Yoshida T, Willardson B M, Wilkins J F, Jensen G J, Thornton B D, Bitensky M W
Physics Division, Los Alamos National Laboratory, University of California, New Mexico 87545.
J Biol Chem. 1994 Sep 30;269(39):24050-7.
Heterotrimeric GTP-binding proteins (G-proteins) serve many different signal transduction pathways. Phosducin, a 28-kDa phosphoprotein, is expressed in a variety of mammalian cell types and blocks activation of several classes of G-proteins. Phosphorylation of phosducin by cyclic AMP-dependent protein kinase prevents phosducin-mediated inhibition of G-protein GTPase activity (Bauer, P. H., Müller, S., Puzicha, M., Pippig, S., Obermaier, B., Helmreich, E. J. M., and Lohse, M. J. (1992) Nature 358, 73-76). In retinal rods, phosducin inhibits transducin (Gt) activation by binding its beta gamma subunits. While rod phosducin is phosphorylated in the dark and dephosphorylated after illumination (Lee, R.-H., Brown, B. M., and Lolley, R. N. (1984) Biochemistry 23, 1972-1977), the significance of these reactions is still unclear. The data presented here permit a more precise characterization of phosducin function and the consequences of its phosphorylation. Dephosphophosducin blocked binding of the Gt alpha 1 subunit to activated rhodopsin in the presence of stoichiometric amounts of Gt beta gamma, whereas phosphophosducin did not. Surprisingly, the binding affinity of phosphophosducin for Gt beta gamma was not significantly reduced compared with the binding affinity of dephosphophosducin. However, the association of phosducin with Gt beta gamma in a size exclusion column matrix was dependent on the phosphorylation state of phosducin. Moreover, the ability of phosducin to compete with Gt alpha for binding to Gt beta gamma was also dependent on the phosphorylation state of phosducin. No interaction was found between phosducin and Gt alpha. These data indicate that phosducin decreases rod responsiveness by binding to the beta gamma subunits of Gt and preventing their interaction with Gt alpha, thereby inhibiting Gt alpha activation by the activated receptor. Moreover, phosphorylation of phosducin blocks its ability to compete with Gt alpha for binding to Gt beta gamma.
异源三聚体GTP结合蛋白（G蛋白）参与多种不同的信号转导途径。磷光蛋白是一种28 kDa的磷蛋白，在多种哺乳动物细胞类型中表达，并能阻断几类G蛋白的激活。环磷酸腺苷依赖性蛋白激酶对磷光蛋白的磷酸化可防止磷光蛋白介导的对G蛋白GTP酶活性的抑制作用（鲍尔，P.H.，米勒，S.，普齐查，M.，皮皮格，S.，奥伯迈尔，B.，赫尔姆赖希，E.J.M.，和洛泽，M.J.（1992年）《自然》358卷，73 - 76页）。在视网膜视杆细胞中，磷光蛋白通过结合转导素（Gt）的βγ亚基来抑制其激活。虽然视杆细胞中的磷光蛋白在黑暗中被磷酸化，光照后去磷酸化（李，R.-H.，布朗，B.M.，和洛利，R.N.（1984年）《生物化学》23卷，1972 - 1977页），但这些反应的意义仍不清楚。本文提供的数据使我们能够更精确地描述磷光蛋白的功能及其磷酸化的后果。在化学计量的Gtβγ存在下，去磷酸化的磷光蛋白可阻断Gtα1亚基与活化视紫红质的结合，而磷酸化的磷光蛋白则不能。令人惊讶的是，与去磷酸化的磷光蛋白的结合亲和力相比，磷酸化磷光蛋白对Gtβγ的结合亲和力并没有显著降低。然而，在尺寸排阻柱基质中，磷光蛋白与Gtβγ的结合取决于磷光蛋白的磷酸化状态。此外，磷光蛋白与Gtα竞争结合Gtβγ的能力也取决于磷光蛋白的磷酸化状态。未发现磷光蛋白与Gtα之间存在相互作用。这些数据表明，磷光蛋白通过与Gt的βγ亚基结合并阻止它们与Gtα相互作用，从而降低视杆细胞的反应性，进而抑制活化受体对Gtα的激活。此外，磷光蛋白的磷酸化会阻断其与Gtα竞争结合Gtβγ的能力。
