Characterization of the physiological ligand and function of a novel opsin RGR from the retinal pigment epithelium.

摘要

The Putative retinal G protein-coupled receptor (RGR) is an abundant opsin that is expressed in the smooth endoplasmic reticulum membranes of the retinal pigment epithelium (RPE) and the Muller cells in the retina (Jiang et al, 1993; Pandey et al, 1994). Fractionation of bovine RPE cells by differential centrifugation led to the preparation of RPE microsomal membranes with enriched RGR. The membrane-bound RGR binds both all-trans-retinal and 11-cis-retinal after reduction by sodium borohydride, but with preference to all-trans isomer.; Digitonin-solubilized RGR from bovine RPE was isolated by the means of immunoaffinity chromatography. The absorption spectrum of RGR revealed pH-dependent photopigments with absorption maxima of 469 +/- 2.4 and 370 +/- 7.3 nm. The effects of hydrogen ion concentration suggest that the blue and near-UV photopigments are tautomeric forms of RGR, in which an all-trans-retinal Schiff base is protonated or unprotonated, respectively.; The endogenous retinaldehyde bound to RGR was extracted from purified RGR in the dark and identified as all-trans-retinal by HPLC analysis. Irradiation of RGR with monochromatic fight at 370 nm and 470 nm isomerizes stereospecifically all-trans-retinal into 11-cis-retinal unless the protein structure was denatured by incubation with 2% SDS. The photosensitivity of RGR 469 photopigment is about 1/3 that of rhodopsin, suggesting that RGR is capable of functioning as an isomerase.; The rgr gene-disrupted mouse is being generated to further investigate the gene function. Mouse RGR cDNA and genomic gene were isolated. A knockout vector that is aimed to disrupt exon 2 by insertion of the neo gene was constructed. Four embryonic stem cell clones from CJ7 strain and 18 clones from R1 strain were demonstrated to have one of two rgr alleles disrupted, giving targeting frequencies of 0.7% and 3% for 0.7 and R1, respectively. Mice heterozygous for mutated rgr alleles were generated from ES clone 1C10. These mice are being mated to produce homozygous mutant mice for phenotypic analysis.