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F Ushikubi, K Nakamura and S Narumiya
Department of Pharmacology, Kyoto University Faculty of Medicine, Japan.
The partially purified thromboxane (TX) A2 receptor was reconstituted with two species of purified heterotrimeric G proteins, Gq and Gi2, in phospholipid vesicles. The receptors reconstituted with Gq and Gi2 showed a single class of [3H]S-145 binding with Kd values of 9.6 +/- 0.7 and 12.1 +/- 1.0 nM, respectively; binding was displaced by GR32191, 9,11-epithio-11, 12-methano-thromboxane A2 (STA2), and U46619, with almost identical Ki values for each compound in the two types of reconstituted vesicles. When the receptor and Gq were reconstituted, the agonist STA2 stimulated guanosine-5'-O-(3-[35S]thio)triphosphate binding. This stimulation was half-maximal at 80 nM and reached a plateau at 1 microM STA2 stimulated the initial rate by 20-30-fold, compared with the basal rate. The stimulation of guanosine-5'-O-(3- [35S]thio)triphosphate binding to Gi2 by the agonist-liganded receptor was seen in the presence of GDP. Under these conditions, 10 microM STA2 stimulated the initial rate by 1.5-2-fold, compared with the basal rate. This effect was half-maximal at 150 nM and reached a plateau at 1 microM. The agonist-liganded receptor also stimulated the GTPase activities of the reconstituted G proteins. The steady state rates of STA2-stimulated [32P]Pi release from [gamma-32P]GTP were 2.21/min.receptor and 0.87/min.receptor in the Gq- and Gi2-reconsituted vesicles, respectively, and the Kcat values of Gq and Gi2 in the presence of STA2 were 0.87 +/- 0.21 min-1 and 2.41 +/- 0.12 min-1, respectively. These results clearly show that the TXA2 receptor functionally couples to both Gq and Gi2. Consistent with this finding, STA2, by acting on the TXA2 receptor in intact platelets, inhibited prostaglandin I2-induced cAMP elevation.
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