Targeting of exon VI-skipping human RGR-opsin to the plasma membrane of pigment epithelium and co-localization with terminal complement complex C5b-9.

PURPOSE Rare mutations in the human RGR gene lead to autosomal recessive retinitis pigmentosa or dominantly inherited peripapillary choroidal atrophy. Here, we analyze a common exon-skipping isoform of the human retinal G protein-coupled receptor opsin (RGR-d) to determine differences in subcellular targeting between RGR-d and normal RGR and possible association with abnormal traits in the human eye. METHODS The terminal complement complex (C5b-9), vitronectin, CD46, syntaxin-4, and RGR-d were analyzed in human eye tissue from young and old donors or in cultured fetal RPE cells by means of immunofluorescent labeling and high-resolution confocal microscopy or immunohistochemical staining. RESULTS We observed that RGR-d is targeted to the basolateral plasma membrane of the RPE. RGR-d, but not normal RGR, is expressed in cultured human fetal RPE cells in which the protein also trafficks to the plasma membrane. In young donors, the amount of RGR-d protein in the basolateral plasma membrane was much higher than that in the RPE cells of older subjects. In older donor eyes, the level of immunoreactive RGR-d within RPE cells was often low or undetectable, and immunostaining of RGR-d was consistently strongest in extracellular deposits in Bruch's membrane. Double immunofluorescent labeling in the basal deposits revealed significant aggregate and small punctate co-localization of RGR-d with C5b-9 and vitronectin. CONCLUSIONS RGR-d may escape endoplasmic reticulum-associated degradation and in contrast to full-length RGR, traffick to the basolateral plasma membrane, particularly in younger subjects. RGR-d in the plasma membrane indicates that the protein is properly folded, as misfolded membrane proteins cannot otherwise sort to the plasma membrane. The close association of extracellular RGR-d with both vitronectin and C5b-9 suggests a potential role of RGR-d-containing deposits in complement activation.