blood flow downstream of the affected vessel. A recent study of both familial and sporadic IA cases identified mutations in the ArhGEF17 gene as associated with increased risk for IA in both European and Asian populations. ArhGEF17 codes for a Dbl-family GEF (guanine nucleotide exchange factor) specific for RhoA. The molecular architecture of ArhGEF17 is unusual for a Dbl-family GEF, in that it is predicted to harbor an unusual split PH-domain (sPH) downstream of the DH domain, and a predicted ?-propeller (WD40) domain. Interestingly, the human genome has two homologues of ArhGEF17 with this architecture: ArhGEF10 and GRINCH-GEF (ArhGEF10L) (see Fig 1). A polymorphism in the ArhGEF10L gene has also been associated with increased risk to ischemic stroke. The structures of these three proteins are not known, and the specific functions of the putative sPH and WD40 domains are not understood. We propose an exploratory project aimed at structural and functional characterization of ArhGEF17/10. We will characterize the structural features of the ArhGEF17/10 family, with the overarching goal to gain understanding of the structure-function relationships in these proteins. Our ultimate objective is to gain detailed understanding of the structure and functions of the three globular domains, and to determine if and how are the GEFs regulated by the supramodular architecture. Focusing on the physiological function of ArhGEF17, we want to determine if it regulates cerebral vascular tone, by down regulating its expression in smooth muscle in mouse mid cerebral arteries, and in endothelial cells, using adenoviral shRNAs. RhoA activity and vasodilation will be monitored under basal conditions and in response to increases in intraluminal pressure and agonists in the arteries. Membrane resistance measurements in endothelial cells will monitor changes in permeability. This will lay the ground work for the future development of a conditional ArhGEF17 knockout mouse to determine whether deletion of ArhGEF17 in either smooth muscle or endothelial cells (or both) leads to increased cerebral vessel dilation and/or increased endothelial permeability, and increases the propensity for cerebral hemorrhage.
The proposed research is relevant to public health as it explores the possibility of new signaling pathways that are relevant to both hemorrhagic and ischemic stroke. We will study the molecular structure and function of proteins, which ? when mutated ? contribute to aneurysm and stroke. Such findings are relevant to the part of NIH?s mission that pertains to developing fundamental knowledge that will help to protect and improve health.
