Activation of the rod G-protein Gt 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 (Gt, a member of the Gi family) was studied in a reconstituted system of membranes from Sf9 cells expressing the thrombin receptor and purified Gt from bovine rod outer segments. TRAP6-agonist-activated PAR1 interacts productively with the distant G-protein. Agonist-dependent Gt 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 (IC50 = 5.2 +/- 1.5 microM at 5 microM GTP[S]), indicating that receptor-Gt coupling, followed by instantaneous GDP release, is rate limiting under the conditions (25 degrees C). Arrhenius plots of the temperature dependence reflect an apparent Ea of 60 +/- 3.5 kJ.mol-1. Evaluation of the pH/rate profiles of Gt activation indicates that the activating conformation of the receptor is determined by protonation of a titratable group with an apparent pKa 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.
Seibert
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C
Institut für Medizinische Physik und Biophysik, Medizinische Fakultät Charité, Humboldt-Universitat, Berlin, Germany.
Harteneck
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C
Ernst
O P
OP
Schultz
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G
Hofmann
K P
KP
D000818:Animals; D002417:Cattle; D002460:Cell Line; D015870:Gene Expression; D006150:Guanine Nucleotides / Q000494:pharmacology; D006801:Humans; D006863:Hydrogen-Ion Concentration; D066298:In Vitro Techniques; D007700:Kinetics; D044463:Receptor, PAR-1; D018179:Receptors, Thrombin / Q000235:genetics / Q000378:metabolism*; D012243:Rhodopsin / Q000378:metabolism; D018411:Spodoptera; D015293:Transducin / Q000378:metabolism*
D006150:Guanine Nucleotides; D044463:Receptor, PAR-1; D018179:Receptors, Thrombin; D012243:Rhodopsin; D015293:Transducin