Regulated shedding of PAR1 N-terminal exodomain from endothelial cells.
G protein-coupled receptors can trigger metalloproteinase-dependent shedding of proteins from the cell surface. We now report that G protein-coupled receptors can themselves undergo regulated metalloproteinase-dependent shedding. The N-terminal exodomain of protease-activated receptor-1 (PAR1), a G protein-coupled receptor for thrombin, displayed regulated shedding in endothelial cells, which normally express this receptor. Cleavage occurred at a site predicted to render the receptor unresponsive to thrombin. A chimeric protein in which the N-terminal exodomain of PAR1 was fused to an unrelated transmembrane segment was shed as efficiently as PAR1, shedding of both proteins was stimulated by phorbol ester and by a PAR1 agonist. TNFalpha protease inhibitor-2 (TAPI-2), phenanthroline, and tissue inhibitor of metalloproteinase-3 (TIMP-3) but not TIMP-1 or -2 inhibited such shedding. These and other data suggest that the information that specifies PAR1 shedding resides within its N-terminal exodomain rather than its heptahelical segment, that activation of protein kinase C or of PAR1 itself can stimulate PAR1 shedding in trans, and that ADAM17/TACE or a metalloproteinase with similar properties mediates PAR1 shedding. Regulated shedding reduced the amount of cell surface PAR1 available for productive cleavage by thrombin by half or more, but thus far we have been unable to demonstrate an effect of PAR1 shedding on cellular responsiveness to thrombin. Nonetheless, regulated shedding of G protein-coupled receptors represents a new mechanism by which signaling by this important class of receptors might be modulated.
Cardiovascular Research Institute, University of California, San Francisco 94143-0130, USA.
Matthew J
MJ
Ludeman
Yao Wu
YW
Zheng
Kenji
K
Ishii
Shaun R
SR
Coughlin
D000469:Alkaline Phosphatase / Q000235:genetics / Q000378:metabolism; D000595:Amino Acid Sequence; D004730:Endothelium, Vascular / Q000166:cytology*; D004789:Enzyme Activation; D006801:Humans; D006868:Hydrolysis; D008024:Ligands; D045726:Metalloproteases / Q000378:metabolism; D011480:Protease Inhibitors / Q000494:pharmacology; D017434:Protein Structure, Tertiary; D044463:Receptor, PAR-1 / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D011993:Recombinant Fusion Proteins; D013917:Thrombin / Q000378:metabolism; D014471:Umbilical Veins
D008024:Ligands; D011480:Protease Inhibitors; D044463:Receptor, PAR-1; D011993:Recombinant Fusion Proteins; D000469:Alkaline Phosphatase; D045726:Metalloproteases; D013917:Thrombin