Molecular Cloning and Expression of Murine and Bovine Endothelial Cell Protein C/Activated Protein C Receptor (EPCR)

Abstract

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.

A Protein C Deficiency Exacerbates Inflammatory and Hypotensive Responses in Mice During Polymicrobial Sepsis in a Cecal Ligation and Puncture Model

“…Expression of EPCR in cell cultures decreased after treatment with mediators of the inflammatory response, eg, TNF-␣, 64 which is in agreement with the impairment in PC activation seen in septic shock patients. However, up-regulation of EPCR gene expression, and increase in the release of a soluble form of EPCR by shedding from the endothelium, were observed in rats as a result of an endotoxin challenge.…”

Differential regulation of matrix metalloproteinase 2 and matrix metalloproteinase 9 by activated protein C: Relevance to inflammation in rheumatoid arthritis

“…EPCR is a 46-kd type I transmembrane glycoprotein that is homologous to class I major histocompatibility complex/CD1 family of proteins (19,23). It is predominantly expressed by endothelial cells, mainly those of larger blood vessels, although recent reports have shown it to be present on some leukocytes of the innate immune system (21,42), including RA monocytes (Xue M, et al: unpublished observations).…”

“…The conversion to APC is augmented in the presence of the specific receptor endothelial protein C receptor (EPCR) (19), which is expressed on the surface of endothelial cells, keratinocytes (20), and some leukocytes, including neutrophils and monocytes (21). In addition, APC is now recognized as playing a key role in the regulation of inflammation by down-regulating leukocyte activation and inflammatory cytokine production (22).…”

Factor VIIa binding to endothelial cell protein C receptor

“…This rules out the possibility that factor X also binds to EPCR. It is interesting to note that both FVII and FVIIa bind to EPCR on non-stimulated HUVEC with a similar affinity (Kd, ~30-50 nM range) as that of protein C/APC [14]. The number of EPCR-specific FVII/FVIIa binding sites on endothelial cells (~50,000 sites/cell) is also very similar to the number of protein C/APC binding sites.…”