The Endothelial Cell Protein C Receptor: CELL SURFACE EXPRESSION AND DIRECT LIGAND BINDING BY THE SOLUBLE RECEPTOR

Kenji Fukudome,Shinichiro Kurosawa,Deborah J Stearns-Kurosawa,Xuhua He,Alireza R Rezaie,Charles T Esmon

doi:10.1074/jbc.271.29.17491

Abstract

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 FLAGtrade mark antibody and APC bound to cells expressing an EPCR construct containing the FLAGtrade mark 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, Kd (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.

Highlights

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
This study provides many lines of evidence that indicate EPCR is expressed as a cell surface protein and that it is directly involved in protein C/APC binding
A monoclonal antibody and affinity purified polyclonal antibodies raised against recombinant EPCR bound to HUVEC and to human or murine cell lines stably transfected with human EPCR but not to the same sham-transfected cell lines

Summary

CELL SURFACE EXPRESSION AND DIRECT LIGAND BINDING BY THE SOLUBLE RECEPTOR*

(Received for publication, February 27, 1996, and in revised form, May 10, 1996). Kenji Fukudome‡, Shinichiro Kurosawa‡, Deborah J. We express a soluble form of EPCR, demonstrate EPCR expression on the cell surface, and direct binding between soluble EPCR and protein C/APC. The unexpected observation that EPCR is homologous, and probably identical, to a previously described intracellular murine protein, CCD41 [14], raised questions about this model of s-EPCR-HPC4, a soluble form of EPCR with the HPC4 epitope inserted in place of the transmembrane domain and cytosolic tail; TFT210HIS, a bacterially expressed EPCR fusion protein with thioredoxin on the amino terminus and the transmembrane and cytosolic tail of EPCR deleted and replaced with a 6-residue His tag; FACS, fluorescenceactivated cell sorter; E-7, 293 cells stably transfected with human EPCR; N-1, control sham transfected 293 cells; HEN-1, NIH3T3 cells stably tranfected with human EPCR; NN-1, control sham transfected NIH3T3 cells; HUVEC, human umbilical vein endothelial cells; PAGE, polyacrylamide gel electrophoresis; PCR, polymerase chain reaction; HBSS, Hank’s buffered salt solution; Fl-APC, fluorescein-labeled APC; FITC, fluorescein isothiocyanate

EPCR Expression and Function

EXPERIMENTAL PROCEDURES

RESULTS

DISCUSSION