The objective of this study is to explore the signal transduction mechanism by which protease-activated receptors (PARs) regulate endothelial nitric oxide synthase (eNOS) phoshorylation and activation in endothelial cells. Proteases play very important roles in endothelial physiology and pathophysiology through activation of PARs. Their functions include regulations of endothelium-dependent contraction/relaxation, endothelial permeability/barrier function, leukocyte adhesion, angiogenesis, endothelial cell migration and proliferation. Thus, PARs are very likely to be involved in the development of cardiovascular diseases. However, the detailed molecular mechanisms by which PARs regulate these functions remain largely unclear. PAR1 and PAR2 are believed to play major roles in endothelial cell function. Recent studies revealed multiple heterotrimeric G protein-coupling (Gq, Gi, G12/13) and several downstream signaling cascades of PAR1 in endothelial cells and other cells. By contrast, much less is known regarding PAR2 signal transduction in endothelial cells, which mainly couples to Gq. Our goal is to study the specific signal transduction pathways utilized by PARs including heterotrimeric G protein- coupling together with the structure-activity relationship of PARs during coupling and their roles in mediating downstream signals that regulate eNOS. We hypothesize that PAR1 and PAR2 reciprocally regulate eNOS through distinct signaling pathways and have proposed the following specific aims to address this hypothesis. We plan: 1. To demonstrate that a distinct receptor C-tail structure associated with distinct G protein-coupling is required for reciprocal eNOS phosphorylation by PARs. 2. To identify eNOS kinases that reciprocally regulate eNOS activity in response to distinct PARs. 3. To identify the distinct PAR C-tail associated proteins that are responsible for reciprocal eNOS regulation. Cultured endothelial cells will be used to detect phosphorylation of eNOS. Kinase assays, site mutations, and novel proteomic approaches will be used to identify other proteins involved in the reciprocal phosphorylation of eNOS. The results of this study will be valuable in developing specific-acting therapeutic drugs that can be used to treat endothelial dysfunctions that lead to cardiovascular diseases. PUBLIC HEALTH RELEVANCE: This project is relevant to public health because of its importance in understanding some of the possible causes of cardiovascular disease, the number one killer in America, especially among the African American community. It will provide information regarding the role of protease-activated receptors in the regulation of the endothelium, which plays an important role in vascular tone. Endothelial dysfunction is a major cause of hypertension, and these studies will contribute to the development of treatment strategies to alleviate or prevent cardiovascular disease.