POSTTRANSLATIONAL 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 throm bin receptor. A human receptor cDNA was engineered to encode an epitop e 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 pro tein with an apparent molecular mass ranging from 68 to 80 kDa by SDS- polyacrylamide gel electrophoresis. Removal of asparagine-linked oligo saccharides with N-glycosidase F leads to the appearance of a 36-40-kD a receptor species. The current model for receptor activation by throm bin involves specific hydrolysis of the arginine-4l/serine-42 (Arg-41/ Ser-42) peptide bond. Cleavage of the receptor by thrombin was demonst rated directly by Western analyses performed on membranes and glycopro tein-enriched lysates from transfected cells. Whereas thrombin treatme nt of cells results in increased mobility of the receptor in SDS-polya crylamide gel electrophoresis, we found that their treatment with the thrombin receptor agonist peptide leads to a decrease in thrombin rece ptor mobility due, in part, to phosphorylation. The serine proteases t rypsin and plasmin also cleave and activate the receptor similar to th rombin, whereas chymotrypsin cleaves the receptor at a site distal to Arg-41, thus rendering it unresponsive to thrombin while still respons ive to thrombin receptor agonist peptide.