Identification of the protein C/activated protein C finding sites on the endothelial cell protein C receptor - Implications for a novel mode of ligand recognition by a major histocompatibility complex class 1-type receptor

The endothelial cell protein C receptor (EPCR) is an endothelial cell-speci fic transmembrane protein that binds both protein C and activated protein C (APC), EPCR regulates the protein C anticoagulant pathway by binding prote in C and augmenting protein C activation by the thrombin-thrombomodulin com plex. EPCR is homologous to the MHC class 1/CD1 family, members of which co ntain two alpha -helices that sit upon an 8-stranded beta- sheet platform. In this study, we identified 10 residues that, when mutated to alanine, res ult in the loss of protein C/APC binding (Arg-81, Leu-82, Val-83, Glu-86, A rg-87, Phe-146, Tyr-154, Thr-157, Arg-158, and Glu-160). Glutamine substitu tions at the four N-linked carbohydrate attachment sites of EPCR have littl e affect on APC binding, suggesting that the carbohydrate moieties of EPCR are not critical for ligand recognition. We then mapped the epitopes for fo ur anti-human EPCR monoclonal antibodies (mAbs), two of which block EPCR/Fl -APC (APC labeled at the active site with fluorescein) interactions, wherea s two do not. These epitopes were localized by generating human-mouse EPCR chimeric proteins, since the mAbs under investigation do not recognize mous e EPCR. We found that 5 of the 10 candidate residues for protein C/APC bind ing (Arg-81, Leu-82, Val-83, Glu-86, Arg-87) colocalize with the epitope fo r one of the blocking mAbs, Three-dimensional molecular modeling of EPCR in dicates that the 10 protein C/APC binding candidate residues are clustered at the distal end of the two alpha -helical segments. Protein C activation studies on 293 cells that coexpress EPCR variants and thrombomodulin demons trate that protein C binding to EPCR is necessary for the EPCR-dependent en hancement in protein activation by the thrombin-thrombomodulin complex. The se studies indicate that EPCR has exploited the MHC class 1 fold for an alt ernative and possibly novel mode of ligand recognition. These studies are a lso the first to identify the protein C/APC binding region of EPCR and may provide useful information about molecular defects in EPCR that could contr ibute to cardiovascular disease susceptibility.