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Abstract
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.
Highlights
In vertebrate rod photoreceptor cells, the photoresponse is mediated by a G-protein-linked pathway
We have shown that ROS PKA is activated in vivo by cAMP and not cGMP
Knowledge of the range and time scale of the changes in cAMP is crucial to a complete understanding of the regulation of Pd phosphorylation
Summary
Phosducin; G, G-protein; Gt, retinal rod G-protein, transducin; PKA, cAMP-dependent protein kinase; Rho, rhodopsin; Rho*, light-activated rhodopsin; PDE, phosphodiesterase; PAGE, polyacrylamide gel electrophoresis; ROS, rod outer segment(s); 8-Br-cAMP, 8-bromo-cyclic AMP; 8-Br-cGMP, 8-bromo-cyclic GMP. Photon capture by rhodopsin (Rho) causes photoisomerization of its prosthetic group, 11-cis-retinal, to the all-trans isomer. This isomerization results in a conformational change that converts Rho to its active form, Meta II rhodopsin (Rho*). In this regime, a single Rho* can initiate hydrolysis of ϳ106 molecules of cGMP. Light-dependent reactions that appear to contribute to light adaptation include phosphorylation of Rho and arrestin binding [11], activation of guanylyl cyclase [12], and dephosphorylation of phosducin [5]. The results provide evidence that the Pd1⁄7Gt␥ complex could encode a molecular memory in retinal rods The duration of this memory results from the stability of the Pd1⁄7Gt␥ complex and the relatively slow rate of its phosphorylation. The properties of Pd suggest that it could play a fundamental role in light adaptation
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