REGULATION OF THE KINETICS OF PHOSDUCIN PHOSPHORYLATION IN RETINAL RODS

Phosducin (Pd) is a widely expressed phosphoprotein that regulates G-p rotein (G) signaling, Unphosphorylated Pd binds to G beta gamma subuni ts and blocks their interaction with G alpha, This binding sequesters G beta gamma and inhibits both receptor-mediated activation of G alpha and direct interactions between G beta gamma and effector enzymes. Wh en phosphorylated by cAMP-dependent protein kinase, Pd does not affect these functions of G beta gamma. To further understand the role of Pd in regulating G-protein signaling in retinal rod photoreceptor cells, we have measured the abundance of Pd in rods and examined factors tha t control the rate of Pd phosphorylation, Pd is expressed at a copy nu mber comparable to that for the rod G-protein, transducin (G(t)). The ratio of rhodopsin (Rho) to Pd is 15.5 +/- 3.5 to 1, The rate of Pd ph osphorylation in rod outer segment preparations was dependent on [cAMP ]. K-1/2 for cAMP was 0.56 +/- 0.09 mu M, and the maximal rate of phos phorylation was similar to 500 pmol PO4 incorporated/min/nmol Rho, In the presence of G(t) beta gamma this rate was decreased similar to 50- fold. From these data, one can estimate a t(1/2) of similar to 3 min f or the rephosphorylation of Pd in rods during the recovery period afte r a light response. This relatively slow rephosphorylation of the Pd . G(t) beta gamma complex may provide a period of molecular memory in w hich sensitivity to further light stimuli is reduced as a result of se questration of G(t) beta gamma by Pd.