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 peptide (TRAP) and to the fibrinogen-binding exosite have been prepared. On Western blots these antibodies bound to TR-1 bands of 65-70 kDa and 44 kDa, similar bands for RTRM, and a doublet of about 10 and 12 kDa for ECD. The free cytosolic Ca++ concentration, measured by Fura-2, increased after thrombin or TRAP stimulation in the cells expressing TR-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.