Thrombin-induced shedding of tumour endothelial marker 5 and exposure of its RGD motif are regulated by cell-surface protein disulfide-isomerase.
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.
Department of Nuclear Medicine, Technische Universität München, Ismaninger Strasse 22, 81675 Munich, Germany. mario.vallon@lrz.tu-muenchen.de
Mario
M
Vallon
Philipp
P
Aubele
Klaus-Peter
KP
Janssen
Markus
M
Essler
D002478:Cells, Cultured; D057809:HEK293 Cells; D061307:Human Umbilical Vein Endothelial Cells / Q000378:metabolism / Q000502:physiology; D006801:Humans; D008565:Membrane Proteins / Q000737:chemistry / Q000378:metabolism / Q000502:physiology; D008954:Models, Biological; D009842:Oligopeptides / Q000737:chemistry / Q000235:genetics / Q000502:physiology; D019704:Protein Disulfide-Isomerases / Q000737:chemistry / Q000378:metabolism / Q000502:physiology*; D054730:Protein Interaction Domains and Motifs / Q000235:genetics / Q000502:physiology; D011499:Protein Processing, Post-Translational / Q000187:drug effects; D021381:Protein Transport / Q000187:drug effects / Q000235:genetics; D059748:Proteolysis / Q000187:drug effects; D043562:Receptors, G-Protein-Coupled / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D013917:Thrombin / Q000494:pharmacology*; D014162:Transfection
C531101:ADGRA2 protein, human; D008565:Membrane Proteins; D009842:Oligopeptides; D043562:Receptors, G-Protein-Coupled; C047981:arginyl-glycyl-aspartic acid; D013917:Thrombin; D019704:Protein Disulfide-Isomerases