β-Arrestin2 is a critical component of the GPCR-eNOS signalosome

Endothelial cell nitric oxide (NO) synthase (eNOS), the enzyme responsible for synthesis of NO in endothelial cells, is regulated by complex posttranslational mechanisms. Sinusoidal portal hypertension, a disorder characterized by liver sinusoidal endothelial cell (SEC) injury with resultant reduced eNOS activity and NO production within the liver, has been associated with defects in eNOS protein-protein interactions and posttranslational modifications. We and others have previously identified novel eNOS interactors, including G protein-coupled receptor (GPCR) kinase interactor 1 (GIT1), which we found to play an unexpected stimulatory role in GPCR-mediated eNOS signaling. Here we report that beta-arrestin 2 (beta-Arr2), a canonical GPCR signaling partner, localizes in SECs with eNOS in a GIT1/eNOS/NO signaling module. Most importantly, we show that beta-Arr2 stimulates eNOS activity, and that beta-Arr2 expression is reduced and formation of the GIT1/eNOS/ NO signaling module is interrupted during liver injury. In beta-Arr2- deficient mice, bile duct ligation injury (BDL) led to significantly reduced eNOS activity and to a dramatic increase in portal hypertension compared to BDL in wild-type mice. Overexpression of beta-Arr2 in injured or beta-Arr2-deficient SECs rescued eNOS function by increasing eNOS complex formation and NO production. We also found that beta-Arr2-mediated GIT1/eNOS complex formation is dependent on Erk1/2 and Src, two kinases known to interact with and be activated by beta-Arr2 in response to GCPR activation. Our data emphasize that beta-Arr2 is an integral component of the GIT1/eNOS/NO signaling pathway and have implications for the pathogenesis of sinusoidal portal hypertension.