FUNCTIONAL EXPRESSION OF A HUMAN THROMBIN RECEPTOR IN SF9 INSECT CELLS - EVIDENCE FOR AN ACTIVE TETHERED LIGAND

Desensitization of recombinant human thrombin receptors expressed in S f9 insect cells was compared with native thrombin receptors in megakar yoblast erythroleukaemia (HEL) cells. Addition of thrombin (2 units/ml ) or agonist peptide SFLLRN (10 mu M) Co HEL cells, or to Sf9 cells in fected with recombinant baculovirus containing the thrombin receptor, cDNA, produced an increase in the free cytosolic Ca2+ concentration ([ Ca2+](i)) as measured by fura-2. The response in HEL cells was transie nt, reflecting a rapid homologous desensitization. In contrast, [Ca2+] (i) in Sf9 cells expressing the thrombin receptor increased rapidly to a peak value that slowly declined, but remained elevated for at least 12 min following stimulation by thrombin. The sustained [Ca2+](i) res ponse to thrombin was not reversed by washout of thrombin or by subseq uent addition of hirudin. Pretreatment of Sf9 cells with either thromb in (2 units/ml) or SFLLRN (10 or 50 mu M) for 5 min produced a shift i n the ED(50) for SFLLRN (added 10 min after washout) from 0.4 mu M to 20 and 7 mu M, respectively, Thus, desensitization of thrombin recepto rs expressed in Sf9 cells occurs slowly and reflects a decrease in rec eptor affinity. The sustained [Ca2+](i) response in Sf9 cells stimulat ed by thrombin may reflect continuous activation by the tethered ligan d. To test this hypothesis, the effect of protease treatment during th e sustained phase of the response was examined. Addition of either ami nopeptidase M or thermolysin reversed the sustained response to SFLLRN , but only thermolysin reversed the sustained response to thrombin. Th ermolysin had no effect on the change in [Ca2+](i) observed following carbachol stimulation of Sf9 cells expressing the M(5) muscarinic rece ptor. Furthermore, following thermolysin treatment, the cells remained responsive to a subsequent application of SFLLRN, These results demon strate that the tethered ligand remains active for extended periods of time after thrombin stimulation and suggests that further hydrolysis by extracellular proteases may represent an important mechanism of rap id receptor deactivation.