Activated protein C promotes β-arrestin-2- and c-Src-dependent caveolin-1 (Cav1) phosphorylation and alters Cav1 association with PAR1 and GRK5.

G protein-coupled receptors (GPCRs) display bias towards either G proteins or GPCR kinase (GRK)-mediated β-arrestin signaling depending on the agonist stabilized receptor conformation and cellular context. The cellular location of GPCRs particularly within plasma membrane microdomains such as lipid rafts contributes to biased signaling through mechanisms that are not well understood. The protease-activated receptor-1 (PAR1) exhibits biased signaling in response to thrombin and activated protein C (APC). APC-induced β-arrestin-2 (βarr2) biased signaling requires PAR1 compartmentalization in caveolae, a subtype of lipid rafts, whereas thrombin-activated PAR1 G protein signaling does not. Caveolin-1 (Cav1) is the principal structural protein of caveolae and regulates signaling through protein-protein interactions. The mechanisms by which Cav1 contributes to APC/PAR1-induced βarr2 biased signaling is not known. Here we report that APC-activated PAR1 modulates Cav1 phosphorylation via a βarr2- and c-Src-dependent pathway. APC also regulates the dynamics of endogenous PAR1-Cav1 and GRK5-Cav1 co-localization examined by single molecule super-resolution stochastic optical reconstruction microscopy imaging in human cultured endothelial cells. We further demonstrate that GRK5 interacts with Cav1 in intact cells through an N-terminus aromatic-rich consensus Cav1 binding motif. Unlike wildtype GRK5, a GRK5 mutant defective in Cav1 binding localized predominantly to the cytoplasm rather than the plasma membrane and failed to promote βarr2 recruitment to APC-activated PAR1. These studies suggest that Cav1 itself contributes to the regulation of APC-activated PAR1 βarr2 biased signaling likely through multiple mechanisms that may converge on GRK5.