Activated protein C drives β-arrestin-2- and c-Src-dependent phosphorylation of Cav1 and modulates Cav1 association with PAR1 and GRK5.

G-protein-coupled receptors (GPCRs) display bias toward either G proteins or GPCR kinase (GRK)-mediated β-arrestin (βarr) signaling depending on the agonist-stabilized receptor conformation. The cellular context and subcellular location of GPCRs can also influence biased signaling through mechanisms that are not well understood. The protease-activated receptor-1 (PAR1) exhibits signaling bias in response to thrombin and activated protein C (APC). APC-induced 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 component of caveolae and regulates protein-protein interactions. The mechanisms by which Cav1 contributes to APC-PAR1-induced βarr2-biased signaling are not known. Here, we report that a substantial population of endogenous PAR1 colocalizes with Cav1 in endothelial cells and is modulated by APC, assessed by single-molecule super-resolution stochastic optical reconstruction microscopy imaging. APC activation of PAR1 also induces Cav1 tyrosine-14 phosphorylation through a βarr2- and c-Src-dependent pathway, which disrupts PAR1-Cav1 coassociation. A smaller population of endogenous GRK5 was also found to colocalize with Cav1 in endothelial cells and was modestly altered by APC activation of PAR1. Moreover, GRK5 was found to interact with Cav1 in intact cells through an N-terminal aromatic-rich Cav1 binding motif. Mutation of this motif disrupts GRK5-Cav1 binding, shifts GRK5 predominantly to the cytoplasm rather than the plasma membrane, and perturbs GRK5-mediated βarr2 recruitment to APC-activated PAR1. Thus, beyond its structural function, Cav1 participates in protein-protein interactions with PAR1 and GRK5, two key effectors that enable APC-induced βarr2 signaling.