The endothelial cell protein C receptor - Cell surface expression and direct ligand binding by the soluble receptor

Expression of the endothelial cell protein C receptor (EPCR) gene in mammalian 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 questions 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, demonstrate EPCR expression on the cell surface, and direct binding between soluble EPCR and protein C/APC. Affinity purified polyclonal and a monoclonal antibody against EPCR bound to the cell surface of EPCR-transfected cells but not to control cells. A 49-kDa protein, a mass similar to soluble EPCR, was immunoprecipitated from the cell surface of endothelium and cells transfected with human EPCR but not from control cells, The FLAG(TM) antibody and APC bound to cells expressing an EPCR construct containing the FLAG(TM) epitope located in a putative extracellular domain, whereas an EPCR construct truncated just before the putative transmembrane domain produced only soluble EPCR antigen, Soluble EPCR inhibited APC binding to EPCR expressing cells in a concentration-dependent fashion, K-d (app) = 29 nM and bound to immobilized protein C in a Ca2+-dependent fashion. Thus, EPCR is a type 1 transmembrane protein that binds directly to APC.