RGR Increases Isomerohydrolase Activity Independently of Light

Abstract

Abstract: : Purpose:To quantify the effect of retinal G protein–coupled receptor (RGR) on regeneration of rhodopsin both during and after light exposure; i.e. in the photic and classical visual cycles. Materials and Methods:RGR^–/– (from H. Fong), wild type and RPE65^+/– mice were analyzed. All mice expressed the Rpe65₄₅₀Met variant only. The rate of rhodopsin regeneration was measured under two conditions: 1) under constant exposure to 100 lux of white light – "regeneration in light"; 2) after exposure to 5000 lux for 10 min, bleaching >90% – "regeneration in darkness". Retinoid profiles were analyzed by HPLC. Protein levels were assayed by Western blotting. Results:1) Regeneration of rhodopsin in light was more than 2 times faster when RGR was present. 2) Regeneration in darkness was even 3 times faster when RGR was present. 3) These differences were not attributable to the reduced amounts RPE65 protein in the RGR^–/– mice, as they also occur between RGR^–/– and RGR^+/+/RPE65^+/– mice with equal amounts of RPE65. 4) Regeneration in light (100 lux) for each genotype was at least 6 times faster than in darkness. 5) The conversion of retinyl esters to 11–cis retinal after bleaching was faster when RGR was present. 6) The rate of rhodopsin regeneration in darkness matched both the rate of reappearance of 11–cis retinal and the rate of disappearance of retinyl esters, but was not correlated with the quantity of retinyl esters present during regeneration. Conclusions:RGR accelerates rhodopsin regeneration about 2.3 to 3–fold regardless of the presence of light. Light of 100 lux accelerates rhodopsin regeneration at least 6–fold regardless of the presence of RGR. These results are not in line with the proposed photoisomerase activity of RGR in a photic visual cycle. Instead, RGR acts as a multiplicative factor on isomerohydrolase activity, in both the classical and photic visual cycles. Surprisingly, light (100 lux) – independently of RGR – accelerates visual pigment regeneration under these conditions by a factor of at least 6 by an unknown mechanism.

Keywords: retinoids/retinoid binding proteins • retinal pigment epithelium • photoreceptors