(664.11) Regulation of activated Protein C/PAR1 endothelial cytoprotective signaling by G protein-coupled receptor kinases

Poster Board Number: A278

Monica Gonzalez Ramirez (University of California San Diego), Ying Lin (University of California San Diego), Huaping Qin (University of California San Diego), Cierra Birch (University of California San Diego), JoAnn Trejo (University of California San Diego)

Endothelial dysfunction is a hallmark of inflammation and associated with vascular diseases. Our goal is to define pathways by which the endothelium can resist injury and disruption to facilitate the advancement of new targets for therapeutic development. Activated Protein C (APC) has been shown in preclinical studies to increase endothelial barrier protection and reduce inflammatory responses and signals via protease-activated receptor-1 (PAR1). APC enhances endothelial cytoprotection through cleavage of PAR1 at an N-terminal arginine (R)-46 site, that is distinct from the canonical thrombin cleavage site at R-41. This results in the generation of a distinct tethered ligand that promotes PAR1 bias signaling. We discovered that APC/PAR1 signals preferentially via b-arrestin2 (b-arr2), rather than heterotrimeric G proteins to promote endothelial barrier protection. The objective of this study is to develop a mechanistic understanding of how APC/PAR1 generates b-arr2 cytoprotective signaling.  G protein-coupled receptor kinases (GRKs) phosphorylate thrombin-activated PAR1 and facilitate β-arrestin-mediated desensitization. However, contribution of GRKs to β-arrestin driven APC/PAR1-induced cytoprotection is unclear. We hypothesize that compartmentalization of APC/PAR1 together with GRK5 is essential to initiate b-arr2 recruitment and biased signaling. Here we will present studies examining the function and mechanism by which GRKs regulate endothelial cytoprotective responses using GRK-specific siRNA to assess function as well as confocal immunofluorescence imaging and biochemical sucrose fraction to examine GRK localization and compartmentalization in caveolae. The work is expected to provide important new insight into the role of GRKs in regulating APC/PAR1 cytoprotection in human cultured endothelial cells.

This work is funded by NIH/NIGMS R35 GM127121, and NIH/NIGMS K12 GM068524.