Abstract
Purpose:
Due to the considerable structural variance of the exon-skipping isoform of the human RPE‑retinal G protein-coupled receptor opsin (RGR-d) from normal RGR, we hypothesize that subcellular targeting and processing of RGR-d will differ substantially from RGR and may be associated with abnormal traits in the human eye.
Methods:
Human eye tissue from young and old donors and cultured human fetal RPE cells were analyzed by means of immunofluorescent labeling and high-resolution confocal microscopy or immunohistochemical (IHC) staining. Monoclonal antibodies were used as primary antibody probes against the neoepitope of the terminal complement complex, C5b‑9, human vitronectin, human CD46, and syntaxin-4. Affinity-purified polyclonal antibodies that were directed against the carboxyl termini of human RGR and RGR-d (HRGR‑DE7), and an RGR-d-specific antibody (DE21) were produced, as described previously (Jiang et al., 1995; Fong et al., 2006). RGR and RGR-d in human fetal RPE cells were analyzed by Western blotting.
Results:
We observed that RGR-d is targeted to the basolateral plasma membrane of 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. The amount of RGR-d protein detected in RPE cells of young subjects is significantly higher than that in the RPE of older donors. In older donor eyes, RGR‑d levels within RPE cells was often low or undetectable, and immunostaining of RGR‑d was consistently strongest in Bruch’s membrane rather than in the RPE. Double immunofluorescence labeling of extracellular basal deposits revealed a close association of RGR-d with C5b-9 and vitronectin.
Conclusions:
Unlike RGR, intracellular human RGR-d protein may sort to the RPE basolateral plasma membrane. Thus, RGR-d may escape endoplasmic reticulum-associated degradation under some conditions. The abundance of RGR-d in young RPE cells is significantly higher than in the RPE of older donors, in which RGR-d is predominantly in extracellular basal deposits. Age-related changes may contribute to the accumulation of RGR-d deposits in the extracellular regions. The close association of RGR-d with both vitronectin and C5b-9 in basal deposits suggests that an important role in complement activation cannot be excluded.