Henry K. W. Fong

A photic visual cycle of rhodopsin regeneration is dependent on Rgr

During visual excitation, rhodopsin undergoes photoactivation and bleaches to opsin and all-trans-retinal. To regenerate rhodopsin and maintain normal visual sensitivity, the all-trans isomer must be metabolized and reisomerized to produce the chromophore 11-cis-retinal in biochemical steps that constitute the visual cycle and involve the retinal pigment epithelium (RPE; refs. 3–8). A key step in the visual cycle is isomerization of an all-trans retinoid to 11-cis-retinol in the RPE (refs. 9–11). It could be that the retinochrome-like opsins, peropsin, or the retinal G protein-coupled receptor (RGR) opsin12–16 are isomerases in the RPE. In contrast to visual pigments, RGR is bound predominantly to endogenous all-trans-retinal, and irradiation of RGR in vitro results in stereospecific conversion of the bound all-trans isomer to 11-cis-retinal. Here we show that RGR is involved in the formation of 11-cis-retinal in mice and functions in a light-dependent pathway of the rod visual cycle. Mutations in the human gene encoding RGR are associated with retinitis pigmentosa.

The endogenous chromophore of retinal G protein-coupled receptor opsin from the pigment epithelium

The recent identification of nonvisual opsins has revealed an expanding family of vertebrate opsin genes. The retinal pigment epithelium (RPE) and Müller cells contain a blue and UV light-absorbing opsin, the RPE retinal G protein-coupled receptor (RGR, or RGR opsin). The spectral properties of RGR purified from bovine RPE suggest that RGR is conjugated in vivo to a retinal chromophore through a covalent Schiff base bond. In this study, the isomeric structure of the endogenous chromophore of RGR was identified by the hydroxylamine derivatization method. The retinaloximes derived from RGR in the dark consisted predominantly of the all-trans isomer. Irradiation of RGR with 470-nm monochromatic or near-UV light resulted in stereospecific isomerization of the bound all-trans-retinal to an 11-cisconfiguration. The stereospecificity of photoisomerization of the all-trans-retinal chromophore of RGR was lost by denaturation of the protein in SDS. Under the in vitroconditions, the photosensitivity of RGR is at least 34% that of bovine rhodopsin. These results provide evidence that RGR is bound in vivo primarily to all-trans-retinal and is capable of operating as a stereospecific photoisomerase that generates 11-cis-retinal in the pigment epithelium.

Localization of the human RGR opsin gene to chromosome 10q23.

The humanRGR gene encodes an opsin protein (retinal G protein-coupled receptor), which is expressed in Müller cells and the retinal pigment epithelium and is thought to play a role in the visual process. To investigate a possible linkage of theRGR gene to retinal dystrophies, the locus of the gene was mapped on human metaphase chromosomes. Genomic and cDNA fragments of the humanRGR gene were used as probes for fluorescence in situ hybridization. Analysis of the fluorescence signals on high-resolution banded chromosomes showed that theRGR gene is localized to human chromosome lOq23. This result now provides for the rapid analysis of this gene with respect to inherited diseases of the retina.

[27] Analysis of chromophore of RGR: Retinal G-protein-coupled receptor from pigment epithelium

Publisher Summary The retinal pigment epithelium (RPE) and Mtiller cells contain an abundant opsin that is distinct from rhodopsin and cone visual pigments. The RPE and Műller cell opsin, first referred to as RPE retinal G-proteincoupled receptor (RGR), represents a distant evolutionary branch of vertebrate opsins that are most closely related in amino acid sequence to invertebrate visual pigments and retinochrome. Despite the unique features of RGR, many established methods are directly applicable to the characterization of this novel opsin. This chapter presents procedures used to analyze the chromophore of RGR to elude various limitations in accessibility, abundance, and biochemical properties. RGR is purified, as described, and may be irradiated at room temperature by a 30-W fiber optic light source for 5 minutes at a distance of 10 cm. It may be irradiated with monchromatic light, using an Oriel light source equipped with a 150-W xenon arc lamp. The reaction of RGR and hydroxylamine may be followed by measurement of the absorbance of RGR at various times after the addition of hydroxylamine.

Alternative splicing in human retinal mRNA transcripts of an opsin-related protein

An opsin-related gene encodes a putative RPE-retinal G-protein-coupled receptor (RGR) that is most homologous to the visual pigments and invertebrate retinochrome. A splice variant of human RGR mRNA can be demonstrated by the sequence of isolated cDNA clones and by the amplification and analysis of human retinal mRNA. The shortened transcript contains a deletion of 114 nucleotides that correspond exactly to the sequence of exon 6 in the human rgr gene. The predicted RGR variant lacks the putative sixth transmembrane domain and has a calculated molecular weight of 27,726. Variable amounts of a 28-kDa protein were found in the retinas of some individuals by immunoblot assay. Since a similar shortened RGR transcript was not detected in bovine retina or RPE, the RGR variant is not essential for vertebrate vision. Analysis of the structure of the rgr gene and of the sequences of cDNA clones indicates that the truncated mRNA may be produced through alternative splicing of pre-mRNA from which a cassette exon is removed and the predicted RGR variant is radically altered in primary structure.

Accumulation of extracellular RGR-d in Bruch's membrane and close association with drusen at intercapillary regions.

Human retinal pigment epithelial (RPE) cells synthesize an extraneous splice isoform of retinal G protein-coupled receptor (RGR). In this study, we analyzed the exon-skipping variant of RGR (RGR-d) that is found in extracellular deposits. RPE-choroid tissue sections were prepared from postmortem human eyes from donors of various ages. RGR-d was analyzed in drusen and Bruchs membrane by immunohistochemical localization. Extracellular RGR-d is present in most drusen, including hard, soft, confluent and early-stage. Initial drusen formation is known to be preferentially associated with the intercapillary regions of Bruchs membrane. We corroborated this significant association of drusen, including early-stage drusen, with the intercapillary regions. The distribution of extracellular RGR-d in Bruchs membrane differs in old and young donors. In older persons, nodes of concentrated RGR-d accumulate at intercapillary loci, predominantly at the lateral edges of the capillaries of the choriocapillaris. RGR-d loci at the lateral capillary wall appear numerous in old, but not young, donors. Intensely immunostained RGR-d loci can be found at the base of early-stage drusen mounds in the older donors and may precede the formation of these drusen.