EXPRESSION OF RAT THROMBIN RECEPTOR AND MUTANTS IN THE BACULOVIRUS-INFECTED INSECT-CELL SYSTEM

The rat thrombin receptor (TR-1) has been expressed in Sf9 cells. Two mutant receptors, one lacking a thrombin cleavage site (RTRM), and the other containing only the extracellular domain of the receptor (ECD), have been expressed. Antibodies to the thrombin receptor activating p eptide (TRAP) and to the fibrinogen-binding exosite have been prepared . On Western blots these antibodies bound to TR-1 bands of 65-70 kDa a nd 44 kDa, similar bands for RTRM, and a doublet of about 10 and 12 kD a for ECD. The free cytosolic Ca++ concentration, measured by Fura-2, increased after thrombin or TRAP stimulation in the cells expressing T R-1. The RTRM cells did not respond to thrombin and had an attenuated response to TRAP. The ECD protein was found in the medium; it was not glycosylated. Both the mutants and the antibodies should be useful for studies of TR-1 structure and function. (C) 1997 Elsevier Science Ltd .