Phosphorylation of Non-bleached Rhodopsin in Intact Retinas and Living Frogs

Brad M Binder,Teresia M O'Connor,M Deric Bownds,Vadim Y Arshavsky

doi:10.1074/jbc.271.33.19826

Abstract

The photoresponse in retinal photoreceptors begins when a molecule of rhodopsin is excited by a photon of light. Photoexcited rhodopsin activates an enzymatic cascade including the G-protein transducin and cyclic GMP phosphodiesterase. As a result, cytoplasmic cyclic GMP concentration is decreased and the photoresponse is initiated. This process is terminated when rhodopsin is phosphorylated by rhodopsin kinase and subsequently blocked by a protein called arrestin. It has been noted by several investigators that light can cause phosphorylation of not only photoexcited but also non-excited rhodopsin in rod photoreceptors. A goal of this study was to determine how much non-bleached rhodopsin is phosphorylated. To determine how the structural integrity of the photoreceptor influences the extent of non-bleached rhodopsin phosphorylation, we studied the reaction in electropermeabilized rod outer segments, in rod outer segments still attached to isolated retinas and in living frogs. In the first two preparations, we found that the maximum extent of non-bleached rhodopsin phosphorylation was approximately 1% of the total rhodopsin pool. In living frogs, the maximal amount of non-bleached rhodopsin phosphorylation was approximately 2% of the total rhodopsin pool and occurred after prolonged illumination by the relatively dim light intensity of 20 lux. These data appear to exclude models for light adaptation that postulate high levels of phosphorylation of non-bleached rhodopsin in rod photoreceptors.

Highlights

When vertebrate ROS1 are illuminated, photoisomerized rhodopsin activates many molecules of retinal G-protein, transducin, which in turn stimulates cyclic GMP hydrolysis by activating cyclic GMP phosphodiesterase
Since the integrity of the ROS structure seems so important for maintaining a high gain of nonbleached rhodopsin phosphorylation, we thought that it is possible that the efficiency of phosphorylation in electropermeabilized ROS might be much lower than in intact photoreceptors
Frog photoreceptors were used in this study because a large amount of rhodopsin can be harvested from a single animal and because the highest gain of the non-bleached rhodopsin phosphorylation has been reported for frog (Binder et al, 1990)

Summary

Introduction

When vertebrate ROS1 are illuminated, photoisomerized rhodopsin activates many molecules of retinal G-protein, transducin, which in turn stimulates cyclic GMP hydrolysis by activating cyclic GMP phosphodiesterase. One of the central reactions responsible for the turnoff of the photoresponse is rhodopsin phosphorylation, catalyzed by the enzyme rhodopsin kinase This phosphorylation reduces the rate of transducin activation by rhodopsin and enhances binding of arrestin to rhodopsin, which completely inhibits further transducin activation. Up to 1400 phosphates were incorporated into non-bleached rhodopsin for each rhodopsin bleached by a flash of dim light (Binder et al, 1990) We have called this phenomenon high gain rhodopsin phosphorylation. The highest level of the non-bleached rhodopsin phosphorylation, up to ϳ3% of the total rhodopsin pool, was observed in rod photoreceptors of living animals exposed to relatively dim levels of background light (ϳ20 lx) for a time period of 30 min

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