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Zhaoxia Zhang, Henry KW Fong; RGR-opsin and exon-6-skipping RGR-d isoform in human cone photoreceptors. Invest. Ophthalmol. Vis. Sci. 2016;57(12):561.
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© ARVO (1962-2015); The Authors (2016-present)
We have observed that cone outer segments in neuroretina sections from some human donors are immunoreactive to an antipeptide antibody (DE21) that is directed against the exon-6-skipping isoform of human retinal G protein-coupled receptor (RGR-d). The purpose of this study is to investigate RGR expression in cone photoreceptors, notwithstanding the finding that carboxyl terminal antibodies (DE7, DE1) against RGR do not appear to immunostain cones.
We produced and validated a rabbit polyclonal antipeptide antibody (DE15), which is directed against a peptide sequence (SSLLRRWPHGSEGC) partly conserved in RGR across a number of different species. Recombinant bovine RGR protein (bRGR) was produced with the Bac-to-Bac Baculovirus Expression System (Invitrogen). RGR was isolated from bovine retinal pigment epithelium (RPE). The bRGR protein standard, purified RGR, and membrane extracts of RPE cells were analyzed by Western immunoblotting. Bovine and human eye tissue sections were analyzed by immunohistochemical staining with affinity-purified primary antibodies.
The DE15 antibody bound positively to over-expressed bRGR in extracts from baculovirus-transduced Sf9 insect cells, but did not react with the protein extracts from untreated control Sf9 cells. This antibody bound positively to purified RGR that was isolated from bovine RPE. In addition, DE15 bound specifically to native RGR in crude membrane extracts of bovine RPE without cross-reaction to other proteins. Immunohistochemical staining of bovine and human retinas with DE15 showed specific labeling of RPE and Müller cells, and also, cone photoreceptors in both species. Strong labeling with DE15 was detected throughout the cone photoreceptor, including outer segment, inner segment, cell body, and synaptic terminus. Immunostaining for human RGR-d was prominent in cone outer segments in some donors, while other donors showed no cone RGR-d. Immunostaining of the carboxyl terminal epitope of RGR appeared to be thoroughly blocked in cone photoreceptors.
These results indicate that bovine and human cone photoreceptors express a member of the Go/RGR opsin group. Human cone photoreceptors may also contain the nonfunctional, or dysfunctional, RGR-d isoform with variation in amount or processing of the RGR-d protein among individuals.
This is an abstract that was submitted for the 2016 ARVO Annual Meeting, held in Seattle, Wash., May 1-5, 2016.
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