The Endogenous Chromophore of Retinal G Protein-coupled Receptor
Opsin from the Pigment Epithelium*
Wenshan
Hao
and
Henry K. W.
Fong
§¶
From the Departments of
Microbiology and
§ Ophthalmology, University of Southern California School of
Medicine and the ¶ Doheny Eye Institute,
Los Angeles, California 90033
 |
ABSTRACT |
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 |
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 (
max = ~466 nm) and near-ultraviolet
(
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.
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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
(
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 (
) and its
quantum efficiency of photoisomerization (
) (32). The quantum
efficiency of rhodopsin (
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,
|
(Eq. 1)
|
where
RGR469 = 62,800,
rhodopsin = 32,900, and
rhodopsin = 0.67. The extinction coefficient of rhodopsin (40,600 at
max) (34) was corrected for the wavelength of the light used (
= 470 nm),
according to Beer's law (A =
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.
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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.
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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.
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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.
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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
(
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 ( ). 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.
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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
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
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.
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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