title: Targeting of exon VI-skipping human RGR-opsin to the plasma membrane of pigment epithelium and co-localization with terminal complement complex C5b-9.
abstract: OBJECTIVE
  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.
authors.affiliation: Doheny Eye Institute, Los Angeles, CA.
authors.forename: Harold
authors.initials: H
authors.lastname: Kochounian
authors.affiliation: Department of Ophthalmology, Keck School of Medicine of the University of Southern California, Los Angeles, CA; USC Eye Institute, Keck School of Medicine of the University of Southern California, Los Angeles, CA.
authors.forename: Zhaoxia
authors.initials: Z
authors.lastname: Zhang
authors.affiliation: Department of Ophthalmology, Keck School of Medicine of the University of Southern California, Los Angeles, CA.
authors.forename: Christine
authors.initials: C
authors.lastname: Spee
authors.affiliation: Department of Ophthalmology, Keck School of Medicine of the University of Southern California, Los Angeles, CA; Department of Pathology, Keck School of Medicine of the University of Southern California, Los Angeles, CA; USC Eye Institute, Keck School of Medicine of the University of Southern California, Los Angeles, CA.
authors.forename: David R
authors.initials: DR
authors.lastname: Hinton
authors.affiliation: Department of Ophthalmology, Keck School of Medicine of the University of Southern California, Los Angeles, CA; Department of Molecular Microbiology and Immunology, Keck School of Medicine of the University of Southern California, Los Angeles, CA; USC Eye Institute, Keck School of Medicine of the University of Southern California, Los Angeles, CA.
authors.forename: Henry K W
authors.initials: HK
authors.lastname: Fong
mesh_terms: D000368:Aged; D017398:Alternative Splicing / Q000502:physiology*; D015153:Blotting, Western; D016570:Bruch Membrane / Q000378:metabolism; D002462:Cell Membrane / Q000378:metabolism*; D002478:Cells, Cultured; D015938:Complement Membrane Attack Complex / Q000378:metabolism*; D005091:Exons / Q000235:genetics; D005136:Eye Proteins / Q000235:genetics / Q000378:metabolism*; D005260:Female; D019084:Fluorescent Antibody Technique, Indirect; D005822:Genetic Vectors; D006801:Humans; D008297:Male; D051916:Membrane Cofactor Protein / Q000378:metabolism; D018613:Microscopy, Confocal; D008875:Middle Aged; D055355:Opsins / Q000378:metabolism*; D012333:RNA, Messenger / Q000235:genetics; D043562:Receptors, G-Protein-Coupled / Q000235:genetics / Q000378:metabolism*; D055213:Retinal Pigment Epithelium / Q000378:metabolism*; D014019:Tissue Donors; D014162:Transfection; D019096:Vitronectin / Q000378:metabolism
chemical_list: C497857:CD46 protein, human; D015938:Complement Membrane Attack Complex; D005136:Eye Proteins; C089754:G protein-coupled receptor RGR; D051916:Membrane Cofactor Protein; D055355:Opsins; D012333:RNA, Messenger; D043562:Receptors, G-Protein-Coupled; D019096:Vitronectin