Functional reconstitution of platelet thromboxane A2 receptors with Gq and Gi2 in phospholipid vesicles.
Functional reconstitution of platelet thromboxane A2 receptors with Gq and Gi2 in phospholipid vesicles.
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发表时间：
1994-11
影响因子：
3.6
通讯作者：
F. Ushikubi;K. Nakamura;S. Narumiya
中科院分区：
文献类型：
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作者：
F. Ushikubi;K. Nakamura;S. Narumiya
部分纯化的血栓烷（TX）A2受体重建与两种纯化的异源三聚体G蛋白，Gq和Gi 2，在磷脂囊泡。用Gq和Gi 2重建的受体显示单一类型的[3 H]S-145结合，Kd值分别为9.6 +/- 0.7和12.1 +/- 1.0 nM;结合被GR 32191、9，11-表硫代-11，12-甲酰基-血栓烷A2（STA 2）和U46619取代，在两种类型的重构囊泡中，每种化合物的Ki值几乎相同。当受体和Gq重建时，激动剂STA 2刺激鸟苷-5 '-O-（3-[35 S]硫代）三磷酸结合。这种刺激在80 nM时达到最大值的一半，在1 μ M时达到平台，与基础速率相比，STA 2刺激初始速率20-30倍。在GDP存在下，观察到激动剂配体受体对鸟苷-5 '-O-（3-[35 S]硫代）三磷酸与Gi 2结合的刺激。在这些条件下，与基础速率相比，10 μ M STA 2刺激初始速率1.5-2倍。这种作用在150 nM时达到一半最大，在1 μ M时达到平台。激动剂配体的受体也刺激了重组G蛋白的GTdR活性。在Gq-和Gi 2-重构的囊泡中，STA 2-刺激的[32 P]Pi从[γ-32 P]GTP释放的稳态速率分别为2.21/min.受体和0.87/min.受体，并且在STA 2存在下，Gq和Gi 2的Kcat值分别为0.87 +/-0.21 min-1和2.41 +/-0.12 min-1。这些结果清楚地表明，TXA 2受体在功能上与Gq和Gi 2偶联。与这一发现一致，STA 2通过作用于完整血小板中的TXA 2受体，抑制前列腺素I2诱导的cAMP升高。
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.