Receptors of the protein C activation and activated protein C signaling pathways are colocalized in lipid rafts of endothelial cells

Abstract

Ever-increasing evidence in the literature suggests that the antiinflammatory and cytoprotective properties of activated protein C (APC) are mediated through its endothelial protein C receptor (EPCR)-dependent cleavage of protease-activated receptor 1 (PAR-1) on endothelial cells. However, recent results monitoring the cleavage rate of PAR-1 on human umbilical vein endothelial cells, transfected with an alkaline phosphatase-PAR-1 fusion reporter construct, have indicated that the catalytic activity of thrombin toward PAR-1 is several orders of magnitude higher than that of APC. Because thrombin is required for generation of APC, and because it also functions in the proinflammatory pathways through the activation of PAR-1, it has been difficult to understand how APC can elicit protective cellular responses through the activation of PAR-1 when thrombin is present. In this study we provide a plausible answer to this question by demonstrating that the critical receptors required for both protein C activation (thrombomodulin and EPCR) and APC cellular signaling (EPCR and PAR-1) pathways are colocalized in the membrane lipid rafts in endothelial cells. We further show that the APC cleavage of PAR-1 on cells transfected with a PAR-1 cleavage reporter construct is not sensitive to the cofactor function of EPCR. Thus, the colocalization of EPCR and PAR-1 in lipid rafts is a key requirement for the cellular signaling activity of APC. Thrombomodulin colocalization with these receptors on the same membrane microdomain can also recruit thrombin to activate the EPCR-bound protein C, thereby eliciting PAR-1 signaling events that are involved in the APC protective pathways. endothelial protein C receptor ͉ protease-activated receptor 1 ͉ thrombin ͉ thrombomodulin

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

“…Our findings further indicate that PAR1 and β-arrestins are preassembled and coexist in caveolar microdomains. These results are consistent with the importance of PAR1 compartmentalization in caveolar microdomains for APCinduced cytoprotective signaling (5,6). This work also indicates that the β-arrestin-2 isoform is the predominant mediator of APC-induced cytoprotective signaling and is consistent with previous studies demonstrating a protective role in vivo for β-arrestin-2 in mouse models of experimentally induced sepsis lethality (23,24).…”

“…1A). These findings confirm that PAR1 is present in caveolar microdomains (5,6). In addition, both β-arrestin-1 and -2 isoforms were detected in caveolin-1-enriched fractions in control cells (Fig.…”

“…partmentalization in caveolae (5,6). We found that endogenous PAR1 and β-arrestins exist in a preassembled complex and localize to caveolar microdomains, consistent with a function in cytoprotective signaling.…”

“…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).…”

“…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.…”