Activated protein C promotes protease-activated receptor-1 cytoprotective signaling through β-arrestin and dishevelled-2 scaffolds

Abstract

Protease-activated receptor-1 (PAR1) is a guanine nucleotidebinding (G) protein-coupled receptor that elicits cellular responses to coagulant and anticoagulant proteases. Activation of PAR1 by the coagulant protease thrombin results in Ras homolog gene family member A (RhoA) activation, disassembly of adherens junctions, and disruption of the endothelial barrier. In contrast, activation of PAR1 with the anticoagulant protease activated protein C (APC) results in activation of Ras-related C3 botulinum toxin substrate 1 (Rac1) and endothelial barrier protection. We previously showed that APC cytoprotective signaling requires the compartmentalization of PAR1 in caveolar microdomains. However, the mechanism by which APC-activated PAR1 promotes cytoprotective signaling in human endothelial cells remains poorly understood. Here we show that APC-activated PAR1 cytoprotective signaling is mediated by β-arrestin recruitment and activation of the dishevelled-2 (Dvl-2) scaffold and not by G protein α inhibiting activity polypeptide 2 (Gα i ) signaling. In human endothelial cells, PAR1 and β-arrestins form a preassembled complex and cosegregate in caveolin-1-enriched fractions. Remarkably, we found that depletion of β-arrestin expression by RNA interference resulted in the loss of APC-induced Rac1 activation but not of thrombin-stimulated RhoA signaling. APC also failed to protect against thrombin-induced endothelial barrier permeability in cells deficient in β-arrestin expression. We further demonstrate that APC activation of PAR1 results in β-arrestin-dependent recruitment of Dvl-2, which is critical for Rac1 signaling and endothelial barrier protection but not for thrombin-induced RhoA signaling. Our findings identify a role for β-arrestin and Dvl-2 scaffolds in APC-activated PAR1 cytoprotective signaling in human endothelial cells.biased agonism | endothelial dysfunction | inflammation | sepsis

Bidirectional Pathologic Effects of Thrombin

“…EPCR acts as a switch for the bidirectional activities of PAR1 [8]. EPCR-dependent cytoprotective signaling is mediated by PAR1 phosphorylation by GRK5 through beta-arrestin, resulting in reduced endothelial permeability due to Rac1 activation [16,19]. The contribution of Gai to these reactions is not well understood, however.…”

“…PAR1 and its cofactors, including G-proteins and EPCR, are localized in lipid rafts, which are specialized cell surface microdomains with or without caveolin [18,19,25]. Endothelial cells are rich in lipid raft.…”

β-Arrestin and dishevelled coordinate biased signaling

“…Such biased agonism can be exploited to develop better drugs acting at the 7TMRs with reduced side effects (5). In PNAS, Soh and Trejo report a cytoprotective β-arrestin-biased signaling pathway emanating from the 7TMR, protease activated receptor-1 (PAR1), which could be important in reducing sepsis-induced inflammation (6).…”

“…As β-arrestin is necessary for APCinduced activation of the small GTPase RAC1, which is crucial for the endothelial barrier protection, the authors investigated the underlying molecular mechanism connecting β-arrestin and RAC1 (6). Remarkably, it turned out that dishevelled 2 (DVL2), a phosphoprotein associated with the nonclassical 7TMR called frizzled (FZD), is the missing link (Fig.…”

“…1). Moreover, APC functions as a β-arrestin-biased agonist, because it does not activate G protein signaling (6).…”