Signaling bias of the protease-activated receptor-1 is dictated by distinct GRK5 and β-arrestin-2 determinants

G protein-coupled receptors (GPCRs) exhibit signaling bias or preferential activation of heterotrimeric G proteins versus GPCR kinase (GRK)-mediated β-arrestin signaling. The protease-activated receptor-1 (PAR1) activates both G protein and β-arrestin in response to thrombin but only β-arrestin in response to activated protein C (APC). Thrombin-activated PAR1-G protein signaling is desensitized by β-arrestin-1, whereas APC-activated PAR1 signaling is propagated by β-arrestin-2. The mechanisms underlying PAR1 biased signaling are not known. Here, using computational modeling combined with cellular and biochemical studies, we reveal the molecular basis of signaling by thrombin- and APC-activated PAR1. Although both thrombin- and APC-induced PAR1 signaling are regulated by the same GRK, GRK5, the two types of signaling are differentially dependent on GRK5 membrane anchoring, PAR1 C-terminal phosphorylation sites, and the binding mode of β-arrestin-2. These differences translate into distinct β-arrestin-2 conformations and define the APC cytoprotective signaling signature, which contrasts with thrombin inflammatory signaling.