From the Department of Pharmacology and the
§ Departments of Chemistry and Biochemistry, University of
Washington, Seattle, Washington 98195
Received for publication, May 31, 2000, and in revised form, September 28, 2000
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ABSTRACT |
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The Retinal photoreceptor cells, rods and cones, can directly sense
and respond to photons of light. In these cells, light induces a highly
regulated and well studied cascade of events that leads to changes in
photoreceptor membrane potential and neurotransmitter release.
PDE61 is an integral part of
this cascade. When a photon of light hits the chromophore in the
transmembrane protein rhodopsin, a conformational change occurs that
allows the G protein, transducin, to be activated. Transducin then
activates PDE6 by binding to its inhibitory Rhodopsin is a transmembrane protein that is found in the internal
membranous disks of photoreceptor cells. Therefore, it is logical that
significant fractions of its effectors, transducin and PDE6, are also
found on disc membranes. This colocalization presumably helps guarantee
the most effective transfer of the light signal from one protein to the
next. The localization of these effector molecules to the membrane is
dependent on their post-translational modification with prenyl groups
(2-4). In the case of PDE6, the modifications are found on the
catalytic subunits: the In addition to the membrane-bound PDE (mPDE), a soluble fraction of rod
PDE6 (sPDE) (5) has been identified. Under isotonic conditions, ~30%
of the PDE6 isolated from rod outer segments is found in this soluble
fraction. sPDE appears identical to mPDE, except that sPDE is complexed
with one or more 17-kDa Several observations led us to hypothesize that the Due to the effects of the Materials
Fresh bovine eyes were purchased from Schenk Packing (Stanwood,
Washington). [ Methods
Delta Subunit Expression and Purification--
Recombinant Peptide Synthesis--
All peptides prepared in this study were
prepared using the previously published general method for peptide
synthesis with or without prenyl groups and C-terminal methyl esters
(15). All peptides were purified to apparent homogeneity by high
performance liquid chromatography on a C18 reverse phase column, and
their structures were confirmed by electrospray ionization mass
spectrometry. Peptides used in this paper were described previously in
detail (42) and include GKQPGGGPASKSC ( pH Assay--
This assay is used to measure
time-dependent cGMP hydrolysis in a mixture of ROS,
nucleotides, and proteins. All procedures were performed in complete
darkness using an infrared viewer unless otherwise indicated. ROS from
fresh retinas (16) were permeablized by trituration 10 times through a
28-gauge needle attached to a 1-ml disposable insulin syringe Becton
Dickinson (Franklin Lakes, NJ). ROS (final [rhodopsin] = 7-10 µM in a total reaction size of 400 µl) were then
added to a solution containing pH assay buffer (140 mM
potassium aspartate, 7 mM KCl, 5 mM NaCl, 5 mM HEPES, 1 mM EGTA, 3.3 mM
MgCl2, 0.986 mM CaCl2, pH 8.0; this
buffer is calculated to give 641 nM free Ca2+),
2 mM ATP, 0.5 mM GTP, and 2 µM
Single Turnover GTPase Assay--
GTPase activity was determined
under the same conditions as those used for the pH assay, except that
[ Immunoblots--
Samples were prepared for immunoblot analysis
by the addition of 6× Laemmli sample buffer. Samples containing ROS
were not boiled. After running these samples on 12 or 15% gels, the
gels were blotted onto nitrocellulose. Nitrocellulose was blocked with 5% milk in TBST (20 mM Tris, pH 8, 140 mM
NaCl, 0.05% (v/v) Tween 20). PDE catalytic subunit immunoreactivity
was measured using the PDE6 cat pAb (1), a rabbit polyclonal antibody,
at 1:3000 dilution. Transducin Based on the localization of the subunit of the rod photoreceptor PDE has
previously been shown to copurify with the soluble form of the enzyme
and to solubilize the membrane-bound form (1). To determine the
physiological effect of the
subunit on the light response of bovine
rod outer segments, we measured the real time accumulation of the
products of cGMP hydrolysis in a preparation of permeablized rod outer segments. The addition of
subunit GST fusion protein (
-GST) to
this preparation caused a reduction in the maximal rate of cGMP
hydrolysis in response to light. The maximal reduction of the light
response was about 80%, and the half-maximal effect occurred at 385 nM
subunit. Several experiments suggest that this
effect was not due to the effects of
-GST on transducin or rhodopsin
kinase. Immunoblots demonstrated that exogenous
-GST solubilized the
majority of the PDE in ROS but did not affect the solubility of
transducin. Therefore, changes in the solubility of transducin cannot
account for the effects of
-GST in the pH assay. The reduction in
cGMP hydrolysis was independent of ATP, which indicates that it was not
due to effects of
-GST on rhodopsin kinase. In addition to the
effect on cGMP hydrolysis, the
-GST fusion protein slowed the
turn-off of the system. This is probably due, at least in part, to an
observed reduction in the GTPase rate of transducin in the presence of
-GST. These results demonstrate that
-GST can modify the activity
of the phototransduction cascade in preparations of broken rod outer
segments, probably due to a functional uncoupling of the transducin to
PDE step of the signal transduction cascade and suggest that the
subunit may play a similar role in the intact outer segment.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
subunits. When active,
PDE6 rapidly hydrolyzes cGMP, causing the closure of cGMP-gated ion
channels in the plasma membrane of the photoreceptor, reduced influx of
sodium and calcium, and hyperpolarization of the photoreceptor cell.
The light signal ends when transducin becomes inactive by hydrolyzing
its GTP, and rhodopsin kinase phosphorylates rhodopsin, blocking
further interaction between transducin and rhodopsin. GDP-bound
transducin releases the
subunits of PDE6, allowing the catalytic
subunits of PDE6 to become reinhibited after transducin has hydrolyzed
its GTP. Reductions in calcium caused by light stimulate guanylate
cyclase to replace the hydrolyzed cGMP.
subunit is farnesylated, and the
subunit is geranylgeranylated. In other proteins, such as small G
proteins, prenylation is sufficient to bind the protein firmly to the
membrane. However, despite having two such hydrophobic prenyl
modifications, PDE6 catalytic subunits can easily be removed from the
membrane by treatment with detergent-free hypotonic buffers.
subunits. Purified sPDE (which has bound
subunit) is activated by transducin with the same dose response as
purified mPDE (without
subunit) in solution (6), so the
subunit
does not appear to directly affect the enzyme's catalytic activity. At
this point, the functional roles of sPDE and the
subunit in the
photoreceptor are not clear; however, it is possible that regulation of
the localization of PDE6's catalytic subunits could modify the
response of the cell to light.
subunit might
modulate the activity of the phototransduction cascade. The
subunit
is highly expressed in retina and is localized in the outer segments of
the photoreceptors (1), so it is in the right location to have an
effect on the cascade. Additionally,
subunit-free PDE6 is activated
to a higher level by transducin when the PDE is membrane-bound than
when it is in solution (7-10). Therefore, the
subunit could alter
PDE6's interaction with the membrane to cause a conformational change
or a destabilization of the PDE6/transducin interaction. Alternatively,
the
subunit might simply move PDE6 away from the membrane where
active transducin is bound, thus reducing the amount of PDE that is
locally available to be activated by transducin. The
subunit has
also been shown to have an effect on the binding of cGMP to
noncatalytic sites on PDE6(11). This could modify the PDE's
interaction with its
subunit (and thus its ability to be activated
by transducin) or modify the
subunit's ability to act as a
GTPase-activating protein cofactor for transducin. In addition, the
subunit can interact with RPGR, a photoreceptor protein that has
homology to the guanine nucleotide exchange factor RCC1 (12) and can act as a guanine nucleotide dissociation inhibitor for Arl3, a small G
protein (13). Similar interactions could affect phototransduction.
subunit on the solubility of PDE6, we
hypothesized that the
subunit would reduce the PDE activity induced
by a flash of light. In this paper, we test this hypothesis and
demonstrate the effect of
subunit-GST fusion protein on light-induced PDE activity in isolated rod outer segments. We use an
assay that measures the real time accumulation of the products of
cyclic nucleotide hydrolysis, so the effect of
-GST on both the rate
of cGMP hydrolysis and the inactivation of the system is shown. Since
this assay measures protons generated as a direct consequence of cGMP
hydrolysis in homogenized rod outer segments, the effects we see cannot
be affected by downstream changes in the phototransduction pathway,
such as Ca2+ influx and channel closure. We also perform
experiments that suggest that the effects of
-GST on cGMP hydrolysis
are probably due to direct effects on the catalytic activity of the PDE
rather than effects on other parts of the phototransduction cascade.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-32P]GTP was purchased from PerkinElmer
Life Sciences. Ultrafree® filters were purchased from
Millipore Corp. (Bedford, MA). Super Signal® West Pico
chemiluminescent substrate was purchased from Pierce. All other
chemicals were purchased from Sigma.
subunit was expressed as a fusion protein with glutathione
S-transferase (
-GST) and purified as described (42). Concentrations of all proteins were determined using the Bradford assay
(14) using bovine serum albumin as a standard.
-13-f-Me), PASKSC
(
-6-f-Me), and PRSSTC (
-6-gg-Me), all with C-terminal methylation
and prenylation. As negative controls,
GKQPGGGPASKSC(S-farnesyl)-COOH without methylation (
-13-f-OH) and PRSSTC-COOMe without prenylation (
-6-np-Me) were used.
-GST or control buffer (see purification procedure, above). This was
incubated at room temperature for 5 min to allow full mixing of the
components. Then cGMP was added to a final concentration of 5 mM, and the pH electrode was placed in the mixture. The
mixture was stirred constantly. After a constant base line (generally
0.1-1 mV/second) was achieved, light flashes were given as indicated
in the figure legends. A 25-ms light flash with the 0.0 neutral density
filter bleached ~0.1% of the rhodopsin. Data from the pH meter were
collected and analyzed using the DataLogger program and the Serial Box
Interface from Vernier Software (Beaverton, OR). Gain was adjusted so
that an increase of 1 mV corresponded to about 3.4 × 10
10 mol of cGMP hydrolyzed. Two data points
were collected per second. Derivatives shown are from data averaged
over seven points. The dark rate of cGMP hydrolysis is subtracted from
all results shown in this paper. The pH of the reaction changed 0.3 pH
units at most, which has little effect on the PDE's activity (17). A long light flash given at the end of every experiment to measure remaining cGMP showed that at least 2.5 mM cGMP remained
after all data was collected, significantly above the
Km for the enzyme. In some experiments, neutral
density filters were used to vary the light intensity of the flash.
These filters are graded on a log scale so that a 1.0 filter lets in
10% as much light as a 0.0 filter. 0.3, 0.6, and 0.9 filters let in
50, 25, and 12.5% as much light, respectively, as the 0.0 filter.
-32P]GTP (~4 × 104 dpm/pmol, 50 nM) was added to the mixture, and ATP was omitted to remove
rhodopsin phosphorylation as a factor in the rate of GTP hydrolysis.
Zero time values were determined by first adding 300 µM
GTP
S and then the [
-32P]GTP. The assay was carried
out as described previously (18). Briefly, ROS from fresh bovine eyes
(7-10 µM rhodopsin) were mixed with
subunit, pH
assay buffer, and cGMP. This mixture was then incubated in the light or
dark for 10 min to hydrolyze endogenous GTP. The reaction was started
by the addition of [
-32P]GTP, and 30-µl aliquots
were taken at the noted times. Reactions were stopped by adding the
aliquots to 115 µl of 6% perchloric acid. After collection, aliquots
were treated with 700 µl of 5% activated charcoal in 50 mM NaH2PO4 (pH 7.5). Free phosphate
remaining in the supernatant after this treatment was counted in a
Tri-Carb scintillation counter (Packard). At the zero time point,
between 3 and 7% of the total radioactivity in these reactions
remained in the supernatant.
-subunit immunoreactivity was
detected using antibody 5552 (kindly provided by Dr. Jim Hurley) at
1:3000 dilution. Transducin
subunit was detected using the G
(M-14) antibody from Santa Cruz Biotechnology at 1:1000 dilution. All
primary antibodies were detected by incubation with a horseradish
peroxidase-linked goat anti-rabbit secondary antibody. The secondary
antibody was visualized with Super Substrate West Pico chemiluminescent substrate.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
subunit and its ability to
remove PDE6 from the membrane, we hypothesized that
-GST would reduce the PDE activity seen in response to a flash of light. To test
the effect of
-GST on the light-induced PDE activity, we used the pH
assay to measure the real time hydrolysis of cGMP in response to light
in a rod outer segment preparation containing key components of the
phototransduction cascade (19, 20). In this assay, a pH meter was used
to measure the generation of H+ produced by the hydrolysis
of cGMP into GMP by PDE. In the absence of
-GST, ROS responded to
light with a transient increase in cGMP hydrolysis due to activation of
the phototransduction cascade (Fig.
1A, top
trace). The rate of cGMP hydrolysis throughout the course of
the reaction was measured by taking the derivative of the raw data
shown in Fig. 1A (Fig. 1B). After the light
flash, the rate of cGMP hydrolysis increased as the PDE was activated by transducin and then after about 50 s returned to the base-line dark rate of hydrolysis as the cascade was shut off by transducin's GTPase activity, the phosphorylation of rhodopsin by rhodopsin kinase,
and reassociation of the inhibitory
subunit of PDE6.
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Fig. 1.
The effect of recombinant subunit (2 µM) on the light response of broken rod
outer segments. ROS membranes ([rhodopsin] = 7 µM
in final reaction) were incubated in the dark in pH assay buffer with
0.5 mM GTP, 2 mM ATP, and 2 µM
subunit-GST fusion protein or control buffer for 5 min at room
temperature. 5 mM cGMP was added, a pH electrode was put
into the reaction mixture, and stirring was begun. After changes in pH
had stabilized, a 25-ms flash of light was given (flash given at
10 s on these graphs). A shows data from these
experiments. An increase of 1 mV corresponds to 3.42 × 10
10 mol of cGMP hydrolyzed. Data shown has
the low dark rate of cGMP hydrolysis (generally 0.1-1 mV/s) subtracted
out. B shows the derivative over time of the data shown in
A. A and B, the response of the
preparation to light in the absence (top trace,
solid) and presence (bottom trace,
dashed) of
-GST. Data shown in all panels is
representative of at least three experiments performed with different
batches of ROS and
subunit.
The addition of -GST to this reaction resulted in a decrease in the
total amount of cGMP hydrolyzed (Fig. 1A, bottom
trace). This was associated with a reduction in the maximal
rate of cGMP hydrolysis in the presence of
-GST (Fig. 1B,
bottom trace).
Peptides that can block -GST's ability to solubilize PDE have been
developed (42). These peptides block the solubilization of PDE6 by 2 µM
-GST with IC50 values ranging from 1 to
10 µM. To determine whether they would also block
-GST's effect in the pH assay, these peptides (10 µM)
were added to the pH assay reaction mixture (Fig.
2). At this concentration, the peptides
do block the effect of
-GST (Fig. 2, A and B).
Peptides alone, without
subunit, do not affect the response to
light (Fig. 2, C and D). This is not surprising,
since immunoblots show that these ROS preparations contain little
endogenous
subunit (data not shown).
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pH assay experiments were repeated to take into account variations
between different preparations of ROS and -GST. Figs. 1 and 2 show
representative data from one experiment; Fig.
3 shows the average maximum rate of cGMP
hydrolysis from three separate experiments. Purified expressed GST
alone had no effect in this assay (data not shown).
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Immunoblots of ROS supernatants and pellets were performed to determine
whether PDE6 catalytic subunits were solubilized under the same
conditions used for the pH assay. These blots demonstrate that PDE6
catalytic subunits become soluble at the concentrations of -GST
used, unless peptide is present to block the effect of
-GST (Fig.
3). Since the hypothesis being tested suggested that the reduction in
cGMP hydrolysis would be caused by this solubilization of the PDE by
-GST, we determined whether both of these effects occur in a similar
concentration range of
-GST (Fig. 4).
Dose-response curves of the solubilization of PDE catalytic subunits by
-GST (Fig. 4A) and the
-GST-associated reduction in
cGMP hydrolysis (Fig. 4B) were performed. Both effects occur
in a similar concentration range. This strongly suggests that the two
effects are causally linked.
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The dose-response curve using various amounts of -GST (Fig.
4B) showed that the IC50 of
-GST's effect in
the pH assay was 385 nM. Based on calculations of the
rhodopsin/PDE ratio in bovine rod outer segments (21), there should be
about 140 nM holo-PDE in this preparation, 280 nM catalytic subunits. Therefore, the IC50 of
-GST was in the range of the amount of PDE catalytic subunits in
this experiment. Since other work has suggested that the
subunit
interacts with the catalytic subunits with high affinity (
14
nM) (1), it is likely that this IC50 represents titration of the catalytic subunits by the
subunit and
underestimates the true affinity of the interaction.
The magnitude of the -GST effect on the maximum rate of cGMP
hydrolysis was dependent on light intensity (Fig.
5). Using a 1.0 neutral density filter,
which blocks 90% of the light that will come through a 0.0 filter
(~0.01% rhodopsin bleached), the maximal rate of cGMP hydrolysis in
the presence of
-GST was 80% of the maximal rate without
-GST
(Fig. 5, A and C). Using a 0.0 neutral density
filter (~0.1% rhodopsin bleached) the maximal rate of cGMP
hydrolysis in the presence of
-GST was 18% of the maximal rate
without
subunit (Fig. 5, A and B).
Immunoblots showed that
-GST brought approximately the same amount
of PDE into the supernatant with and without light exposure (data not shown), so the variable effect of
-GST was not due to different amounts of PDE being solubilized under different light conditions. One
interpretation of these results is that in the presence of
subunit,
the response of the ROS is basically independent of light; a similar,
low level response is always seen. Another possibility is that the
dose-response curve for light has been shifted well to the right. Due
to high noise at low light intensities and the lack of a brighter light
source, we could not distinguish between these two possibilities by
using higher or lower light levels.
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In addition to its effect on the maximal rate of cGMP hydrolysis,
-GST prolonged the time to turn-off of the system (Fig. 6). The amount of time that it took for
the rate of cGMP hydrolysis to be reduced from its maximal value to
half of the maximal value (over base line) was calculated. In the
presence of 2 µM
-GST, it took ~2.5 times as long
for the signal from a 0.0 flash to be reduced by half. Due to the
larger amount of noise in derivatives of lower light flashes, it was
not possible to determine whether this effect of
-GST was also
reduced at lower levels of light.
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It was important to determine whether the effects on cGMP hydrolysis
and inactivation were due to action on the PDE itself, to the effects
of -GST on some other part of the signal transduction cascade, or a
combination of these two possibilities. Previous studies have shown
that
-GST can solubilize mPDE and that the
subunit appears to
have no direct effect on the catalytic activity of the PDE. Fig. 4
shows that solubilization of PDE is associated with the reduction of
cGMP hydrolysis by
-GST, and both effects of
-GST were negated by
preincubation with peptides that block the solubilization of PDE
activity (42) by
-GST (Figs. 2 and 3). These observations suggest
that the effects of
-GST in the pH assay may be due to PDE
solubilization, but they do not rule out that
-GST's effects could
be due to actions on other proteins in the signal transduction cascade.
To investigate these possibilities further, we performed experiments
designed to determine whether reduction of cGMP hydrolysis by
-GST
could be due to effects on transducin or rhodopsin kinase.
If -GST moved transducin away from the membrane, it might be
expected to reduce the gain of the signal transduction pathway and
result in reduced cGMP hydrolysis. This was a distinct possibility, since part of the
-GST binding site on the PDE catalytic subunits are the prenylated C termini (42), and the
subunit of transducin is
prenylated (farnesylated) as well. We performed immunoblots on aliquots
of ROS incubated with various concentrations of
-GST (Fig.
7). At concentrations where PDE is moved
almost completely into the supernatant by
-GST, transducin's
and
subunits are unaffected. Therefore, solubilization of
transducin is unlikely to be the mechanism for the reduction in cGMP
hydrolysis by
-GST. This result is also interesting, since it
demonstrates the specificity of
-GST action.
-GST selectively
solubilizes some prenylated proteins and not others.
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To determine whether changes in the ability of transducin to hydrolyze
GTP were involved in the effects on the light response, the effect of
-GST on the GTPase rate of transducin was examined directly. Single
turnover GTPase activity was measured under conditions of limiting GTP,
which was added to start the reaction. In the light under these
conditions, only a small fraction of the activated transducin should
have been able to bind and hydrolyze GTP, so there was only one round
of GTP hydrolysis by transducin (18). GTP was hydrolyzed more slowly in
the presence of
-GST (Fig. 8). The
rate constants calculated from multiple experiments show that
-GST
slowed the rate of GTP hydrolysis to 43% of its original rate, from an
average half-time of 5.25 s to an average half-time of 11.9 s
(Fig. 8B). This effect of
-GST was also blocked by preincubation with peptides that block the ability of
-GST to make
the catalytic subunits soluble. The dark rate of GTP hydrolysis, which
is probably due to other GTPases such as small G proteins as well as
transducin, was not significantly altered by
-GST (Fig.
8A). These results show that
-GST does not cause its
reduction in cGMP hydrolysis by accelerating transducin's rate of GTP
hydrolysis; however, this result could explain the increased amount of
time it takes the system to turn off in the presence of
-GST (Fig. 6).
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To determine whether -GST might reduce the maximum rate of cGMP
hydrolysis by increasing the activity of rhodopsin kinase, the pH assay
was performed without ATP (Fig. 9). After
a light flash under these conditions, the phototransduction cascade
should activate as usual. However, in the absence of ATP, rhodopsin is not phosphorylated, so the cascade will not turn off until the limiting
substrate, in this case cGMP, is depleted. Incubation with
-GST
decreased the maximum rate of cGMP hydrolysis to a similar extent as
was seen in the presence of ATP. Therefore, the effect of
-GST on
cGMP hydrolysis in response to light was not ATP-dependent
and was unlikely to be mediated by a kinase.
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DISCUSSION |
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It has been established that the recombinant purified subunit
solubilizes membrane-bound type 6 PDE (1), but the physiological implications of this solubilization were unknown. Much recent research
has pointed out the importance of compartmentalization of signal
transduction components for proper function of regulatory cascades. For
these reasons, it was interesting to determine the physiological
effects of the
subunit in the photoreceptor. It is likely that the
subunit, which is expressed in a number of other tissues outside of
the retina and is highly conserved, may play similar roles in tissues
outside of the photoreceptor.
Localization studies (1) demonstrate that the subunit in the retina
is primarily localized in the outer segments of the rod cells, where
other components of the phototransduction cascade are also present at
high concentrations. A significant fraction (~30%) of the PDE
purified from rod outer segments is soluble. These results suggest that
one of the
subunit's primary actions in the retina may be to
modify the phototransduction cascade by affecting PDE activity. One
obvious possibility was that by moving PDE away from the membrane, the
subunit would reduce the ability of transducin to activate the PDE,
thus reducing the gain of the system in response to a flash of light.
One must keep in mind, however, that in the intact rod, the
concentration of membranes is much higher than it is in the experiments
shown in this paper. Therefore, it is possible that in the intact
photoreceptor, the
subunit does not actually make the PDE catalytic
subunits soluble. Nonetheless, it is well documented that the ability
of transducin to activate the PDE is sensitively dependent on the
presence and/or composition of membranes in the assay (8, 10, 22).
Several studies have also shown that PDE is more tightly membrane bound when activated by transducin (22, 23). Therefore, it is very likely
that the
subunit could affect the transducin/PDE interaction by
altering the PDE's interaction with the membrane even without making
the PDE soluble. We decided to examine the effects of
-GST on the
photoreceptor's light response, and to confirm that peptides that
block the solubilization of the PDE (42) could block these physiological effects. Data from the pH assay shows that
-GST had
several effects on the rod's response to light. As we expected, it
reduced the total amount of cGMP that was hydrolyzed in response to a
flash of light and decreased the maximum rate of cGMP hydrolysis. The
subunit had a larger-fold effect at high (although not saturating) levels of light than at lower levels of light. This may represent a
shift in the sensitivity of the light response. Unexpectedly,
-GST
also increased the amount of time it took the system to turn off.
Based on both previously published data and data presented in this
paper, we feel it is likely that the reduction in cGMP hydrolysis in
the presence of -GST was due to the interaction of
-GST with the
PDE and not some other component of the signal transduction pathway.
The
subunit has not been reported to be copurified with any of the
other proteins of the signal transduction pathway, suggesting that the
PDE may be the
subunit's most common partner in this pathway, or
at least its most tightly interacting partner. Peptides that block the
ability of
-GST to make the PDE soluble (42) also blocked the
reduction in the rate of cGMP hydrolysis. However, the
subunit may
also interact with other prenylated proteins in the photoreceptor, and
if
-GST's effects were due to actions on these proteins, the
peptides might be expected to block these effects as well. Therefore,
we made more direct measurements of
-GST's effects on transducin
and rhodopsin kinase, the two other components of the signal
transduction pathway that could reduce cGMP hydrolysis in this assay.
The effects of
-GST on these other elements of the phototransduction
cascade cannot explain the reduction in cGMP hydrolysis presented here.
Namely, reduction of the GTPase activity of transducin by
-GST would be expected to increase, not decrease, the amount of cGMP hydrolyzed by
increasing the lifetime of active transducin. The reduction in cGMP
hydrolysis also occurred in the absence of ATP, which shows that an
effect on rhodopsin kinase was not responsible for this result. We
therefore feel that the reduction in the rate of cGMP hydrolysis was
most likely due to a functional uncoupling of the
rhodopsin-transducin-PDE pathway and a resultant reduction in the gain
of the transducin to PDE step of the phototransduction cascade.
One circumstance in which the gain of the phototransduction cascade is
reduced is during light adaptation. Two types of light adaptation have
been identified: background adaptation and bleaching adaptation.
Background light adaptation in the rod outer segment occurs when a rod
is exposed to a very low level (<0.01% rhodopsin bleached) of
background light. Bleaching adaptation occurs after a bright flash of
light that bleaches significantly more of the rhodopsin. In both cases,
adaptation results in a response with a smaller amplitude (reduced
gain) than the same flash given to a nonadapted rod. Background
adaptation has a large calcium-dependent component (24).
One model to explain background adaptation proposes that it occurs as a
result of guanylyl cyclase activation at low levels of calcium
(25-27). This calcium-dependent change in the recovery
phase of the cascade resets the background level of cGMP to a higher
concentration and probably accounts for much of the change seen with
adaptation. However, there are data to suggest that a slowing of the
excitation stages of the flash response may also be involved in light
adaptation. For example, a reduction in the gain of rhodopsin to PDE
activation (28-30) has been noted during light adaptation. It has also
been shown that during the rising phase of the light-adapted response,
which occurs before significant changes in intracellular calcium have
occurred, there is a reduced rate of activation of the PDE as compared
with the dark-adapted response (31). A molecular mechanism for this
slowed activation has not yet been determined, but the reduction in the hydrolysis of cGMP in response to a flash of light that we demonstrate in the presence of -GST could be responsible.
Bleaching adaptation appears to occur due to an activation of the
phototransduction cascade by bleached photopigment (32, 33) or
arrestin-bound meta-II rhodopsin (34). The "inactivated" rhodopsin
activates the visual signal transduction cascade, but to a lesser
extent than unphosphorylated meta II rhodopsin does. The low level
activation of the cascade mimics the changes seen in background light
adaptation. However, not all of the characteristics of bleaching
adaptation are the same as those seen as background adaptation. For
instance, bleached rhodopsin does not turn on the signal transduction
cascade as well as unbleached rhodopsin in equivalent background light
(31, 35). It is not clear whether this reduction in gain is
calcium-dependent. This could potentially be caused by a
reduction in the pool of activable PDE by the subunit. Further
experiments to determine whether the effects of the
subunit are
calcium-dependent may help to discern whether the
subunit may be involved in background adaptation, bleaching adaptation,
or some other process in the retina.
Although the reduced hydrolysis of cGMP that is seen in the presence of
-GST is suggestive of a role for the subunit in light adaptation,
there are some aspects of
-GST's effects that do not incorporate
well into this model. Prolongation of the turn-off of the system is not
one of the electrophysiological features of light adaptation. Several
factors could contribute to this prolongation. The reduction in GTP
hydrolysis in the presence of
-GST could contribute to prolongation
of the light signal. This reduced rate of GTPase may be a secondary
effect that results from reduced interaction between transducin's
subunit and the PDE
subunit, which can promote acceleration of the
GTPase activity of transducin (36, 37). Other work has shown that
-GST may reduce the PDE catalytic subunit's affinity for the
subunit (11). This could potentially prolong the amount of time that it
takes for the PDE to be reinhibited. The prolongation of turn-off could
also be due to the interaction of
-GST with other proteins in the
retina, such as RPGR.
If the subunit is involved in light adaptation or some other
dynamic process in the outer segment, it would be expected that its
interaction with the catalytic subunits of PDE6 would be regulated.
Recent work by our group (42) has suggested that the
subunit
interacts more strongly with methylated PDE than with demethylated PDE.
Since methylation is a reversible process, it could regulate the
PDE/
subunit interaction. However, at this point this model is
purely hypothetical, since light-induced regulation of neither
methylation nor PDE solubility has been reported.
In conclusion, data presented in this paper demonstrate that -GST
can have significant effects on the phototransduction cascade and that
it may be involved in the light adaptation process. The action of the
subunit is probably not limited to its effects on the visual signal
transduction cascade. The
subunit is present in a number of tissues
outside of the retina (1,
38),2 where none of the other
components of the visual signal transduction pathway are known to
localize. It has been cloned from organisms as varied as mouse, human,
and Caenorhabditis elegans (39, 40) and is very
highly conserved, which suggests that its functional role may be quite
important. It is possible that the
subunit may regulate the gain of
other signal transduction pathways, such as those involving small G
proteins (12, 13, 38), by changing the localization of their necessary
components; alternately, the
subunit could have a different role in
these other tissues. We hope that future studies can more definitively
determine the physiological purpose of this subunit in the retina as
well as in other tissues.
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ACKNOWLEDGEMENTS |
---|
We are extremely grateful to Mark Gray-Keller for instruction in the pH assay and Peter Detwiler for the use of a darkroom and equipment as well as helpful discussions. ROS from fresh retinas were a kind gift from Preston VanHoosier and Dr. Krzysztof Palczewski. We thank Dr. Rick Cote and Dr. Krzysztof Palczewski for comments.
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FOOTNOTES |
---|
* This work was supported by National Institutes of Health (NIH) Grants EY08197 and EY01730 and NIH Grant 2 T32 EY07031-21 (to T. A. C.).
¶ Present address: ICOS Corp., Bothell, Washington.
To whom correspondence should be addressed: Dept. of
Pharmacology, Box 357280, University of Washington, Seattle, WA 98195. Tel.: 206-543-9006.
Published, JBC Papers in Press, October 26, 2000, DOI 10.1074/jbc.M004690200
2 T. A. Cook, unpublished observations.
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ABBREVIATIONS |
---|
The abbreviations used are:
PDE6, type 6 3'-5'
cyclic nucleotide phosphodiesterase;
GST, glutathione
S-transferase;
GTPS, guanosine
5'-O-(3-thiotriphosphate);
G protein, GTP-binding protein;
mPDE, type 6 3'-5' cyclic nucleotide phosphodiesterase found bound to
the membrane;
ROS, rod outer segment(s);
sPDE, type 6 3'-5' cyclic
nucleotide phosphodiesterase found in the soluble fraction.
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