Activation of the rod G-protein G(t) by the thrombin receptor (PAR1) expressed in Sf9 cells

Functional coupling of the human thrombin receptor PAR1 (protease- activated receptor 1) with the retinal rod G-protein transducin (G(l), a member of the G(i) family) was studied in a reconstituted system of membranes from Sf9 cells expressing the thrombin receptor and purified G(t) from bovine rod outer segments. TRAP6-agonist-activated PAR1 interacts productively with the distant G-protein. Agonist-dependent G(t) activation was measured using a real-time fluorimetric GTP[S]-binding assay and membranes from Sf9 cells. To characterize nucleotide-exchange catalysis by PAR1, we analyzed dependence on nucleotides, temperature and pH. Activation was inhibited by low GDP concentrations (IC = 5.2 ± 1.5 μM at 5 μM GTP[S]), indicating that receptor-G(t) coupling, followed by instantaneous GDP release, is rate limiting under the conditions (25 °C). Arrhenius plots of the temperature dependence reflect an apparent E(a) of 60 ± 3.5 kJ·mol. Evaluation of the pH/rate profiles of G(t) activation indicates that the activating conformation of the receptor is determined by protonation of a titratable group with an apparent pK(a) of 6.4. This supports the idea that the active state of agonist-bound PAR1 depends on forced protonation, indicating possible analogies to the scheme established for rhodopsin.