Ushikubi F, Nakamura K, Narumiya S
Department of Pharmacology, Kyoto University Faculty of Medicine, Japan.
Mol Pharmacol. 1994 Nov;46(5):808-16.
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.
将部分纯化的血栓素（TX）A2受体与两种纯化的异源三聚体G蛋白Gq和Gi2在磷脂囊泡中进行重组。用Gq和Gi2重组的受体显示出单一类别的[3H]S-145结合，其Kd值分别为9.6±0.7和12.1±1.0 nM；GR32191、9,11-环氧-11,12-甲撑-血栓素A2（STA2）和U46619可使结合发生位移，两种类型的重组囊泡中每种化合物的Ki值几乎相同。当受体与Gq重组时，激动剂STA2刺激鸟苷-5'-O-(3-[35S]硫代)三磷酸结合。这种刺激在80 nM时达到半数最大效应，在1 μM STA2时达到平台期，与基础速率相比，刺激初始速率提高了20 - 30倍。在GDP存在的情况下，激动剂结合的受体可观察到对Gi2的鸟苷-5'-O-(3-[35S]硫代)三磷酸结合的刺激。在这些条件下，与基础速率相比，10 μM STA2刺激初始速率提高了1.5 - 2倍。这种效应在150 nM时达到半数最大效应，在1 μM时达到平台期。激动剂结合的受体还刺激了重组G蛋白的GTP酶活性。在Gq和Gi2重组的囊泡中，STA2刺激的[γ-32P]GTP释放[32P]Pi的稳态速率分别为2.21/分钟·受体和0.87/分钟·受体，在STA2存在的情况下，Gq和Gi2的Kcat值分别为0.87±0.21分钟-1和2.41±0.12分钟-1。这些结果清楚地表明，TXA2受体在功能上与Gq和Gi2均偶联。与此发现一致的是，STA2通过作用于完整血小板中的TXA2受体，抑制了前列腺素I2诱导的cAMP升高。
