Opsin Phosphorylation in Rpe65–/– Mice Reduces Retinal Degeneration

Abstract

Abstract: : Purpose:To determine if the kinase responsible for this opsin phosphorylation in the Rpe65^–/– is rhodopsin kinase (RK) and, if so, to study the effect on retinal function and morphology of removing this phosphorylation. Methods:Rpe65^–/– /RK^–/– mice were generated and genotyped. Rod function was analyzed in vivo using scotopic single–flash electroretinograms (ERGs) with different light intensities. For phosphorylation measurements, retinae were homogenized in urea and rhodopsin C– termini were cleaved by endoproteinase Asp–N in Tris buffer. The phosphorylation level of the rhodopsin C–terminal peptide was determined by mass spectrometry. Protein expression in the retinae of Rpe65^–/–/RK^–/–, Rpe65^–/– and C57 mice was examined by semi–quantitative Western blot analysis using antibodies specific for RPE65, RK, opsin, IRBP, and ß–actin. Quantification of opsin was determined by obtaining the UV–Visible spectra after regeneration of rhodopsin by adding excess of 11–cis retinal. Rod morphology was assessed in histological sections. Results: Western blot analysis confirmed that no RPE65 or RK was expressed in Rpe65^–/–/RK^–/– mice. Compared with Rpe65^–/– mice, Rpe65^–/–/RK^–/– mice showed reduced scotopic ERGs signals, which indicated less photoreceptor function. In 2–month–old dark adapted Rpe65^–/–/RK^–/– mice, <2% of opsin was monophosphorylated, while in age–matched Rpe65^–/– mice, 15% –20% of monophosphorylation was detected. Compared with Rpe65^–/– mice, significant lower levels of opsin and IRBP were observed in Rpe65^–/–/RK^–/– mice. In addition, opsin levels dropped from 69.37 ± 5.62 pmol/retina at 1 month to 4.13 ± 0.87 pmol/retina at 6 month in Rpe65^–/–/RK^–/– mice, while in Rpe65^–/– mice, opsin levels remained unchanged. These results suggested that serious retinal degeneration has occurred in Rpe65^–/–/RK^–/– mice. This hypothesis was confirmed in histological sections, in which the Rpe65^–/–/RK^–/– retinae were found to have fewer rows of photoreceptor nuclei and more disorganized outer segments than age–matched Rpe65^–/– or RK^–/– mice. Conclusions:The phosphorylation of opsin by RK partially protects the photoreceptors against retinal degeneration, presumably by inhibiting, at least partially, the normal constitutive activity of the apoprotein.

Keywords: phosphorylation • retinal degenerations: cell biology • opsins