Post-translational and activation-dependent modifications of the G protein-coupled thrombin receptor.
The purpose of the present study was to analyze the post-translational and activation-dependent modifications of the G protein-coupled thrombin receptor. A human receptor cDNA was engineered to encode an epitope tag derived from the vesicular stomatitis virus glycoprotein at the COOH terminus of the receptor and expressed in human embryonic kidney 293 cells. We show here that the mature receptor is a glycosylated protein with an apparent molecular mass ranging from 68 to 80 kDa by SDS-polyacrylamide gel electrophoresis. Removal of asparagine-linked oligosaccharides with N-glycosidase F leads to the appearance of a 36-40-kDa receptor species. The current model for receptor activation by thrombin involves specific hydrolysis of the arginine-41/serine-42 (Arg-41/Ser-42) peptide bond. Cleavage of the receptor by thrombin was demonstrated directly by Western analyses performed on membranes and glycoprotein-enriched lysates from transfected cells. Whereas thrombin treatment of cells results in increased mobility of the receptor in SDS-polyacrylamide gel electrophoresis, we found that their treatment with the thrombin receptor agonist peptide leads to a decrease in thrombin receptor mobility due, in part, to phosphorylation. The serine proteases trypsin and plasmin also cleave and activate the receptor similar to thrombin, whereas chymotrypsin cleaves the receptor at a site distal to Arg-41, thus rendering it unresponsive to thrombin while still responsive to thrombin receptor agonist peptide.
Vouret-Craviari
V
V
Centre de Biochimie, CNRS UMR134, Nice, France.
Grall
D
D
Chambard
J C
JC
Rasmussen
U B
UB
Pouysségur
J
J
Van Obberghen-Schilling
E
E
D000595:Amino Acid Sequence; D002460:Cell Line; D005341:Fibrinolysin / Q000378:metabolism; D019204:GTP-Binding Proteins / Q000378:metabolism*; D006031:Glycosylation; D006801:Humans; D006868:Hydrolysis; D008969:Molecular Sequence Data; D010766:Phosphorylation; D011499:Protein Processing, Post-Translational; D018179:Receptors, Thrombin / Q000378:metabolism*; D014162:Transfection; D014357:Trypsin / Q000378:metabolism
D018179:Receptors, Thrombin; D014357:Trypsin; D005341:Fibrinolysin; D019204:GTP-Binding Proteins