Coexpression of nonvisual opsin, retinal G protein-coupled receptor, and visual pigments in human and bovine cone photoreceptors

Purpose: Retinal G protein-coupled receptor (RGR) mRNA is transcribed in the outer nuclear layer of human retinas; however, it is not known whether the RGR gene is expressed in the rod or cone photoreceptors In this study, we investigate broader expression of the normal RGR isoform in photoreceptors of human and bovine retinas. Methods: We produced and validated a rabbit polyclonal antipeptide antibody (DE15) that is directed against a peptide sequence (SSLLRRWPHGSEGC) partly conserved in RGR across several species. Bovine and human retina sections were analyzed with immunohistochemical and double-label immunofluorescent staining. Results: The DE15 antibody bound specifically to overexpressed recombinant RGR, purified RGR from bovine RPE, and RGR in crude RPE membrane extracts without cross-reaction to other proteins. Immunostaining of diurnal bovine and human retinas with DE15 showed labeling of long-wavelength-sensitive and short-wavelength-sensitive cone photoreceptors and some retinal ganglion cells in both species. Strong labeling with DE15 was detected throughout the cone photoreceptor, including the outer segment, inner segment, cell body, axon, and cone pedicle, while rod outer segments were negative. Immunostaining for human exon-6-skipping RGR (RGR-d) was found primarily at the tips of the outer segment of the cones. Conclusions: The results indicate that the cone photoreceptors in these mammals express a nonvisual opsin of the Go/ RGR or tetraopsin group RGR and the visual pigments are predominantly colocalized in the cone outer segment, which suggests functional interaction among these opsins Human cone photoreceptors may also contain normal RGR and the aberrant RGR-d splice isoform.