The phosphorylation state of phosducin determines its ability to block transducin subunit interactions and inhibit transducin binding to activated rhodopsin
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 (G_t) activation by binding its β 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_tα^1 subunit to activated rhodopsin in the presence of stoichiometric amounts of G_tβγ, whereas phosphophosducin did not. Surprisingly, the binding affinity of phosphophosducin for G_tβγ was not significantly reduced compared with the binding affinity of dephosphophosducin. However, the association of phosducin with G_tβγ in a size exclusion column matrix was dependent on the phosphorylation state of phosducin. Moreover, the ability of phosducin to compete with G_tα for binding to G_tβγ was also dependent on the phosphorylation state of phosducin. No interaction was found between phosducin and G_tα. These data indicate that phosducin decreases rod responsiveness by binding to the βγ subunits of G_t and preventing their interaction with G_tα, thereby inhibiting G_tα activation by the activated receptor. Moreover, phosphorylation of phosducin blocks its ability to compete with G_tα for binding to G_tβγ.
© 1994 American Society for Biochemistry and Molecular Biology. (Received for publication, May 31, 1994, and in revised form, July 15, 1994) This work was supported by National Institutes of Health Grant EY 06816. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Published - J._Biol._Chem.-1994-Yoshida-24050-7.pdf