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

• Published in: Molecular vision Vol. 22; pp. 213 - 223
• Main Authors: Kochounian, Harold, Zhang, Zhaoxia, Spee, Christine, Hinton, David R, Fong, Henry K W
• Format: Journal Article
• Language: English
• Published: United States Molecular Vision 03.03.2016
• Subjects: Aged; Alternative Splicing - physiology; Blotting, Western; Bruch Membrane - metabolism; Cell Membrane - metabolism; Cells, Cultured; Complement Membrane Attack Complex - metabolism; Exons - genetics; Eye Proteins - genetics; Eye Proteins - metabolism; Female; Fluorescent Antibody Technique, Indirect; Genetic Vectors; Humans; Male; Membrane Cofactor Protein - metabolism; Microscopy, Confocal; Middle Aged; Opsins - metabolism; Receptors, G-Protein-Coupled - genetics; Receptors, G-Protein-Coupled - metabolism; Retinal Pigment Epithelium - metabolism; RNA, Messenger - genetics; Tissue Donors; Transfection; Vitronectin - metabolism
• ISSN: 1090-0535; 1090-0535

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. 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. 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. 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.