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., Muller, 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 (G(t)) 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 G(t) alpha(1) subunit to activated rhodopsin in the presence of stoichiometric amounts of G(t) beta gamma, whereas phosphophosducin did not. Surprisingly, the binding affinity of phosphophosducin for G(t) beta gamma was not significantly reduced compared with the binding affinity of dephosphophosducin. However, the association of phosducin with G(t) beta gamma in a size exclusion column matrix was dependent on the phosphorylation state of phosducin. Moreover, the ability of phosducin to compete with G(t) alpha for binding to G(t) beta gamma was also dependent on the phosphorylation state of phosducin. No interaction was found between phosducin and G(t) alpha. These data indicate that phosducin decreases rod responsiveness by binding to the beta gamma subunits of G(t) and preventing their interaction with G(t) alpha, thereby inhibiting G(t) alpha activation by the activated receptor. Moreover phosphorylation of phosducin blocks its ability to compete with G(t) alpha for binding to G(t) beta gamma.
