Phospholipase C-β1 directly accelerates GTP hydrolysis by Gαq and acceleration is inhibited by Gβγ subunits
Phospholipase C-β1 directly accelerates GTP hydrolysis by Gαq and acceleration is inhibited by Gβγ subunits
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DOI：
10.1074/jbc.274.28.19639
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发表时间：
1999-07-09
影响因子：
4.8
通讯作者：
Ross, EM
中科院分区：
文献类型：
--
作者：
Chidiac, P;Ross, EM
磷脂酶c - β是由G α (q)调节的主要效应蛋白，已被证明可以增加含有异三聚体G(q)和ml毒蕈碱受体的蛋白脂质体中G(q)的激动剂刺激的稳态GTPase活性。我们现在使用与GTP结合的R183C G α (q)的中等稳定复合物来证明plc - β 1在无膜系统中直接作为分离的G α (q)的gtpase激活蛋白（GAP）。plc - β 1加速了G α (qR183C)的水解。GTP高达20倍。K-m为1.5 nM，这与R183C和野生型G α (q)激活plc - β 1的EC50和plc - β 1在基于囊泡的实验中作为G(q) GAP的EC50相似。RGS4的G α (q) GAP活性也可以通过该方法定量测定；它使结合GTP的水解速度加快约100倍。plc - β 1和RGS4的G(q) GAP活性都被G β γ亚基阻断，这可能是一种竞争机制。这些数据表明，在稳态GTP水解过程中，受体催化的GDP/GTP交换不需要G β γ亚基，或者plc - β或RGS蛋白的gap可以在这组反应中替代G β γ。
Phospholipase C-beta, the principal effector protein regulated by G alpha(q), has been shown to increase the agonist-stimulated, steady-state GTPase activity of G(q) in proteoliposomes that contain both heterotrimeric G(q) and ml muscarinic receptor. We now use a moderately stable complex of R183C G alpha(q) bound to GTP to show that PLC-beta 1 acts directly as a GTPase-activating protein (GAP) for isolated G alpha(q) in a membrane-free system. PLC-beta 1 accelerated the hydrolysis of G alpha(qR183C).GTP up to 20-fold. The K-m was 1.5 nM, which is similar both to the EC50 with which R183C and wild type G alpha(q) activate PLC-beta 1 and to the EC50 with which PLC-beta 1 acts as a G(q) GAP in the vesicle-based assay. The G alpha(q) GAP activity of RGS4 can also be quantitated by this assay; it accelerated hydrolysis of bound GTP about 100-fold. The G(q) GAP activities of both PLC-beta 1 and RGS4 are blocked by G beta gamma subunits, probably by a competitive mechanism. These data suggest either that the G beta gamma subunits are not continuously required for receptor-catalyzed GDP/GTP exchange during steady-state GTP hydrolysis or that GAPs, either PLC-beta or RGS proteins, can substitute for G beta gamma in this set of reactions.