(Received for publication, January 16, 1997, and in revised form, March 3, 1997)
From the Department of Physiology and Biophysics, University of Iowa College of Medicine, Iowa City, Iowa 52242
Cyclic GMP phosphodiesterase (PDE) is the
effector enzyme in the visual transduction cascade of vertebrate
photoreceptor cells. In the dark, the activity of the enzyme catalytic
and
subunits (P
) is inhibited by two
subunits (P
).
Previous results have established that approximately 5-7 C-terminal
residues of P
comprise the inhibitory domain. To study the
interaction between the P
C-terminal region and P
, the P
mutant (Cys68
Ser, and the last 4 C-terminal
residues replaced with cysteine, P
-1-83Cys) was labeled with the
fluorescent probe 3-(bromoacetyl)-7-diethylaminocoumarin (BC) at the
cysteine residue (P
-1-83BC). P
-1-83BC was a more potent
inhibitor of PDE activity than the unlabeled mutant, suggesting that
the fluorescent probe in part substitutes for the P
C terminus in
PDE inhibition. HoloPDE (P
2) had no effect on the
P
-1-83BC fluorescence, but the addition of P
to P
-1-83BC
resulted in an approximately 8-fold maximal fluorescence increase. A
Kd for the P
-1-83BC-P
interaction was
4.0 ± 0.5 nM. Zaprinast, a specific competitive
inhibitor of PDE, effectively displaced the P
-1-83BC C terminus
from its binding site on P
(IC50 = 0.9 µM). cGMP and its analogs, 8-Br-cGMP and 2
-butyryl-cGMP, also competed with the P
-1-83BC C terminus for binding to P
. Our results provide new insight into the mechanism of PDE inhibition by
showing that P
blocks the binding of cGMP to the PDE catalytic site.
In the visual transduction cascade of rod photoreceptor cells, the
photoexcited visual receptor, rhodopsin, interacts with the rod
G-protein, transducin, and stimulates the exchange of GTP for bound
GDP. The GTP-bound subunit of transducin dissociates from rhodopsin
and the transducin
subunits and activates the effector enzyme,
cGMP phosphodiesterase (PDE),1 by relieving
the inhibitory constraint imposed by two identical inhibitory subunits
of PDE (P
) on the enzyme
catalytic subunits (P
) (for
review see Refs. 1-4).
Insights into the P-P
interaction are critical for
understanding the mechanisms of PDE inhibition by P
and PDE
activation by transducin. Approximately 5-7 C-terminal amino acid
residues of P
are involved in the inhibitory interaction with
P
(5-9). Recently, using a cross-linking approach we have
identified a site on P
for binding of the P
C terminus as a
region P
-751-763 within the PDE catalytic domain (10). The finding
suggests that the P
C terminus either occupies the site for binding
and catalysis of cGMP or induces local conformational changes of the
PDE catalytic site that block cGMP hydrolysis. Here, we study the
interaction between the C terminus of P
and P
using a novel
fluorescence assay to elucidate the mechanism of PDE inhibition by
P
.
cGMP was obtained from Boehringer Mannheim. 3-(bromoacetyl)-7-diethylaminocoumarin (BC) was purchased from Molecular Probes, Inc. Trypsin and soybean trypsin inhibitor were from Worthington. Zaprinast and all other reagents were purchased from Sigma.
Preparation of Trypsin-activated PDE, PBovine rod outer segment (ROS) membranes were prepared by
the method of Papermaster and Dreyer (11). PDE was extracted from ROS
membranes as described in Ref. 12. PDE and trypsin-activated PDE (tPDE)
were prepared and purified as described previously (6). The purified
proteins were kept in 40% glycerol at 20 °C. The P
subunit was
expressed in Escherichia coli and purified as described in
Ref. 9. The P
mutants P
Cys68
Ser and
P
-1-83Cys (Cys68
Ser, and the last 4 C-terminal
residues, Tyr-Gly-Ile-Ile, replaced with a single cysteine) were
obtained as described in Ref. 10.
To
obtain P-1-83BC and P
BC, a 5 mM stock solution of BC
(final concentration, 200 µM) was added to either 100 µM P
-1-83Cys or 100 µM P
, each in 20 mM HEPES buffer (pH 7.6), and the mixture was incubated for
30 min at room temperature. P
-1-83BC and P
BC were then passed
through a PD-10 column (Pharmacia Biotech Inc.) equilibrated with 20 mM HEPES buffer (pH 7.6) containing 100 mM NaCl
and purified by reversed-phase HPLC on a C-4 column Microsorb-MW (Rainin) using a 0-100% gradient of acetonitrile, 0.1%
trifluoroacetic acid. The preparations of P
-1-83BC and P
BC
contained no free BC. Using
445 = 53,000 for BC, the
molar ratio of BC incorporation into P
and P
-1-83BC was greater
than 0.8 mol/mol. P
-24-45BC was prepared by labeling of peptide
P
-24-45Cys and purified as described in Ref. 13. A P
mutant,
P
Cys68
Ser, and a peptide, P
-24-45, that contain
no cysteine were not derivatized with BC under similar conditions,
suggesting the selectivity of cysteine labeling.
Fluorescent assays were performed on a
F-2000 Fluorescence Spectrophotometer (Hitachi) in 1 ml of 80 mM Tris-HCl buffer (pH 7.6) containing 2 mM
MgCl2. Fluorescence of P-1-83BC, P
BC, or P
-24-45BC was monitored with excitation at 445 nm and emission at
495 nm. The assays were carried out at equilibrium, which was typically
reached less than 3 s after mixing of the components. The
concentration of labeled polypeptides was determined using
445 = 53,000. Where indicated, zaprinast was added from
1 mM stock solution to an assay buffer. The
Kd values in Figs. 2 and 4 were calculated by
fitting the data to Equation 1,
![]() |
(Eq. 1) |
The IC50 values in Figs. 3 and 5 were calculated by fitting the data to the one site competition equation with variable slope:
![]() |
(Eq. 2) |
Analytical Methods
The PDE activity was measured using the proton evolution assay of Liebman and Evanczuk (14). The assay was performed at room temperature in 200 µl of 10 mM HEPES (pH 7.8) containing 100 mM NaCl and 1 mM MgCl2. The reaction was initiated by the addition of cGMP. The pH was monitored with a pH microelectrode (Microelectrode, Inc.). Protein concentrations were determined by the method of Bradford (15) using IgG as a standard or using calculated extinction coefficients at 280 nm. SDS-polyacrylamide gel electrophoresis was performed by the method of Laemmli (16) in 12% acrylamide gels. Fitting of the experimental data was performed with nonlinear least squares criteria using GraphPad Prizm Software. The Ki, Kd, and IC50 values are expressed as the means ± S.E. of three independent measurements.
To study the inhibitory interaction between the P C
terminus and P
, we have replaced C-terminal amino acid residues
of P
with a fluorescent probe by labeling the P
-1-83Cys mutant with BC. To determine if the fluorescent probe can substitute for the
P
C terminus, the ability of the labeled and unlabeled mutant to
inhibit tPDE was tested. Limited proteolysis of holoPDE with trypsin
removes intrinsic P
subunits and small farnesylated and
geranyl-geranylated C-terminal fragments of P
and P
,
respectively, leaving mainly intact active catalytic P
subunits
(17, 18). The P
-1-83Cys mutant was, as expected, a weak inhibitor
of tPDE activity. It inhibited maximally only ~50% of tPDE activity
with an apparent Ki value of 13 nM (not
shown). The labeled mutant, P
-1-83BC, was a significantly more
potent inhibitor of tPDE than P
-1-83Cys. P
-1-83BC almost
completely inhibited tPDE activity with an apparent
Ki of 5.4 ± 0.5 nM (Fig.
1A). Free BC had no effect on tPDE activity
at tested concentrations up to 20 µM (not shown). This
suggests that when the P
C-terminal residues, Gly-Ile-Ile, are
replaced with the fluorescent probe BC, the probe binds into a
complimentary hydrophobic pocket on P
and substantially restores
the inhibitory potential of the P
-1-83Cys mutant. Furthermore, the
apparent Ki for tPDE inhibition by P
-1-83BC was
influenced by the cGMP concentration in the assay. Decrease in cGMP
concentration from 3 to 0.3 mM resulted in a reduction of
the apparent Ki value from 5.4 to 2.1 nM
(Fig. 1A). In control experiments, labeling of wild-type P
with BC at Cys68 did not notably affect the ability of
P
to inhibit tPDE (not shown). Both P
and P
BC fully inhibited
tPDE with Ki values less than 0.25 nM
(not shown), and the Ki values were not affected by
the concentration of cGMP in the assay (not shown). Next we examined
effects of P
-1-83BC and P
on Km values of
cGMP hydrolysis by tPDE. A Km of 90 µM
was calculated from the Michaelis-Menten plot for tPDE (Fig.
1B). This Km is consistent with earlier
estimates (17). In the presence of 3.5 and 8 nM of
P
-1-83BC, the apparent Km values were 215 and
480 µM, and Vmax values were 95 and 80%, respectively (Fig. 1B). The inhibitory interaction
between P
-1-83BC and P
was not purely competitive with cGMP,
because the Vmax values were also affected by
P
-1-83BC (Fig. 1B). In agreement with previous data
(17), the addition of P
did not significantly change the Km for cGMP hydrolysis by tPDE (not shown).
Binding of P
Addition of tPDE to
P-1-83BC increased the fluorescence of the probe many fold in a
dose-dependent manner (Fig. 2). The binding curve shows a single class of binding sites with Kd = 4.0 ± 0.5 nM and a maximal fluorescence enhancement
F/F0 = 8.4 ± 0.2. No change in the fluorescence of
P
-1-83BC was detected upon the addition of holoPDE or tPDE
reconstituted with P
. Furthermore, addition of P
to the
P
-1-83BC-P
complex readily reduced fluorescence to a basal
F0 level, suggesting that the fluorescent increase reflects
a specific interaction between P
and P
-1-83BC (not shown).
Fluorescence of P
BC was not notably affected in the presence of
tPDE. It appears that in the P
BC-P
complex the probe is oriented away from the P
subunits, whereas in the
P
-1-83BC-P
complex the probe occupies the pocket for binding
of the P
C terminus.
Zaprinast is a well characterized competitive inhibitor
of photoreceptor PDEs and cGMP-binding, cGMP-specific PDE (19, 20). We
have investigated effects of zaprinast on the interaction between P-1-83BC and P
. Zaprinast had no effect on the basal
fluorescence of P
-1-83BC. Addition of increasing concentrations of
zaprinast resulted in a complete reversal of the fluorescent
enhancement of P
-1-83BC bound to P
(IC50 of 0.9 µM) (Fig. 3). Zaprinast was effective in
blocking the P
-1-83BC-P
interaction within a
pharmacologically relevant range of concentrations. A
Kd value of 140 nM for zaprinast binding
to rod PDE catalytic sites was calculated based on inhibition of PDE
activity by zaprinast (19). Our assay does not allow us to calculate
the true Kd value for the zaprinast binding from the
curve in Fig. 3 because zaprinast does not compete for the
P
-24-45BC-P
interaction (see below) and cannot completely
displace P
-1-83BC from P
. A Kd value for
the zaprinast-PDE interaction lower than an IC50 value of
0.9 µM (Fig. 3) can be predicted.
To examine the effects of zaprinast on the apparent
Kd of P-1-83BC binding to P
, the binding
curves were obtained in the presence of different concentrations of
zaprinast (Fig. 4). Increasing concentrations of
zaprinast reduced the fluorescent enhancement of P
-1-83BC by
P
and increased the apparent Kd of the
P
-1-83BC binding to P
. The apparent Kd
calculated from the binding curves in the presence of 2 and 4 µM of zaprinast were 14.7 ± 0.9 and 22.6 ± 1.3 nM, respectively. The decrease in fluorescence of the
P
-1-83BC-P
complex and the increase in apparent
Kd values suggest that zaprinast competitively displaces the fluorescently labeled P
-1-83BC C terminus from the
binding pocket on P
. Dipyridamole, another potent competitive inhibitor of photoreceptor PDEs (19), was unsuitable for studies using
our assay. Dipyridamole is highly fluorescent with maximal emission at
480 nm.
To investigate if zaprinast can compete for binding
between the polycationic region of P, P
-24-45, and P
, we
utilized an assay of interaction between a synthetic peptide,
P
-24-45Cys, labeled with BC and tPDE (13). Zaprinast at
concentrations that completely reversed the fluorescent enhancement of
the P
-1-83BC-P
complex had no effect on the fluorescent
increase of P
-24-45BC caused by its binding to tPDE (not shown). At
a higher concentration (10 µM), zaprinast reduced the
maximal increase in fluorescence of the P
-24-45BC-P
complex
by only ~15% without affecting the Kd
. The Kd values for P
-24-45BC
binding to P
in the presence of 10 µM of zaprinast
or with no zaprinast added were indistinguishable (~26
nM) (not shown). Interestingly, the apparent
Kd values for P
-1-83BC binding to P
at higher concentrations of zaprinast (Fig. 4) approached the
Kd for the P
-24-45BC-P
complex. This
supports the notion that zaprinast competes with the P
-1-83BC C
terminus for binding to P
and does not affect the interaction of
the polycationic region, P
-24-45, with P
.
The relative potency of zaprinast in
competition with the P-1-83BC C terminus for binding to P
indicated that cGMP and its analogs might be effective as well. Effects
of cGMP on the P
-1-83BC-P
interaction were tested in the
presence or the absence of Mg2+. Mg2+
participates in binding of cGMP to the PDE catalytic site and is
critical for cGMP hydrolysis. In the presence of Mg2+, cGMP
reversed the fluorescent enhancement of P
-1-83BC bound to P
with an apparent IC50 of 0.77 mM (Fig.
5A). Presumably, cGMP would be significantly
more effective in the absence of cGMP hydrolysis. In the presence of
EDTA, cGMP was ~2.5-fold less potent (IC50 of 1.9 mM, Fig. 5A). Because cGMP is not hydrolyzed in
the presence of EDTA, the data suggest that Mg2+ enhances
affinity of cGMP for the catalytic site by more than 2.5-fold. We also
tested two cGMP analogs, 8-Br-cGMP and 2
-butyryl-cGMP, because they
block ROS-PDE activity with a relatively high affinity (21). Both cGMP
analogs competed with the P
-1-83BC C terminus for binding to
P
(Fig. 5B). The IC50 values from the
competition curves for 8-Br-cGMP and 2
-butyryl-cGMP were 0.42 mM and 0.58 mM, respectively. Perhaps, an
IC50 for 2
-butyryl-cGMP was higher than an
IC50 for 8-Br-cGMP because tPDE hydrolyzed 2
-butyryl-cGMP with a rate of ~15% of cGMP hydrolysis, whereas 8-Br-cGMP was resistant to the hydrolysis (not shown).
Interaction between the inhibitory subunits of PDE and the
catalytic P
subunits is essential for blocking PDE activity in
the dark and for inactivation of the enzyme upon recovery of the
photoreceptor cell from light stimulation. The P
subunits bind to
P
with very high affinity (Kd< 100 pM) (22). The high affinity of the P
-P
interaction
is provided by two major binding sites on P
, the central
polycationic region, P
-24-45, and the C-terminal 5-7 amino acid
residues (5-9). The main role of the P
-24-45 region is to enhance
the affinity of P
interaction with P
. The C terminus of P
is critical for PDE inhibition. Truncations of the P
C-terminal
residues lead to a loss of the P
inhibitory function (5, 8, 9).
Peptides corresponding to the C-terminal region of P
can fully
inhibit PDE activity (6, 7, 9). Recently, we have shown that the
C-terminal region of P
binds within the catalytic domain of PDE
(10). This finding raised the possibility that P
may inhibit PDE
activity by physically blocking the binding site for cGMP. An
alternative mechanism of PDE inhibition by P
would be a local
conformational change of the PDE catalytic site that prevents cGMP
hydrolysis. Standard analysis for competitive (noncompetitive)
inhibition of cGMP hydrolysis may not discriminate between the two
mechanisms because P
binds to P
very tightly
(Kd <100 pM) compared with cGMP binding
(Km for cGMP is within 17-80 µM
range) (17, 19, 22). The large differences in affinity and very slow
off-rates of P
from P
(>10 min) (8, 22) may not allow cGMP to
compete with P
bound to P
. It has been shown previously that
the addition of P
to tPDE caused very little change in the apparent
Km value (17). Indeed, in our experiments the
Km value of 90 µM was unaffected by
the addition of P
.
To study the mechanism of PDE inhibition by P, we developed an assay
that reports binding of the P
C terminus to P
. The assay
utilizes a P
mutant with the C-terminal amino acid residues replaced
with a fluorescent probe, BC. The fluorescently labeled mutant,
P
-1-83BC, was a more potent inhibitor of PDE activity than the
unlabeled mutant, P
-1-83Cys, suggesting that the probe interacts
with the inhibitory pocket on P
. Addition of P
-1-83BC to
P
led to a dose-dependent increase of the apparent
Km values for cGMP hydrolysis. Binding of
P
-1-83BC to P
produced a large ~8-fold increase in the
probe fluorescence. Zaprinast, a specific competitive inhibitor of
photoreceptor PDEs, effectively competed for the interaction between
P
-1-83BC and P
, but had no effect on binding of the
polycationic region, P
-24-45, to P
. Perhaps the fact that
P
-1-83BC binds to P
(Kd of 4 nM) less tightly than P
has helped zaprinast to compete for the P
-1-83BC-P
interaction. cGMP and its analogs,
8-Br-cGMP and 2
-butyryl-cGMP, were also effective in blocking the
interaction between the P
-1-83BC C terminus and P
using the
fluorescent assay. Effects of cGMP and its analogs on the P
-1-83BC
binding to P
cannot be attributed to the noncatalytic
cGMP-binding sites of PDE, because bovine rod PDE contains two
molecules of tightly bound cGMP with an extremely slow off-rate
(t1/2 = ~4 h) (23).
Overall, our data strongly suggest that P inhibits PDE activity by
physically blocking access of the substrate, cGMP, to the PDE catalytic
site. The region of P
, P
-751-763, that interacts with the C
terminus of P
(10) is adjacent to the NKXD motif. In
G-proteins, the NKXD motif specifies binding of the GTP
guanine ring (24, 25). Based on our results, it is likely that the NKXD motif is involved in the binding of cGMP by
photoreceptor PDEs.