Transgenic Analysis of Cys150Ser Mutation in Rds: Distinct Effects on Rod and Cone Photoreceptors

Abstract

Abstract: : Purpose:Cysteine at the position 150 (C150) of Rds has been suggested to mediate the intermolecular disulfide bond involved in the Rds complex assembly. This study was designed to examine the role of this cysteine in Rds/Rom–1 complex formation in rods versus cones. Methods: The transgene containing the full–length mouse Rds cDNA with the C150S mutation followed by the SV40 polyA tail was directed to rods by the mouse opsin promoter (MOPS) and to cones by the human red/green opsin promoter (COPS). Transgenic mice crossed onto wild–type (WT), Rds^+/– and Rds^–/– genetic backgrounds. Transgene expression was verified by Northern and Western blot. Morphological and functional assessments of transgenic retinas were performed by light microscopy and electroretinography (ERG), respectively. Biochemical properties of C150S–Rds were evaluated by Western blotting under reducing and non–reducing conditions and by velocity sedimentation. Results: The C150S–Rds was expressed separately in rods (MOPS–C150S) and cones (COPS–C150S). Functional analysis showed that, in WT and Rds^+/– backgrounds, expression of COPS–C150S significantly diminished the photopic ERG amplitudes. On the contrary, expression of MOPS–C150S in rods showed no dominant negative effect on the rod function in the WT background while it rescued the scotopic ERG amplitude in the Rds^+/– mice. No structural anomalies were detected in the transgenic retinas by light microscopy. Co–immunoprecipitation analysis demonstrated that C150S Rds interacts with Rom–1 in rods, but not in cones. Higher order oligomerization of C150S Rds was defective, as shown by sedimentation velocity analysis. Conclusions: The C150S Rds exerts a dominant negative effect specifically on cones, but not on rods. The apparent outer segment structural differences between rods and cones may render cones more susceptible to the abolition of C150S–mediated intermolecular disulfide bonding necessary for Rds oligomerization.

Keywords: photoreceptors • transgenics/knock-outs • protein structure/function