title: Occupancy of human EPCR by protein C induces β-arrestin-2 biased PAR1 signaling by both APC and thrombin.
abstract: Activation of protease-activated receptor 1 (PAR1) by activated protein C (APC) and thrombin elicits paradoxical cytoprotective and cytotoxic signaling responses in vascular endothelial cells through cleavage of the receptor at Arg-46 and Arg-41 protease recognition sites, respectively. It has been reported that unlike a disruptive G-protein-mediated PAR1 signaling by thrombin, APC induces a protective β-arrestin-2 biased PAR1 signaling by unknown mechanisms. We hypothesize that the occupancy of endothelial protein C receptor (EPCR) by the Gla-domain of protein C/APC is responsible for the β-arrestin-2 biased PAR1 signaling independent of the protease cleavage site. To test this hypothesis, we monitored the signaling specificity of thrombin in endothelial cells in response to lipopolysaccharide (LPS) with or without pretreatment of cells with protein C-S195A. The PAR1-dependent recruitment of β-arrestin-2 in response to LPS by both APC and thrombin was analyzed by functional, gene silencing, and signaling assays. Results indicate that similar to APC, thrombin exerts cytoprotective effects via β-arrestin-2 biased PAR1 signaling. Similar to APC, thrombin triggered β-arrestin-2-dependent recruitment of disheveled 2 (Dvl-2) in PC-S195A pretreated cells. Further studies in HeLa cells transfected with PAR1 constructs revealed that EPCR occupancy initiates β-arrestin-2 biased PAR1 signaling independent of the protease cleavage sites. We demonstrate that EPCR occupancy recruits G-protein coupled receptor kinase 5, thereby inducing β-arrestin-2 biased PAR1 signaling by both APC and thrombin. In support of a physiological relevance for these results, intraperitoneal administration of PC-S195A conferred a cytoprotective effect for thrombin in an in vivo inflammatory model.
authors: Edward A. Doisy Department of Biochemistry and Molecular Biology, Saint Louis University School of Medicine, Saint Louis, MO.
authors: Ram Vinod
authors: RV
authors: Roy
authors: Edward A. Doisy Department of Biochemistry and Molecular Biology, Saint Louis University School of Medicine, Saint Louis, MO.
authors: Abdolreza
authors: A
authors: Ardeshirylajimi
authors: Edward A. Doisy Department of Biochemistry and Molecular Biology, Saint Louis University School of Medicine, Saint Louis, MO.
authors: Peyman
authors: P
authors: Dinarvand
authors: Edward A. Doisy Department of Biochemistry and Molecular Biology, Saint Louis University School of Medicine, Saint Louis, MO.
authors: Likui
authors: L
authors: Yang
authors: Edward A. Doisy Department of Biochemistry and Molecular Biology, Saint Louis University School of Medicine, Saint Louis, MO.
authors: Alireza R
authors: AR
authors: Rezaie
mesh_terms: D000818:Animals; D015703:Antigens, CD / Q000378:metabolism*; D019610:Cytoprotection; D000072261:Dishevelled Proteins / Q000378:metabolism; D042783:Endothelial Cells / Q000378:metabolism; D000074283:Endothelial Protein C Receptor; D054774:G-Protein-Coupled Receptor Kinase 5 / Q000378:metabolism; D019206:GTP-Binding Protein alpha Subunits, Gi-Go / Q000378:metabolism; D024243:HMGB1 Protein / Q000378:metabolism; D006367:HeLa Cells; D006801:Humans; D007249:Inflammation / Q000378:metabolism / Q000473:pathology; D008297:Male; D021962:Membrane Microdomains / Q000378:metabolism; D008810:Mice, Inbred C57BL; D050505:Mutant Proteins / Q000378:metabolism; D011486:Protein C / Q000378:metabolism*; D021381:Protein Transport; D044463:Receptor, PAR-1 / Q000378:metabolism*; D011956:Receptors, Cell Surface / Q000378:metabolism*; D049349:Receptors, Lysosphingolipid / Q000378:metabolism; D015398:Signal Transduction; D013917:Thrombin / Q000378:metabolism*; D051176:beta Catenin / Q000378:metabolism; D000071560:beta-Arrestin 2 / Q000378:metabolism*
chemical_list: D015703:Antigens, CD; C000606581:DVL2 protein, human; D000072261:Dishevelled Proteins; D000074283:Endothelial Protein C Receptor; D024243:HMGB1 Protein; D050505:Mutant Proteins; C478370:PROCR protein, human; D011486:Protein C; D044463:Receptor, PAR-1; D011956:Receptors, Cell Surface; D049349:Receptors, Lysosphingolipid; D051176:beta Catenin; D000071560:beta-Arrestin 2; D054774:G-Protein-Coupled Receptor Kinase 5; C517898:GRK5 protein, human; D013917:Thrombin; D019206:GTP-Binding Protein alpha Subunits, Gi-Go