Light-stimulated phosphorylation of rhodopsin in the retina: the presence of a protein kinase that is specific for photobleached rhodopsin.

Abstract

A protein kinase has been extiacted from bovine rod outer segments by a mild procedure. The enzyme acts specifically on photobleached, not unbleached, rhodopsin and will not catalyze the phosphorylation of histones, phosvitin, or casein. We propose the name "opsin kinase" for the enzyme, which is not affected by cyclic nucleotides but which is inhibited by theophylline. Preparations of purified rod outer segments, however, appear to contain only low concentration of opsin phosphatase activity.It has recently been shown in several laboratories that when rod outer segments (ROS) prepared from frog (1, 2) or ox (3-5) retinas are incubated with ATP and Mg2+ rhodopsin is phosphorylated and the reaction is markedly stimulated by light.There seem to be three ways in which light could act. First, by directly stimulating the activity of the kinase, second, by altering the conformation of the rhodopsin so that it becomes a substrate for the kinase, and third, by altering the concentration of some cofactor which changes the activity of the kinase.In support of the third possibility, it has been observed that the rate of production of 3': 5'-cAMP and cGMP in ROS is lowered on exposure to light (6-10) and, since rates of protein phosphorylation are, in so many cases, controlled by cyclic nucleotides, it was at first thought that the effect of light on cyclic nucleotide concentration could be correlated with the effect on protein phosphorylation (3, 4). A decrease in cyclic nucleotide concentration could increase the net rate of protein phosphorylation if the cyclic nucleotide inhibited a protein kinase or, alternatively, stimulated a protein phosphatase.Cyclic-AMP-inhibited protein kinases have been discovered in the slime mold (11) and cAMP-stimulated protein phosphatases in the toad bladder cell membrane (12).In this paper, we show that neither cAMP nor cGMP have any effect on the phosphorylation of ROS protein. The protein kinase in ROS is specific to photobleached, not unbleached rhodopsin, and the increase in substrate concentration which results from exposure of ROS to light is alone sufficient to account for the increase in protein phosphorylation. MATERIALS AND METHODSPreparation of Rod Outer Segments from Bovine Retinas was as previously described (13). The segments routinely showed a ratio of absorbance at 280 to 500 nm of about 2.2. Incubation with 1 1-cis-retinaldehyde only increased the absorption at 500 nm by 5-10%, indicating that the material was only 5-10% bleached.Abbreviation: ROS, rod outer segments. Reactions were terminated by the addition of 2 ml of ice-cold 10% trichloroacetic acid (or 20% to precipitate histones) and 0.1 mg (0.1 ml) of bovine serum albumin added as protein carrier. The protein precipitate was washed twice with 10% trichloroacetic acid, M orthophosphoric acid, then resuspended in 0.5 ml of 0.1 N NaOH and incubated for 10 min at 37°, after which 2 ml of 10 or 20% trichloroacetic acid was added and the protein was spun down. The protein precipitates were washed once in 1 ml of e...

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“…The first description of phosphorylation-mediated deactivation of GPCRs was provided in the 1970s where the photoreceptor rhodopsin, a prototypical GPCR, was rapidly deactivated by phosphorylation by "opsin or rhodopsin kinase" (also known as GRK1) (28,133,270). Later, the same deactivation mechanism was proposed in many other GPCRs (reviewed in Ref.…”

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