Regulation of the kinetics of phosducin phosphorylation in retinal rods.
Phosducin (Pd) is a widely expressed phosphoprotein that regulates G-protein (G) signaling. Unphosphorylated Pd binds to Gbetagamma subunits and blocks their interaction with Galpha. This binding sequesters Gbetagamma and inhibits both receptor-mediated activation of Galpha and direct interactions between Gbetagamma and effector enzymes. When phosphorylated by cAMP-dependent protein kinase, Pd does not affect these functions of Gbetagamma. 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 that control the rate of Pd phosphorylation. Pd is expressed at a copy number comparable to that for the rod G-protein, transducin (Gt). The ratio of rhodopsin (Rho) to Pd is 15. 5 +/- 3.5 to 1. The rate of Pd phosphorylation in rod outer segment preparations was dependent on [cAMP]. K1/2 for cAMP was 0.56 +/- 0. 09 microM, and the maximal rate of phosphorylation was approximately 500 pmol PO4 incorporated/min/nmol Rho. In the presence of Gtbetagamma this rate was decreased approximately 50-fold. From these data, one can estimate a t1/2 of approximately 3 min for the rephosphorylation of Pd in rods during the recovery period after a light response. This relatively slow rephosphorylation of the Pd.Gtbetagamma complex may provide a period of molecular memory in which sensitivity to further light stimuli is reduced as a result of sequestration of Gtbetagamma by Pd.
Wilkins
J F
JF
Biophysics Group, Physics Division, Los Alamos National Laboratory, University of California, Los Alamos, New Mexico 87545, USA.
Bitensky
M W
MW
Willardson
B M
BM
D000222:Adaptation, Physiological; D000818:Animals; D000941:Antigens / Q000378:metabolism; D019393:Arrestin; D002417:Cattle; D017868:Cyclic AMP-Dependent Protein Kinases / Q000378:metabolism; D004789:Enzyme Activation; D005136:Eye Proteins / Q000378:metabolism*; D020555:GTP-Binding Protein Regulators; D019204:GTP-Binding Proteins / Q000378:metabolism; D007700:Kinetics; D008027:Light; D010750:Phosphoproteins / Q000378:metabolism*; D010766:Phosphorylation; D012374:Rod Cell Outer Segment / Q000201:enzymology / Q000378:metabolism* / Q000502:physiology
D000941:Antigens; D019393:Arrestin; D005136:Eye Proteins; D020555:GTP-Binding Protein Regulators; D010750:Phosphoproteins; C065631:phosducin; D017868:Cyclic AMP-Dependent Protein Kinases; D019204:GTP-Binding Proteins