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H. Kochounian, H. K. W. Fong; Intracellular Trafficking of RGR and RGR-d in Transfected Cells. Invest. Ophthalmol. Vis. Sci. 2010;51(13):6170.
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© ARVO (1962-2015); The Authors (2016-present)
Human retinal pigment epithelium (RPE) produces two major forms of retinal G protein-coupled receptor (RGR) protein. In human donor RPE, normal RGR resides in the smooth endoplasmic reticulum (ER), whereas the exon-skipping variant RGR-d sorts to the basal region of RPE cells. Extracellular RGR-d accumulates in Bruch’s membrane and in most types of drusen in older donors. To initiate study of how RGR-d trafficks through and exits the basal pole of RPE cells, we investigated the intracellular processing and sorting pathways of human RGR and splice isoform RGR-d in transfected cell lines.
ARPE-19 and LN-229 cell lines were transfected with Flag-RGR and -RGR-d expression vectors having identical CMV promoter, polyadenylation site, 5’-UTR, 3’-UTR and vector sequences. Stably transformed cells were selected in the presence of G418. Nocadozole was used to inhibit intracellular microtubule assembly. Western blots were probed with an anti-Flag antibody or with antibody HRGR-DE7, which is directed against the carboxyl terminus of RGR and RGR-d. Cells were cultured in chamber slides for immunofluorescent analysis of RGR, RGR-d and various subcellular organelles. The immunofluorescence of antibody-conjugated FITC or Cy3 was viewed by spinning disk laser confocal microscopy.
The amount of RGR-d protein at steady state was significantly lower than that of normal RGR in all cell lines, as determined by Western blot assay of whole cell extracts or by the intensity of immunofluorescence of transfected cells. Normal RGR localized substantially with calnexin, an ER marker. In contrast, the RGR-d isoform was found predominantly in non-calnexin sites. RGR-d localized to conspicuous punctate speckles that had a separate distribution from that of calnexin and other subcellular markers. Cells treated with nocodazole showed major rearrangement of the ER but maintained the co-localization of RGR and calnexin. In contrast, localization of RGR-d remained quite distinct from that of calnexin in the nocodazole-treated cells.
The subcellular localization of RGR-d is markedly different from that of normal RGR in both human donor RPE and transfected cells in culture. The results show that the RGR-d protein lacks a dominant ER retention signal and is not stably retained in the ER. The intracellular trafficking of RGR-d may differ from that of RGR via an alternative protein sorting or degradative pathway.
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