Thrombin-induced shedding of tumour endothelial marker 5 and exposure of its RGD motif are regulated by cell-surface protein disulfide-isomerase

Biochem J. 2012 Feb 1;441(3):937-44. doi: 10.1042/BJ20111682.

Abstract

TEM5 (tumour endothelial marker 5; also known as GPR124) is an adhesion G-protein-coupled receptor containing a cryptic RGD motif in its extracellular domain. TEM5 is expressed in endothelial cells and pericytes during angiogenesis. In the present paper, we report that thrombin mediates shedding of an N-terminal TEM5 fragment of 60 kDa (termed N60) containing the RGD motif in an open conformation. Thrombin directly cleaved rsTEM5 (recombinant soluble TEM5) 5 and 34 residues downstream of the RGD motif, resulting in formation of N60 and its C-terminal counterpart (termed C50). Interestingly, N60 derived from thrombin cleavage of rsTEM5 was covalently linked to C50 by disulfide bonds, whereas N60 shed from thrombin-treated cells was not associated with its membrane-bound C-terminal counterpart. Inhibition of the reducing function of cell-surface PDI (protein disulfide-isomerase) abrogated thrombin-induced N60 shedding. Conversely, addition of reduced PDI enhanced N60 shedding. Furthermore, thrombin cleavage of rsTEM5 was increased by reduced PDI and resulted in dissociation of the N60-C50 heterodimer. We conclude that PDI regulates thrombin-induced shedding of N60 and exposure of the TEM5 RGD motif by catalysing the reduction of crucial disulfide bonds of TEM5 on the cell surface. Binding of N60 to RGD-dependent integrins may modulate cellular functions such as adhesion and migration during angiogenesis.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Cells, Cultured
  • HEK293 Cells
  • Human Umbilical Vein Endothelial Cells / metabolism
  • Human Umbilical Vein Endothelial Cells / physiology
  • Humans
  • Membrane Proteins / chemistry
  • Membrane Proteins / metabolism
  • Membrane Proteins / physiology
  • Models, Biological
  • Oligopeptides / chemistry
  • Oligopeptides / genetics
  • Oligopeptides / physiology
  • Protein Disulfide-Isomerases / chemistry
  • Protein Disulfide-Isomerases / metabolism
  • Protein Disulfide-Isomerases / physiology*
  • Protein Interaction Domains and Motifs / genetics
  • Protein Interaction Domains and Motifs / physiology
  • Protein Processing, Post-Translational / drug effects
  • Protein Transport / drug effects
  • Protein Transport / genetics
  • Proteolysis / drug effects
  • Receptors, G-Protein-Coupled / chemistry*
  • Receptors, G-Protein-Coupled / genetics
  • Receptors, G-Protein-Coupled / metabolism*
  • Thrombin / pharmacology*
  • Transfection

Substances

  • ADGRA2 protein, human
  • Membrane Proteins
  • Oligopeptides
  • Receptors, G-Protein-Coupled
  • arginyl-glycyl-aspartic acid
  • Thrombin
  • Protein Disulfide-Isomerases