Caveolae are required for protease-selective signaling by protease-activated receptor–1

Abstract

Protease-activated receptor-1 (PAR1) is a G-protein-coupled receptor uniquely activated by proteolysis. Thrombin, a coagulant protease, induces inflammatory responses and endothelial barrier permeability through the activation of PAR 1. Activated protein C (APC), an anti-coagulant protease, also activates PAR 1. However, unlike thrombin, APC elicits anti-inflammatory responses and protects against endothelial barrier dysfunction induced by thrombin. We found that thrombin and APC signaling were lost in PAR 1-deficient endothelial cells, indicating that PAR 1 is the major effector of protease signaling. To delineate the mechanism responsible for protease-selective signaling by PAR 1, we examined the effect of APC and thrombin on the activation of RhoA and Rac1, small GTPases that differentially regulate endothelial barrier permeability. Thrombin caused robust RhoA signaling but not Rac1 activation, whereas APC stimulated a marked increase in Rac1 activation but not RhoA signaling, consistent with the opposing functions of these proteases on endothelial barrier integrity. Strikingly, APC signaling and endothelial barrier protection effects were abolished in cells lacking caveolin-1, whereas thrombin signaling remained intact. These findings suggest that compartmentalization of PAR 1 in caveolae is critical for APC selective signaling to Rac1 activation and endothelial barrier protection. We further report that APC induces PAR 1 phosphorylation and desensitizes endothelial cells to thrombin signaling but promotes limited receptor cleavage and negligible internalization and degradation even after prolonged APC exposure. Thus, APC selective signaling and endothelial barrier protective effects are mediated through compartmentalization of PAR 1 in caveolae and a novel mechanism of PAR1 signal regulation.

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

“…In addition, APC bound to EPCR on endothelial cells promotes anti-inflammatory and cytoprotective signaling through the activation of PAR1 (4). APC cytoprotective signaling also requires the compartmentalization of PAR1 and its coreceptor EPCR in cholesterol-enriched caveolar microdomains (5,6). A previous study further showed that the cytoprotective activity of a noncoagulant APC is critical for reducing mortality associated with sepsis in an experimental mouse model (7).…”

“…In cultured human endothelial cells, thrombin activation of PAR1 promotes coupling to G protein α subunits 12 and 13 (Gα 12/13 ) and Gα q ; activation of Ras homolog gene family, member A (RhoA) and other signaling effectors that cause disassembly of adherens junctions; reorganization of the actin cytoskeleton; and transient disruption of the endothelial barrier (9,10). Conversely, APC cleaves and activates PAR1, albeit less efficiently than thrombin, and induces endothelial barrier stabilization through selective activation of Ras-related C3 botulinum toxin substrate 1 (Rac1) signaling (5,11). Unlike thrombin, APC activation of PAR1 does not promote coupling to Gα 12/13 or Gα q signaling but instead appears to require G protein α inhibiting activity polypeptide (Gα i ) signaling for cytoprotection.…”

“…These findings reveal a unique function for β-arrestin and Dvl-2 scaffolds in APC-promoted PAR1 cytoprotective signaling in cultured human endothelial cells and provide further support for β-arrestins as negative regulators of sepsis-induced inflammatory responses observed in vivo (23,24). The compartmentalization of PAR1 in caveolar microdomains is critical for APC-promoted Rac1 activation and endothelial barrier protection (5,6). To define the mediators of APC-activated PAR1 signaling, we examined whether Gα i and/or β-arrestins localize to caveolar microdomains together with PAR1 in human umbilical vein endothelial cell-derived EA.hy926 cells using sucrose densitygradient fractionation.…”

β-Arrestin and dishevelled coordinate biased signaling

“…APC-activated EPCR and PAR1 localize to membrane regions enriched in caveolin and promote cytoprotective pathways, thus decreasing mortality in animal models of sepsis (8,10). Earlier studies have shown that APC and thrombin show different effects at the PAR1: thrombin does not engage a coreceptor, induces complete cleavage of PAR1, and promotes PAR1 internalization and degradation; APC engages the coreceptor EPCR, induces limited cleavage of PAR1, and does not promote receptor internalization and degradation (8,11). Furthermore, thrombin is more potent than APC and promotes internalization of APC-bound PAR1 (11,12).…”

“…Earlier studies have shown that APC and thrombin show different effects at the PAR1: thrombin does not engage a coreceptor, induces complete cleavage of PAR1, and promotes PAR1 internalization and degradation; APC engages the coreceptor EPCR, induces limited cleavage of PAR1, and does not promote receptor internalization and degradation (8,11). Furthermore, thrombin is more potent than APC and promotes internalization of APC-bound PAR1 (11,12). Previously, Trejo and coworkers reported that thrombin and APC stimulated different small GTPases, namely RHOA (by thrombin) and RAC1 (by APC), and only APC-induced effects required caveolin (11).…”