The Endogenous Chromophore of Retinal G Protein-coupled Receptor Opsin from the Pigment Epithelium*

Wenshan HaoDagger and Henry K. W. FongDagger §parallel

From the Departments of Dagger  Microbiology and § Ophthalmology, University of Southern California School of Medicine and the  Doheny Eye Institute, Los Angeles, California 90033

    ABSTRACT
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Abstract
Introduction
References

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-cis configuration. The stereospecificity of photoisomerization of the all-trans-retinal chromophore of RGR was lost by denaturation of the protein in SDS. Under the in vitro conditions, 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.

    INTRODUCTION
Top
Abstract
Introduction
References

A number of visual pigment homologues have been identified outside of photoreceptor cells in vertebrates. Nonvisual opsins reside in the pineal gland, melanophores, Müller cells, and the retinal pigment epithelium (RPE)1 (1-5). The RPE and Müller cell opsin, a putative RPE retinal G protein-coupled receptor (RGR, or RGR opsin), is most similar in amino acid sequence to retinochrome, a photoisomerase that catalyzes the conversion of all-trans- to 11-cis-retinal in squid photoreceptors (6, 7). RGR has been isolated from bovine RPE microsomal membranes under dark conditions, and its absorption spectrum reveals two pH-dependent species with absorption maxima in the blue (lambda max = ~466 nm) and near-ultraviolet (lambda max = ~364 nm) regions of light (8). The shape of the absorption peaks and the biochemical properties of the photopigment are consistent with those of a retinylidene Schiff base chromophore, the pKa of which is markedly different from those of the visual pigments. RGR is localized to intracellular membranes in the RPE and Müller cells (9) and is able to bind exogenously added all-trans-retinal more efficiently than 11-cis-retinal (10); however, the structure of its endogenous chromophore is unknown.

In addition to RGR, the RPE contains the visual pigment homologues peropsin and melanopsin (4, 5). Peropsin is another retinochrome-like opsin, and melanopsin most closely resembles cephalopod visual pigments. The presence of multiple opsins in the RPE signals that the RPE may consist of primary photoreceptive cells. This monolayer of highly differentiated epithelial cells is essential for the normal function of adjoining photoreceptors. Its diverse and unique roles in the visual process include the removal by phagocytosis of the discarded tips of photoreceptor outer segments (11), storage of retinoids (12, 13), and the isomerization of all-trans- to 11-cis-retinoids for regeneration of visual pigments (14, 15). To process the flow of retinoids through the visual cycle, the RPE contains an abundance of specialized proteins (16-26). Regulation of the multistep retinoid pathways in the RPE is highly coordinated with visual pigment status and lighting conditions through complex and unknown mechanisms (27).

On the basis of its subcellular localization in RPE microsomes, ability to form a stable photopigment with bound all-trans-retinal, and amino acid sequence homology to retinochrome, RGR may be involved in a retinochrome-like mechanism of the vertebrate visual cycle or in a novel form of phototransduction. All retinaldehyde-based opsins involve stereospecific cis,trans photoisomerization of the bound chromophore as a central step in their biological function. In this paper, we describe studies to directly identify the endogenous chromophore of RGR from the RPE and to investigate the photochemistry of RGR by analyzing the effect of light on the isomeric configuration of the bound retinal.

    EXPERIMENTAL PROCEDURES

Materials-- Digitonin was obtained from Acros Organics (Geel, Belgium). Hydroxylamine and all-trans-retinal were purchased from Sigma. 11-cis-Retinal was a gift from Dr. Rosalie Crouch (Medical University of South Carolina, Charleston, SC). The all-trans- and 11-cis-retinal isomers were analyzed for purity by high-performance liquid chromatography (HPLC) before use, as described previously (10). All organic solvents used in this study were HPLC grade. Dichloromethane and diethyl ether were obtained from J. T. Baker Inc., and hexane was from Fisher.

Isolation of Bovine RGR-- RGR was isolated as described previously (8), except for slight modifications in some experiments. Fresh bovine eyes were obtained from a local abattoir and kept in darkness at ambient temperature for ~1.5 h before dissection. After excision of anterior segments, the RPE cells were scraped gently from the eyecups under dim yellow light. The isolation of RPE microsomal membranes and the purification of RGR were performed under darkness or dim red light, as described previously (8). The membranes were extracted thrice for 1 h at 4 °C with 1.2% digitonin in 10 mM sodium phosphate buffer, pH 6.5, containing 150 mM NaCl and 0.5 mM EDTA. After centrifugation of the extract at 100,000 × g for 20 min, the supernatant was incubated overnight at 4 °C with Affi-Gel 10 resin (Bio-Rad) conjugated to anti-bovine RGR monoclonal antibody 2F4 (10). The immunoaffinity resin was transferred to a column for washing with 25 bed volumes of 10 mM sodium phosphate buffer, pH 6.5, containing 0.1% digitonin, 150 mM NaCl, and 0.5 mM EDTA. The column was then loaded 10 times with 0.5 bed volumes of wash buffer containing 50 µM bovine RGR carboxyl-terminal peptide (CLSPQRREHSREQ). The eluates were pooled and concentrated ~4-fold using a Centricon-3 concentrator (Amicon, Inc., Beverly, MA).

Preparation of Rhodopsin-- Bovine rod outer segment (ROS) membranes were prepared under dim red light, as described by Hong et al. (28), and stored at -80 °C. For measurement of the rate of photoisomerization of 11-cis-retinal, rhodopsin was solubilized by incubation of the ROS membranes in 1.2% digitonin, 10 mM sodium phosphate, pH 6.5, 150 mM NaCl, and 0.5 mM EDTA for 1 h at 4 °C. After centrifugation at 100,000 × g, the pellet was extracted twice more. The supernatants were pooled, and photoisomerization of the rhodopsin chromophore was investigated immediately.

Extraction of Opsin-bound Retinal Isomers by Hydroxylamine Derivatization-- The retinal chromophore of purified bovine RGR was extracted under dim red light and analyzed by the method of hydroxylamine derivatization, as described by Groenendijk et al. (29, 30). In a typical extraction procedure, 100-300 µl of purified RGR, ROS membrane suspension, or purified retinal isomers was supplemented with 2 M hydroxylamine, pH 6.5 (10%, v/v), followed by 300 µl of methanol and 300 µl of dichloromethane. Sodium phosphate buffer, pH 6.5, was added to bring the sample volume to 900 µl. The extraction with dichloromethane (aqueous buffer/methanol/dichloromethane, 1:1:1 by volume) was performed by vortexing the mixture for 30 s, followed by centrifugation at 12,000 × g for 1 min. The lower organic phase was removed carefully, and the upper phase was extracted twice more with 300 µl of dichloromethane. The organic layers were pooled and dried down under a nitrogen stream. The extracted retinaloximes were then solubilized in hexane, filtered through glass wool held in a pipette tip, and dried again. The samples were either stored in darkness at -80 °C or analyzed immediately by HPLC. Rhodopsin and purified retinals were extracted as controls, and the resultant distribution of retinaloxime isomers was analyzed by HPLC to monitor the occurrence of any nonspecific isomerization during extraction. The all-trans- and 11-cis-retinal standards were first solubilized in 0.1% digitonin in 10 mM sodium phosphate buffer, pH 6.5, 150 mM NaCl, and 0.5 mM EDTA.

Analysis of Retinaloximes by HPLC-- The isomers of retinaloximes were analyzed by HPLC, as described previously (29, 31). The extracted retinaloximes were dissolved in hexane and applied to a LiChrosorb RT Si60 silica column (4 × 250 mm, 5 µm; E. Merck, Darmstadt, Germany). The HPLC system was equipped with a Beckman Model 126 solvent module and a Model 166 UV-visible detector (Beckman Instruments). The samples were injected in a volume of 20 µl and separated by an eluent consisting of hexane supplemented with 8% diethyl ether and 0.33% ethanol (31). The HPLC column was precalibrated using the reaction products of hydroxylamine and purified all-trans- or 11-cis-retinal standards. Identification of the retinaloxime isomers was based on the retention times of the known retinaloxime products and was in agreement with the results of previous chromatograms (29-31). Absorbance was measured at 360 nm, and the absorbance peaks from the chromatograph were analyzed with the Gold Nouveau software (Beckman Instruments). The proportion of each isomer in the loading sample was determined from the total peak area of both its syn- and anti-retinaloximes and calculated according to the following extinction coefficients (epsilon 360, in hexane): syn all-trans = 54,900, anti-all-trans = 51,600, syn-11-cis = 35,000, anti-11-cis = 29,600, syn-13-cis = 49,000, and anti-13-cis = 52,100 (29, 31).

Irradiation of Photopigments-- RGR or ROS membranes were irradiated with an Oriel light source equipped with a 150-watt xenon arc lamp. The lamp produces uniform irradiance from 300 to 800 nm. Monochromatic light beams at 370 or 470 nm were formed by passing the light through a 370-nm interference filter (Oriel No. 53415) or both a 470-nm interference filter (Oriel No. 53845) and 455-nm long pass filter (Oriel No. 51284), respectively. The protein samples were held at room temperature in a quartz cuvette positioned 60 cm from the lamp. After delivery of the intended amount of light, the retinal chromophores were extracted by hydroxylamine derivatization and analyzed by HPLC, as described above.

Rate of Photoisomerization of Retinal-- RGR was purified, as described previously (8), with the following modifications. A preparation of RPE microsomes from 20 bovine eyes was incubated in the dark with 50 µM all-trans-retinal to saturate the retinal-binding site of RGR. Prior to elution of RGR from the immunoaffinity column, the beads were resuspended and washed with 15 volumes of 1% bovine serum albumin in wash buffer consisting of 0.1% digitonin, 10 mM sodium phosphate, pH 6.5, 150 mM NaCl, and 0.5 mM EDTA, followed by washing with 15 volumes of wash buffer without bovine serum albumin. RGR was then eluted from a column in 7 ml of peptide-containing wash buffer. The eluate was concentrated to ~0.6 ml using a Centricon-3 concentrator and then increased to 1.5 ml with 10 mM sodium phosphate, pH 6.5, 150 mM NaCl, and 0.5 mM EDTA. The pH of the sample was lowered to 4.2 by the addition of 150 µl of 1 M sodium citrate buffer, pH 3.8. Equal aliquots of RGR were irradiated with 470-nm monochromatic light at an illuminance of 410 lux for various periods of time. Each sample was mixed with 0.2 M hydroxylamine immediately upon the end of irradiation and kept in darkness on ice until the retinal chromophores were extracted from all samples and analyzed by HPLC, as described above. The results are plotted as -ln a/a0 versus time, where a0 is the initial amount of all-trans-retinal in non-irradiated RGR and a is the amount of the all-trans isomer in the irradiated sample. The first-order rate constant was calculated using the data from time points at 0-30 s.

Rhodopsin (ROS) was illuminated in solution containing 1.2% digitonin, 10 mM sodium phosphate, pH 6.5, 150 mM NaCl, 0.5 mM EDTA, and 20 mM hydroxylamine. Equal aliquots were irradiated with 470-nm monochromatic light for various times, and the absorption spectrum of each sample was determined immediately after irradiation. There were no further changes in the absorption spectrum by 20 min of exposure to light. Difference spectra were obtained by subtraction of the absorption spectrum at 20 min from the spectrum at each time point. The rate of photoisomerization of 11-cis-retinal was measured from the difference spectra by the decrease in absorbance at 500 nm. The photoisomerization of 11-cis-retinal in rhodopsin followed a typical first-order reaction, and a rate constant was derived from the plot of -ln a/a0 versus time, where a0 and a are the absorbance of non-irradiated and irradiated rhodopsin, respectively, at 500 nm.

Calculation of Extinction Coefficients and pKa of the Retinylidene Schiff Base of RGR-- The pKa of the retinylidene Schiff base of RGR and the extinction coefficients of blue and UV light-absorbing RGR (RGR469 and RGR370, respectively) were calculated from previous data (8). From difference spectra that indicate the absorbance of RGR and its retinaloxime bleaching product (Fig. 7 in Ref. 8), it was determined that the molar extinction coefficient of RGR469 is ~62,800 cm-1 M-1. From the difference in the absorption spectrum of RGR at pH 8.0 and 4.2 (Fig. 4 in Ref. 8), it was determined that the extinction coefficient of RGR370 is ~66,100 cm-1 M-1.

Determination of the Quantum Efficiency of Photoisomerization and Photosensitivity of RGR-- The photosensitivity of visual pigments is equal to the product of its extinction coefficient (epsilon ) and its quantum efficiency of photoisomerization (gamma ) (32). The quantum efficiency of rhodopsin (gamma rhodopsin) is the number of all-trans-retinal molecules formed per number of photons absorbed and has been calculated previously (32). To determine the quantum efficiency of photoisomerization for RGR, we followed a method analogous to that used by Saari and Bredberg (33) to evaluate the photosensitivity of CRALBP (cellular retinaldehyde-binding protein). In this experiment, RGR was converted essentially to RGR469 by lowering the pH of the sample solution to 4.2. The first-order rate constants for photoisomerization of all-trans-retinal bound to RGR469 (kRGR469) and for photoisomerization of 11-cis-retinal bound to rhodopsin (krhodopsin) were obtained from the experiments on kinetics of photoisomerization, as described above. It is assumed that the ratio kRGR469/krhodopsin is proportional to the ratio of their photosensitivity, hence Equation 1 follows,
<FR><NU>k<SUB><UP>RGR</UP><SUB>469</SUB></SUB></NU><DE>k<SUB><UP>rhodopsin</UP></SUB></DE></FR>=<FR><NU>ϵ<SUB><UP>RGR</UP><SUB>469</SUB></SUB></NU><DE>ϵ<SUB><UP>rhodopsin</UP></SUB></DE></FR> · <FR><NU>&ggr;<SUB><UP>RGR</UP><SUB>469</SUB></SUB></NU><DE>&ggr;<SUB><UP>rhodopsin</UP></SUB></DE></FR> (Eq. 1)
where epsilon RGR469 = 62,800, epsilon rhodopsin = 32,900, and gamma rhodopsin = 0.67. The extinction coefficient of rhodopsin (40,600 at lambda max) (34) was corrected for the wavelength of the light used (lambda  = 470 nm), according to Beer's law (A = epsilon lc).

    RESULTS

Endogenous Chromophore of RGR-- RGR is highly sensitive to hydroxylamine in the dark and reacts completely (8). For each retinal isomer, conjugation with hydroxylamine results in retinaloxime products of syn and anti configurations. The syn and anti configurations refer to the cis and trans positions, respectively, of the hydroxyl group with respect to the hydrogen at C-15 (29). Three isomers of retinal were identified by HPLC and consisted of 11-cis (6%), 13-cis (9%), and all-trans (85%) forms (Fig. 1). The syn/anti ratios for the 11-cis- and all-trans- retinaloximes were 3.9 and 11.4, respectively. In a parallel control experiment, extraction of retinal from rhodopsin yielded 11-cis (91%), 13-cis (2%), and all-trans (7%) isomers and the expected predominant representation of 11-cis-retinal (Fig. 1). The syn/anti ratios of the 11-cis- and all-trans-retinaloxime derivatives from rhodopsin were 1.8 and 2.0, respectively, which are consistent with results described previously (30). When pure 11-cis- or all-trans-retinal standards were extracted under similar conditions, no isomerization of the retinals occurred in the dark (data not shown).


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Fig. 1.   Endogenous chromophore of RGR and rhodopsin. RGR and ROS membranes were isolated from fresh bovine eyes under dim red light. The ROS membranes were stored at -80 °C before use, and RGR was used immediately after preparation. The bound chromophores of RGR (left panel) and rhodopsin (right panel) were reacted with hydroxylamine and analyzed by HPLC. The syn isomers of the retinaloxime reaction products were eluted from the HPLC column first, followed by the group of anti isomers. Absorbance was measured at 360 nm, and the amounts of both syn and anti isomers of retinaloxime were added to determine the percent quantity of each retinal isomer. all, all-trans-retinaloxime; 13, 13-cis-retinaloxime; 11, 11-cis-retinaloxime.

The all-trans isomer of retinal was the predominant chromophore extracted from RGR. The possibility that all-trans-retinal arose from other isomers by nonspecific isomerization during the purification of RGR was investigated. To monitor changes in the levels of individual isomers during purification of RGR, the percent quantity of retinal isomers was measured at various steps throughout the procedure. The experiments are outlined in a flowchart, and the results from three separate purifications are summarized in Fig. 2. In the first experiment, the retinal isomers in RPE microsomal membranes (M) and in the combined retinoid extracts of the 100,000 × g pellet (P), flow-through fraction (F), and purified RGR (E) were analyzed. The results showed that the composition of retinal isomers in the starting material and in the terminal steps of RGR purification did not vary substantially; only 3% of the retinal appeared to be converted from 11-cis- to all-trans-retinal. The absolute amount of 11-cis-retinal that declined could not account for the total amount of all-trans-retinal in RGR. In the second experiment, the retinals were analyzed in RPE microsomal membranes and in the combined retinoid extracts of the 100,000 ×g pellet and supernatant (S). The composition of retinal isomers in the starting material and in the steps after solubilization in 1.2% digitonin varied again just slightly; ~3% of the retinal appeared to be converted from the 11-cis isomer to all-trans-retinal. In the third experiment, retinals were analyzed in the supernatant and in the combined retinoid extracts of the flow-through fraction and purified RGR. The results showed that little or no change occurred in the relative levels of retinal isomers during the step of immunoaffinity chromatography.


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Fig. 2.   Distribution of retinal isomers at various steps in the isolation of RGR. A, diagram of the procedure for isolation of RGR. The boxed letters represent the various fractions resulting from individual purification steps. M, bovine RPE microsomal membranes; S, 100,000 × g supernatant of the digitonin-solubilized extract; P, 100,000 × g pellet of the digitonin-solubilized extract; F, combined flow-through fraction and washes from the immunoaffinity column; E, purified RGR eluted with the bovine RGR carboxyl-terminal peptide. B, summary of the results of three independent experiments performed to determine the distribution of retinal isomers during isolation of RGR. The retinal isomers were extracted separately from the saved fractions and then pooled together for HPLC analysis, as indicated. In Experiment I, retinal isomers were analyzed from the microsomal membranes (M) and from the pooled retinoid extracts of the pellet (P), flow-through fraction (F), and purified RGR (E). In Experiment II, retinals were analyzed from microsomal membranes and from the pooled retinoid extracts of the pellet and supernatant (S). In Experiment III, retinals were analyzed from the supernatant and from the pooled retinoid extracts of the flow-through fraction and purified RGR. The results are expressed as the percent quantity of individual retinal isomers. The amount of all-trans-retinaloxime from purified RGR was ~170 pmol for a typical isolation.

Photoisomerization of All-trans-retinal Bound to RGR-- The effect of light on the chromophore of RGR was investigated after solubilization and purification of the protein. RGR was irradiated or not with 470-nm monochromatic light for 3 min at pH 6.5, and retinal was extracted by hydroxylamine derivatization. The retinal isomers in non-irradiated RGR consisted of 11-cis (8%), 13-cis (7%), and all-trans (85%) forms (Fig. 3). The configuration of retinal isomers shifted in irradiated RGR, which contained 11-cis (40%), 13-cis (8%), and all-trans (52%) forms. In other experiments, the irradiation of RGR with 370-nm monochromatic light also resulted in photoisomerization of all-trans- to 11-cis-retinal without a significant alteration of the relative amount of 13-cis and other isomers (Fig. 4).


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Fig. 3.   Photoisomerization of the retinal chromophore of RGR. The chromophore of purified RGR was analyzed after incubation in darkness (left panel) or irradiation with 470-nm monochromatic light (right panel). The protein at pH 6.5 was irradiated for 3 min at room temperature in a quartz cuvette positioned 60 cm from the lamp. all, all-trans-retinaloxime; 13, 13-cis-retinaloxime; 11, 11-cis-retinaloxime.


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Fig. 4.   Irradiation of RGR by near-UV light. The chromophore of RGR was analyzed after irradiation of the protein at pH 6.5 for 3 min at room temperature with near-UV light passing through the Oriel No. 53415 370-nm interference filter. all, all-trans-retinaloxime; 13, 13-cis-retinaloxime; 11, 11-cis-retinaloxime.

To test protein-dependent stereospecificity of photoisomerization, the effect of light on the chromophore of RGR was analyzed after treatment of the protein with SDS. Denaturation of RGR in 2% SDS is accompanied by an alteration of its absorption spectrum (8). The retinal isomers in non-irradiated SDS-treated RGR consisted of 11-cis (9%), 13-cis (14%), and all-trans (77%) forms (Fig. 5). The resulting syn/anti ratio of all-trans-retinaloxime was 1.3. After irradiation of the denatured protein with white light, the distribution of isomers was 11-cis (27%)-, 13-cis (26%)-, and all-trans (47%)-retinaloximes; the relative levels of both 11-cis- and 13-cis-retinals rose with the decline in all-trans-retinal. Irradiation of the denatured protein with 470-nm monochromatic light also resulted in nonspecific formation of both 11-cis- and 13-cis-retinal isomers. Before irradiation, SDS-treated RGR contained 11-cis (2%)-, 13-cis (11%)-, and all-trans (87%)-retinals. After irradiation with 470-nm light, the distribution of isomers changed to 11-cis (21%), 13-cis (21%), and all-trans (58%) forms.


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Fig. 5.   Photoisomerization of the retinal chromophore of SDS-denatured RGR. RGR at pH 6.5 was incubated in the dark at room temperature for 25 min in the presence of 2% SDS to denature the protein. The protein was then irradiated or not immediately before extraction of the retinal isomers. Upper trace, SDS-denatured RGR irradiated with light from a 150-watt xenon arc lamp for 2 min; lower trace, SDS-denatured RGR kept in the dark. all, all-trans-retinaloxime; 13, 13-cis-retinaloxime; 11, 11-cis-retinaloxime.

Photosensitivity of RGR-- To characterize the photosensitivity of RGR, its rate of photoisomerization of retinal was compared with that of the visual pigment rhodopsin. Steady illumination of RGR resulted in increasing conversion of all-trans- to 11-cis-retinal over a period of 5 min (Fig. 6). The proportion of 13-cis-retinal extracted from the irradiated samples remained essentially constant at all time points. A relatively fast initial rate of decay of all-trans-retinal was maintained for ~30 s, after which the decay of all-trans-retinal occurred at a slower rate. For comparison, the kinetics of photoisomerization of 11-cis-retinal in rhodopsin followed a first-order reaction rate. Under our experimental conditions, the first-order rate constants for RGR from 0 to 30 s and for rhodopsin from 0 to 60 s were kRGR469 = 8.2 × 10-3 s-1 and krhodopsin = 24.2 × 10-3 s-1 (Fig. 6). From Equation 1 (see "Experimental Procedures"), the quantum efficiency of RGR (gamma RGR469) equaled 0.12. The photosensitivity of RGR469 was then determined to be 7540 cm-1 M-1. The average rate constant and quantum efficiency of RGR from three separate experiments were (7.5 ± 2.1) × 10-3 s-1 and 0.11 ± 0.026 (mean ± S.D.), respectively. In each experiment, the first-order rate constant for RGR was calculated from 0 to 30 s.


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Fig. 6.   Kinetics of photoisomerization of the retinal chromophore of RGR and rhodopsin. Before purification of RGR, RPE microsomal membranes were incubated in the dark with 50 µM all-trans-retinal to maximize the yield of photopigment. The pH of the purified protein was lowered from 6.5 to 4.2 to convert RGR into its blue light-absorbing form (RGR469), and equal aliquots were then exposed to 470-nm monochromatic light for various lengths of time. The same light source was used to irradiate digitonin-solubilized ROS at various times in the presence of 20 mM hydroxylamine. The initial amount of all-trans-retinal in non-irradiated RGR (or the absorbance of non-irradiated rhodopsin) at t = 0 is a0. The amount of all-trans-retinal in RGR (or the absorbance of rhodopsin) after a given period of illumination is a. The results are plotted as -ln a/a0 versus time for RGR () and rhodopsin (black-triangle). In this experiment, the first-order rate constants for RGR from 0 to 30 s and for rhodopsin from 0 to 60 s were kRGR469 = 8.2 × 10-3 s-1 and krhodopsin = 24.2 × 10-3 s-1, respectively. At t = 0 s, RGR contained 11-cis (0%), 13-cis (13%), and all-trans (87%) isomers. At t = 300 s, RGR contained 11-cis (50%), 13-cis (11%), and all-trans (39%) isomers.

The 11-cis isomer was a consistent component of the retinaloxime extracts of purified RGR, except when RPE microsomal membranes were treated in the dark with exogenously added all-trans-retinal. After incubation with excess all-trans-retinal to saturate the chromophore-binding sites, the 11-cis isomer was not detected in purified RGR. The distribution of retinal isomers from retinaldehyde-treated RGR before irradiation (t = 0 s) was 11-cis (0%), 13-cis (13%), and all-trans (87%) forms (Fig. 6). After 5 min of irradiation (t = 300 s), the distribution of retinal isomers from RGR was 11-cis (50%), 13-cis (11%), and all-trans (39%) forms (Fig. 6).

    DISCUSSION

The spectral properties of RGR purified from bovine RPE suggest that RGR is conjugated in vivo to a retinal chromophore by means of a covalent Schiff base bond. In this study, the endogenous chromophore of RGR was extracted and identified following hydroxylamine derivatization. Our findings indicate that the predominant chromophore of RGR is all-trans-retinal. Irradiation of RGR results in stereospecific conversion of the bound all-trans isomer to 11-cis-retinal. These results support the notion that RGR functions as a stereospecific photoisomerase in the RPE and Müller cells.

Chromophore of RGR-- In addition to all-trans-retinal, a minor constant amount of 13-cis-retinal was observed routinely in the retinoid extracts from RGR. The origin of 13-cis-retinal is unclear. Persistent small amounts of this isomer have been observed likewise in retinoid extracts of retinochrome (31). A small amount of the 13-cis isomer was also present in our retinoid extract of rhodopsin following the extraction conditions. As a non-physiological constituent, the 13-cis isomer may be formed by thermal isomerization during the protein denaturation. 11-cis-Retinal also was extracted in the dark from RGR. Since RGR photoisomerizes bound all-trans- to 11-cis-retinal, it is conceivable that the 11-cis-retinaloxime originated from a population of physiologically relevant 11-cis-retinal·RGR complex. In relation to this assumption, when the RPE microsomal membranes were preincubated with exogenous all-trans-retinal, 11-cis-retinal was completely absent in purified RGR, although 13-cis-retinal was still present. It is probable that a small fraction of RGR is bound in situ to 11-cis-retinal, which can be quantitatively displaced from the protein by an excess of the all-trans isomer. In a control experiment, the endogenous chromophore of rhodopsin was extracted and analyzed in a similar manner. As expected, the physiologically relevant 11-cis- and all-trans-retinal isomers were identified in rhodopsin, along with the small amount of 13-cis-retinal.

The procedure for purification of RGR extends over 20 h, and during this time, there is a potential for spontaneous thermal isomerization of the retinal chromophore in RGR from a hypothetical 11-cis to all-trans configuration. The actual extent of thermal isomerization of 11-cis-retinal was investigated in three separate purifications of RGR. Overall, there were only slight differences in the distribution of retinal isomers between that in microsomes and the final steps of purification. A small ~3% decrease in 11-cis-retinal and a slight increase in all-trans-retinal were associated with the step of solubilization of RPE microsomal membranes in the presence of digitonin. The actual amount of 11-cis-retinal that underwent artifactual isomerization was significantly less than the total amount of all-trans-retinal in RGR. Thus, the results exclude the possibility that the major endogenous chromophore of RGR from microsomes was originally 11-cis-retinal, which converted to the all-trans configuration during purification of RGR in the dark.

Interestingly, the reaction of the chromophore of RGR with hydroxylamine produced syn and anti isomers of all-trans-retinaloxime in a syn/anti ratio of ~9, which is significantly higher than the syn/anti- retinaloxime ratios of ~2 and 3.4 observed in reactions with illuminated rhodopsin and free all-trans-retinal, respectively (30). The singular syn/anti-oxime ratio for RGR suggests that the protein retains a folded conformation. The interaction between chromophore and hydroxylamine may be constrained by the structure of the binding site for all-trans-retinal, such that the production of the syn isomer of all-trans-retinaloxime is strongly favored in the microenvironment of the reaction. The denaturation of RGR in the presence of SDS then lowers the syn/anti-all-trans-retinaloxime product ratio to ~1.3 (Fig. 5).

Photoisomerase Activity-- Irradiation of the all-trans-retinal·RGR complex modifies the relative amounts of only the two physiologically relevant isomers, i.e. all-trans- and 11-cis-retinals. When irradiated with monochromatic light at 470 or 370 nm, all-trans-retinal in RGR, as in retinochrome, isomerizes stereospecifically to 11-cis-retinal. Only 11-cis-retinal is newly formed in RGR, and relative levels of 13-cis and other isomers of retinal are unchanged. The photoisomerization of all-trans-retinal in RGR was dependent on a folded protein structure since denaturation of RGR in SDS abolished the cis,trans stereospecificity.

Although the chromophore in RGR was converted specifically to 11-cis-retinal, the highest level to which the 11-cis isomer increased was ~50% of total retinals under our experimental conditions. The formation of 11-cis-retinal in RGR contrasts with photoisomerization of the chromophore in retinochrome, in which the 11-cis isomer may increase to as much as 80% of total retinals after illumination (35). The extent to which 11-cis-retinal is formed in RGR may be reduced by stereospecific photoreversal of the chromophore back to the all-trans isomer. Irradiation of RGR does not lead to bleaching or a large shift in the lambda max of the blue light-absorbing form of RGR (8). We conclude that 11-cis-retinal does not dissociate freely from the purified protein in the presence of light. Therefore, continuous light exposure may result in photoisomerization of the 11-cis-retinal bound to irradiated RGR and, eventually, a photoequilibrium of RGR bound to both 11-cis and all-trans isomers. The larger extent to which 11-cis-retinal can be formed in retinochrome upon illumination with orange light may be attributed to the difference of ~26 nm between the lambda max of retinochrome (496 nm) and that of meta retinochrome (470 nm).

The extinction coefficients of RGR469 (62,800) and RGR370 (66,100) are slightly higher than that of retinochrome (60,800) (36) and 1.5- and 1.6-fold greater than that of rhodopsin (40,600), respectively. Although RGR has a higher extinction coefficient, its photosensitivity appears to be lower than that of rhodopsin. Under our experimental conditions, RGR469 is at least one-third as efficient as rhodopsin in using the energy of photons. The photosensitivity of RGR469 is ~7-fold greater than that of CRALBP (1078 cm-1 M-1) (33). The data support the argument that photoisomerization of the bound chromophore is physiologically relevant in the function of RGR. It is possible that RGR is a still more efficient photoisomerase under in vivo conditions or within an optimal lipid membrane environment. Retinal-binding proteins may also serve to transfer retinals to and from RGR and to accelerate the net production of 11-cis-retinal. Recently, CRALBP has been shown to increase the rate of 11-cis-retinol synthesis by RPE isomerohydrolase (37). In squid, RALBP (retinaldehyde-binding protein) downloads 11-cis-retinal from meta retinochrome to meta rhodopsin through an exchange of isomers (38).

A mechanism for rapid synthesis of 11-cis-retinal must exist for the regeneration of visual pigments under photopic conditions. The synthesis of 11-cis-retinol by the RPE isomerohydrolase provides one such mechanism that also can act during dark adaptation. Although it can be demonstrated that RGR is a stereospecific photoisomerase that favors conversion of all-trans-retinal to the 11-cis isomer, there is yet no direct evidence that the 11-cis-retinal from RGR dissociates and enters the pathway for regeneration of visual pigments. The RPE appears to have divergent pools of 11-cis-retinal, the function of which may be to participate in a novel phototransduction system of the RPE, to supply a chromophore for peropsin or melanopsin, or to regenerate rhodopsin and the cone pigments under scotopic and intense photopic lighting conditions. Further characterization of RPE opsins and the precise flow of retinoids through the RPE, including analysis of retinoid metabolism in mouse mutants that lack RGR, will test the hypothesis that RGR functions in a retinochrome-like arm of the vertebrate visual cycle.

    ACKNOWLEDGEMENTS

We thank Pu Chen for technical assistance and Dr. Rosalie Crouch for the generous gift of 11-cis-retinal.

    FOOTNOTES

* This work was supported by grants from the Hoover Foundation and United States Public Health Service Grants EY08364 and EY03040.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

parallel To whom correspondence should be addressed: Doheny Eye Inst., 1355 San Pablo St., Los Angeles, CA 90033. Tel.: 323-442-6675; Fax: 323-442-6688; E-mail: hfong{at}hsc.usc.edu.

    ABBREVIATIONS

The abbreviations used are: RPE, retinal pigment epithelium; RGR, RPE retinal G protein-coupled receptor; HPLC, high-performance liquid chromatography; ROS, rod outer segment(s).

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Abstract
Introduction
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