Molecular cloning and expression of murine and bovine endothelial cell protein C/activated protein C receptor (EPCR). The structural and functional conservation in human, bovine, and murine EPCR.
Recently, we identified and cloned a human endothelial cell protein C/activated protein C receptor (EPCR). EPCR was predicted to be a type 1 transmembrane glycoprotein and a novel member of the CD1/major histocompatibility complex superfamily with 28% identity with CD1d. Even greater homology (62% identity) was detected with the murine protein, CCD41, which was previously characterized as a centrosome-associated, cell cycle-dependent protein. This raised the possibility that CCD41 was the murine homologue of EPCR. To address this possibility, to better understand structure-function relationships, and to facilitate physiological experiments on EPCR function, we cloned and sequenced murine and bovine EPCR from endothelial cell cDNA libraries. The nucleotide sequence of murine EPCR and CCD41 exhibited five differences corresponding to one base change, three single-base insertions, and one base deletion in the protein coding region. As a result, the predicted structures of EPCR and CCD41 differed in their amino and carboxyl termini but were identical in the central portion of the coding sequence. Based on comparison of the murine, bovine, and human EPCR sequences and the regions where discrepancies between murine EPCR and CCD41 were detected, we believe that CCD41 is probably identical to murine EPCR and that the reported sequence differences are likely the result of compression on the sequencing gel. Compared with human EPCR, the murine and bovine sequences were 69 and 73% identical, respectively, and 57% of the residues were identical between all three species. Both bovine and murine EPCR could bind human activated protein C when the cDNA clones were transfected into 293T cells. Like human EPCR, of the cell lines tested, the murine EPCR message was restricted to endothelium. Cloning of the murine and bovine homologue of EPCR will facilitate in vivo and in vitro studies of the role of EPCR in the protein C pathway.
Fukudome
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Howard Hughes Medical Institute, University of Oklahoma Health Sciences Center, Oklahoma City.
Esmon
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D000595:Amino Acid Sequence; D000818:Animals; D001011:Aorta; D001483:Base Sequence; D001779:Blood Coagulation Factors; D015152:Blotting, Northern; D002417:Cattle; D002460:Cell Line; D003001:Cloning, Molecular; D017124:Conserved Sequence; D017931:DNA Primers; D018076:DNA, Complementary; D004730:Endothelium, Vascular / Q000378:metabolism*; D005434:Flow Cytometry; D015870:Gene Expression; D015723:Gene Library; D006801:Humans; D008285:Major Histocompatibility Complex; D051379:Mice; D008969:Molecular Sequence Data; D021382:Protein Sorting Signals / Q000096:biosynthesis; D011956:Receptors, Cell Surface / Q000096:biosynthesis* / Q000737:chemistry / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D014407:Tumor Cells, Cultured
D001779:Blood Coagulation Factors; D017931:DNA Primers; D018076:DNA, Complementary; D021382:Protein Sorting Signals; D011956:Receptors, Cell Surface; C091963:activated protein C receptor