THE ENDOTHELIAL-CELL PROTEIN-C RECEPTOR - CELL-SURFACE EXPRESSION ANDDIRECT LIGAND-BINDING BY THE SOLUBLE RECEPTOR

Expression of the endothelial cell protein C receptor (EPCR) gene in m ammalian cells imparts the capacity to bind activated protein C (APC) or protein C. Immunochemical analysis of CCD41, apparently the murine homologue of EPCR, suggested centrosomal localization, raising questio ns about the location of the EPCR gene product and its role in protein C binding, In this study, we express a soluble form of EPCR, demonstr ate EPCR expression on the cell surface, and direct binding between so luble EPCR and protein C/APC. Affinity purified polyclonal and a monoc lonal antibody against EPCR bound to the cell surface of EPCR-transfec ted cells but not to control cells. A 49-kDa protein, a mass similar t o soluble EPCR, was immunoprecipitated from the cell surface of endoth elium and cells transfected with human EPCR but not from control cells , The FLAG(TM) antibody and APC bound to cells expressing an EPCR cons truct containing the FLAG(TM) epitope located in a putative extracellu lar domain, whereas an EPCR construct truncated just before the putati ve transmembrane domain produced only soluble EPCR antigen, Soluble EP CR inhibited APC binding to EPCR expressing cells in a concentration-d ependent fashion, K-d (app) = 29 nM and bound to immobilized protein C in a Ca2+-dependent fashion. Thus, EPCR is a type 1 transmembrane pro tein that binds directly to APC.