Abstract
Purpose :
The human RGR gene is expressed in retinal pigment epithelium (RPE), Müller cells, cone photoreceptors, and a population of retinal ganglion cells. Since the exon-VI-skipping isoform of RGR opsin (RGR-d) is a drusen-associated protein marker, we are interested in intracellular trafficking, degradation, and extracellular release of this protein. In this study we have investigated variation in the relative abundance or processing of RGR-d, and sensitivity to GW4869, an inhibitor of N-sphingomyelinase and exosome generation.
Methods :
RPE cells were isolated from human postmortem eyes. Mutant mice with mouse RGR-d (mRGR-d) were generated by CRISPR gene editing. Human fetal RPE cells (hfRPE) were cultured, as described previously. Stably transformed LN-229 human glioblastoma cells that express FLAG-RGR and FLAG-RGR-d were cultured in media supplemented with GW4869, or DMSO only. RGR and RGR-d specific antibodies were used as probes for immunostaining and Western blotting.
Results :
In human donor RPE cells, the amount of RGR-d is detectable on Western blots as a major ~28-kDa protein and a few high molecular weight (MW) bands. In marked contrast, the amount of mRGR-d protein in the eyes of homozygous RGR-d mutant mice is extremely low. The human RGR-d isoform is expressed effectively in cultured hfRPE cells and transfected LN-229 cells. In polarized hfRPE cells, RGR-d trafficks to the plasma membrane which requires that the protein be in a folded conformation. In transfected LN-229 cells, RGR-d localizes instead to intracellular sites as punctate bodies, rather than to the plasma membrane. When RGR and RGR-d transfected LN-229 cells were treated with GW4869, low MW bands (<6500 Da) appeared only in extracts from RGR-d cells. The <6500-Da RGR-d band increased in abundance with time in the presence of GW4869. The bands were not present in the vehicle control lanes. The low MW bands were also not present in any of the RGR samples, even when the blot was overexposed.
Conclusions :
The evidence indicates notable variation in the amount and processing of RGR-d under diverse conditions. Major influencing factors appear to be species, age, cell type, trafficking, and protein degradation. Since proteolytic processing of RGR-d is sensitive to GW4869, it is possible that inhibition of exosome formation results in intracellular accumulation of packaged cargo with truncated RGR-d.
This abstract was presented at the 2019 ARVO Annual Meeting, held in Vancouver, Canada, April 28 - May 2, 2019.