Determinants of the specificity of protease-activated receptors 1 and 2 signaling by factor Xa and thrombin.
Factor Xa (FXa) elicits intracellular signaling responses through the activation of protease-activated receptor 2 (PAR2) and possibly also through PAR1 in endothelial cells. In this study, we investigated FXa signaling in endothelial cells when the protease was either in free form or assembled into the prothrombinase complex. Furthermore, we prepared several wild-type and mutant PAR1 and PAR2 cleavage-reporter constructs in which their exodomains were fused to cDNA encoding for a soluble alkaline phosphatase (ALP). In the mutants, P2 residues were exchanged between PAR1 and PAR2 cleavage-reporter constructs and the hirudin-like binding site (HLBS) of PAR1 was inserted into the homologous site of PAR2. In non-transfected cells, FXa elicited a protective response which could be blocked by a specific anti-PAR2 but not by an anti-PAR1 antibody. A similar protective activity was observed for FXa in the prothrombinase complex. Further studies revealed that neither the Gla- nor EGF1-domain of FXa is required for its signaling activity, however, the N-terminus Arg-86 and Lys-87 of the EGF2-domain were essential. In the cleavage-reporter transfected cells, FXa cleaved the PAR2 construct effectively, however, replacing its P2-Gly with P2-Pro of PAR1 impaired its cleavage by FXa but improved it by thrombin. A PAR2 construct containing both P2-Pro and HLBS of PAR1 was poorly cleaved by FXa, but effectively by thrombin. A PAR1 construct containing P2 and P3 residues of PAR2 was poorly cleaved by thrombin but effectively by FXa. These results provide new insight into mechanisms through which coagulation proteases specifically interact with their target PAR receptors.
Edward A. Doisy Department of Biochemistry and Molecular Biology, Saint Louis University School of Medicine, Saint Louis, Missouri 63104, USA.
Soumendra
S
Rana
Likui
L
Yang
Seyed Mahdi
SM
Hassanian
Alireza R
AR
Rezaie
D000469:Alkaline Phosphatase / Q000032:analysis / Q000235:genetics; D000595:Amino Acid Sequence; D002478:Cells, Cultured; D042783:Endothelial Cells / Q000187:drug effects / Q000378:metabolism; D015951:Factor Xa / Q000737:chemistry / Q000378:metabolism*; D006801:Humans; D008969:Molecular Sequence Data; D017434:Protein Structure, Tertiary; D044463:Receptor, PAR-1 / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D044464:Receptor, PAR-2 / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D016415:Sequence Alignment; D015398:Signal Transduction; D013917:Thrombin / Q000378:metabolism*
D044463:Receptor, PAR-1; D044464:Receptor, PAR-2; D000469:Alkaline Phosphatase; D013917:Thrombin; D015951:Factor Xa