Bauer P H, Lohse M J
Institut für Pharmakologie und Toxikologie der Universität Würzburg, Germany.
Naunyn Schmiedebergs Arch Pharmacol. 1998 Apr;357(4):371-7. doi: 10.1007/pl00005181.
The cytosolic phosphoprotein phosducin is an inhibitor of G-protein GTPase activity and G-protein-mediated signalling. Here we investigate the effects of phosducin on individual steps of the GTPase cycle of Go, and the role of the G-protein betagamma subunits in mediating these effects. Phosducin was expressed in E. coli and purified to apparent homogeneity. Phosducin inhibited the MAS-7-stimulated as well as basal steady-state GTPase activity of Go, but did not affect the GTP-hydrolytic step. It slowed the release of GDP from Go in the presence of high Mg2+ concentrations (25 mM), and enhanced GDP release at low Mg2+ concentrations (100 microM). Likewise, phosducin inhibited basal GTPase activity at 25 mM Mg2+ and stimulated at 100 microM Mg2+. All of these effects were lost following phosphorylation of phosducin by protein kinase A (PKA). These observations are compatible with the hypothesis that phosducin antagonizes the influence of betagamma subunits on alpha(o). Titration of the effects of phosducin on the GDP release and GTPase activity of Go and on the betagamma subunit-dependent ADP-ribosylation of alpha(o) by pertussis toxin indicated an apparent affinity of approximately 20 nM. We conclude that via high-affinity interactions with G-protein betagamma subunits phosducin decreases the proportion of active GTP-bound G-proteins by slowing GDP-release without affecting GTP-hydrolysis, and that thereby it inhibits G-protein-mediated signalling.
胞质磷蛋白光导蛋白是G蛋白GTP酶活性和G蛋白介导信号传导的抑制剂。在此，我们研究了光导蛋白对Go的GTP酶循环各个步骤的影响，以及G蛋白βγ亚基在介导这些影响中的作用。光导蛋白在大肠杆菌中表达并纯化至表观均一。光导蛋白抑制了MAS-7刺激的以及基础稳态的Go的GTP酶活性，但不影响GTP水解步骤。在高镁离子浓度（25 mM）存在下，它减缓了GDP从Go的释放，而在低镁离子浓度（100 μM）下增强了GDP释放。同样，光导蛋白在25 mM镁离子浓度下抑制基础GTP酶活性，而在100 μM镁离子浓度下刺激该活性。在用蛋白激酶A（PKA）对光导蛋白进行磷酸化后，所有这些作用都消失了。这些观察结果与光导蛋白拮抗βγ亚基对α(o)的影响这一假设相符。通过滴定光导蛋白对Go的GDP释放和GTP酶活性以及对百日咳毒素介导的βγ亚基依赖性α(o)的ADP核糖基化的影响，表明其表观亲和力约为20 nM。我们得出结论，光导蛋白通过与G蛋白βγ亚基的高亲和力相互作用，通过减缓GDP释放而不影响GTP水解，降低了结合GTP的活性G蛋白的比例，从而抑制了G蛋白介导的信号传导。
