Regulated Endothelial Protein C Receptor Shedding Is Mediated by Tumor Necrosis Factor-Alpha Converting enzyme/ADAM17

J Thromb Haemost. 2007 Feb;5(2):395-402. doi: 10.1111/j.1538-7836.2007.02347.x. Epub 2006 Dec 7.

Abstract

Endothelial protein C receptor (EPCR) plays an important role in the protein C anticoagulation pathway. Previously, we have reported that EPCR can be shed from the cell surface, and that this is mediated by an unidentified metalloproteinase. In this study, we demonstrate that tumor necrosis factor-alpha converting enzyme/ADAM17 (TACE) is responsible for EPCR shedding. Phorbol-12-myristate 13-acetate (PMA)-stimulated EPCR shedding is reduced by approximately 50% in HEK293 cells transfected with human EPCR cDNA and by 60% in human umbilical vein endothelial cells after transfection of TACE small interfering RNA (siRNA) into these cells. PMA-stimulated EPCR shedding is completely blocked in fibroblasts from TACE-deficient mice transfected with human EPCR cDNA, and restored by transfection of TACE cDNA into this cell line. To characterize the EPCR sequence requirement for shedding, we generated several mutants of EPCR. Replacing amino acids from residue 193 to residue 200 with the FLAG sequence (DYKDDDDK) completely blocks EPCR shedding, whereas a single amino acid substitution in this region has less effect on EPCR shedding.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • ADAM Proteins / metabolism*
  • ADAM Proteins / physiology
  • ADAM17 Protein
  • Animals
  • Antigens, CD / genetics
  • Antigens, CD / metabolism*
  • Cell Line
  • Endothelial Protein C Receptor
  • Endothelium, Vascular / cytology
  • Endothelium, Vascular / metabolism*
  • Humans
  • Mice
  • Mice, Transgenic
  • Receptors, Cell Surface / genetics
  • Receptors, Cell Surface / metabolism*
  • Tetradecanoylphorbol Acetate / pharmacology
  • Transfection
  • Tumor Necrosis Factor-alpha

Substances

  • Antigens, CD
  • Endothelial Protein C Receptor
  • PROCR protein, human
  • Receptors, Cell Surface
  • Tumor Necrosis Factor-alpha
  • ADAM Proteins
  • ADAM17 Protein
  • ADAM17 protein, human
  • Adam17 protein, mouse
  • Tetradecanoylphorbol Acetate