The gamma  Subunit of Rod cGMP-Phosphodiesterase Blocks the Enzyme Catalytic Site*

(Received for publication, January 16, 1997, and in revised form, March 3, 1997)

Alexey E. Granovsky , Michael Natochin and Nikolai O. Artemyev Dagger

From the Department of Physiology and Biophysics, University of Iowa College of Medicine, Iowa City, Iowa 52242

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
REFERENCES


ABSTRACT

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 alpha  and beta  subunits (Palpha beta ) is inhibited by two gamma  subunits (Pgamma ). Previous results have established that approximately 5-7 C-terminal residues of Pgamma comprise the inhibitory domain. To study the interaction between the Pgamma C-terminal region and Palpha beta , the Pgamma mutant (Cys68 right-arrow Ser, and the last 4 C-terminal residues replaced with cysteine, Pgamma -1-83Cys) was labeled with the fluorescent probe 3-(bromoacetyl)-7-diethylaminocoumarin (BC) at the cysteine residue (Pgamma -1-83BC). Pgamma -1-83BC was a more potent inhibitor of PDE activity than the unlabeled mutant, suggesting that the fluorescent probe in part substitutes for the Pgamma C terminus in PDE inhibition. HoloPDE (Palpha beta gamma 2) had no effect on the Pgamma -1-83BC fluorescence, but the addition of Palpha beta to Pgamma -1-83BC resulted in an approximately 8-fold maximal fluorescence increase. A Kd for the Pgamma -1-83BC-Palpha beta interaction was 4.0 ± 0.5 nM. Zaprinast, a specific competitive inhibitor of PDE, effectively displaced the Pgamma -1-83BC C terminus from its binding site on Palpha beta (IC50 = 0.9 µM). cGMP and its analogs, 8-Br-cGMP and 2'-butyryl-cGMP, also competed with the Pgamma -1-83BC C terminus for binding to Palpha beta . Our results provide new insight into the mechanism of PDE inhibition by showing that Pgamma blocks the binding of cGMP to the PDE catalytic site.


INTRODUCTION

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 alpha  subunit of transducin dissociates from rhodopsin and the transducin beta gamma subunits and activates the effector enzyme, cGMP phosphodiesterase (PDE),1 by relieving the inhibitory constraint imposed by two identical inhibitory subunits of PDE (Pgamma ) on the enzyme alpha beta catalytic subunits (Palpha beta ) (for review see Refs. 1-4).

Insights into the Pgamma -Palpha beta interaction are critical for understanding the mechanisms of PDE inhibition by Pgamma and PDE activation by transducin. Approximately 5-7 C-terminal amino acid residues of Pgamma are involved in the inhibitory interaction with Palpha beta (5-9). Recently, using a cross-linking approach we have identified a site on Palpha for binding of the Pgamma C terminus as a region Palpha -751-763 within the PDE catalytic domain (10). The finding suggests that the Pgamma 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 Pgamma and Palpha beta using a novel fluorescence assay to elucidate the mechanism of PDE inhibition by Pgamma .


EXPERIMENTAL PROCEDURES

Materials

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, Pgamma , and Pgamma Mutants

Bovine 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 Pgamma subunit was expressed in Escherichia coli and purified as described in Ref. 9. The Pgamma mutants Pgamma Cys68 right-arrow Ser and Pgamma -1-83Cys (Cys68 right-arrow Ser, and the last 4 C-terminal residues, Tyr-Gly-Ile-Ile, replaced with a single cysteine) were obtained as described in Ref. 10.

Preparation of Pgamma -1-83BC, Pgamma BC, and Pgamma -24-45BC

To obtain Pgamma -1-83BC and Pgamma BC, a 5 mM stock solution of BC (final concentration, 200 µM) was added to either 100 µM Pgamma -1-83Cys or 100 µM Pgamma , each in 20 mM HEPES buffer (pH 7.6), and the mixture was incubated for 30 min at room temperature. Pgamma -1-83BC and Pgamma 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 Pgamma -1-83BC and Pgamma BC contained no free BC. Using epsilon 445 = 53,000 for BC, the molar ratio of BC incorporation into Pgamma and Pgamma -1-83BC was greater than 0.8 mol/mol. Pgamma -24-45BC was prepared by labeling of peptide Pgamma -24-45Cys and purified as described in Ref. 13. A Pgamma mutant, Pgamma Cys68 right-arrow Ser, and a peptide, Pgamma -24-45, that contain no cysteine were not derivatized with BC under similar conditions, suggesting the selectivity of cysteine labeling.

Fluorescent Assays

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 Pgamma -1-83BC, Pgamma BC, or Pgamma -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 epsilon 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,
<FR><NU><UP>F</UP></NU><DE><UP>F</UP><SUB>0</SUB></DE></FR>=1+<FR><NU><FENCE><FR><NU><UP>F</UP></NU><DE><UP>F</UP><SUB>0</SUB></DE></FR><SUB><UP>max</UP></SUB>−1</FENCE>×X</NU><DE>K<SUB>d</SUB>+X</DE></FR> (Eq. 1)
where F0 is a basal fluorescence of Pgamma -1-83BC or Pgamma -24-45BC, F is the fluorescence after additions of tPDE, F/F0max is the maximal relative increase of fluorescence, and X is a concentration of free tPDE.


Fig. 2. Binding of Pgamma -1-83BC to tPDE. The relative increase in fluorescence (F/F0) of Pgamma -1-83BC (10 nM) was determined after the addition of increasing concentrations of tPDE and is plotted as a function of the free tPDE concentration. The binding curve (Kd = 4.0 ± 0.5 nM, maximum F/F0 = 8.4 ± 0.2) fits the data with r = 0.98.
[View Larger Version of this Image (0K GIF file)]


Fig. 4. Effect of zaprinast on apparent Kd for Pgamma -1-83BC binding to Palpha beta . Zaprinast was added to Pgamma -1-83BC (10 nM) at indicated concentrations, and then the relative increase in fluorescence (F/F0) of Pgamma -1-83BC was determined after the addition of increasing concentrations of tPDE. The calculated apparent Kd values for Pgamma -1-83BC binding to tPDE in the presence of 0.5, 2 and 4 µM of zaprinast were 5.1 ± 0.7, 14.7 ± 0.9, and 22.6 ± 1.3 nM, respectively. black-diamond , 5 × 10-7 zaprinast; black-triangle, 2 × 10-6 zaprinast; black-down-triangle , 4 × 10-6 zaprinast.
[View Larger Version of this Image (0K GIF file)]

The IC50 values in Figs. 3 and 5 were calculated by fitting the data to the one site competition equation with variable slope:
<FR><NU><UP>F</UP></NU><DE><UP>F</UP><SUB>0</SUB></DE></FR>=1+<FR><NU><FR><NU><UP>F</UP></NU><DE><UP>F</UP><SUB>0</SUB></DE></FR><SUB><UP>max</UP></SUB>−1</NU><DE>1+10<SUP>[X-<UP>logIC</UP><SUB>50</SUB>]<SUP>×</SUP>H</SUP></DE></FR> (Eq. 2)
where X is a concentration of a competing ligand (zaprinast, cGMP, or cGMP analogs) and H is a Hill slope.


Fig. 3. Competition between Pgamma -1-83BC and zaprinast for binding to Palpha beta . tPDE (total concentration, 6 nM) was added to Pgamma -1-83BC (10 nM), and then the fluorescence was measured before and after the addition of increasing concentrations of zaprinast. The relative fluorescent change (F/F0) is plotted as a function of zaprinast concentration. The curve (IC50 = 0.90 ± 0.02 µM and Hill slope = 1.1) fits the data with r = 0.98.
[View Larger Version of this Image (0K GIF file)]


Fig. 5. Effects of cGMP and its analogs on Pgamma -1-83BC binding to Palpha beta . A, fluorescence of Pgamma -1-83BC (10 nM) in the presence of tPDE (total concentration, 6 nM) was measured before and after addition of increasing concentrations of cGMP. An assay buffer contained 2 mM Mg2+ (square ) or 1 mM EDTA instead of Mg2+ (black-square). The curves in the presence of Mg2+ (IC50 = 0.77 ± 0.01 mM; Hill slope = 0.9) and in the presence of EDTA (IC50 = 1.9 ± 0.3 mM; Hill slope = 1.1) fit the data with r values of 1.0 and 0.96, respectively. B, fluorescence of Pgamma -1-83BC (10 nM) in the presence of tPDE (total concentration, 6 nM) was measured before and after addition of increasing concentrations of 8-Br-cGMP (black-triangle) or 2'-butyryl-cGMP (triangle ). The curves for 8-Br-cGMP (IC50 = 0.42 ± 0.03 mM; Hill slope = 1) and for 2'-butyryl-cGMP (IC50 = 0.58 ± 0.06 mM; Hill slope = 0.85) each fit the data with r value of 0.99.
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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.


RESULTS

Inhibition of tPDE Activity by Pgamma -1-83Cys, Pgamma -1-83BC, and Pgamma BC

To study the inhibitory interaction between the Pgamma C terminus and Palpha beta , we have replaced C-terminal amino acid residues of Pgamma with a fluorescent probe by labeling the Pgamma -1-83Cys mutant with BC. To determine if the fluorescent probe can substitute for the Pgamma C terminus, the ability of the labeled and unlabeled mutant to inhibit tPDE was tested. Limited proteolysis of holoPDE with trypsin removes intrinsic Pgamma subunits and small farnesylated and geranyl-geranylated C-terminal fragments of Palpha and Pbeta , respectively, leaving mainly intact active catalytic Palpha beta subunits (17, 18). The Pgamma -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, Pgamma -1-83BC, was a significantly more potent inhibitor of tPDE than Pgamma -1-83Cys. Pgamma -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 Pgamma C-terminal residues, Gly-Ile-Ile, are replaced with the fluorescent probe BC, the probe binds into a complimentary hydrophobic pocket on Palpha beta and substantially restores the inhibitory potential of the Pgamma -1-83Cys mutant. Furthermore, the apparent Ki for tPDE inhibition by Pgamma -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 Pgamma with BC at Cys68 did not notably affect the ability of Pgamma to inhibit tPDE (not shown). Both Pgamma and Pgamma 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 Pgamma -1-83BC and Pgamma 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 Pgamma -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 Pgamma -1-83BC and Palpha beta was not purely competitive with cGMP, because the Vmax values were also affected by Pgamma -1-83BC (Fig. 1B). In agreement with previous data (17), the addition of Pgamma did not significantly change the Km for cGMP hydrolysis by tPDE (not shown).


Fig. 1. A, inhibition of tPDE activity by Pgamma -1-83BC. The activity of tPDE (5 nM) was determined upon addition of increasing concentrations of Pgamma -1-83BC. The reaction was initiated by addition of 3 (black-square) or 0.3 mM cGMP (square ). The percentage of maximal tPDE activity (3500 mol cGMP·s-1·mol PDE-1 at 3 mM cGMP and 2700 mol cGMP·s-1·mol PDE-1 at 0.3 mM cGMP) is plotted as a function of the free Pgamma concentration. The inhibition curves (black-square, Ki = 5.4 ± 0.5 nM; square , Ki = 2.1 ± 0.2 nM) each fit the data with r value of 0.98. B, effect of Pgamma -1-83BC on the apparent Km values for cGMP hydrolysis by tPDE. The activity of tPDE (2.5 nM) alone (black-triangle) or in the presence of 3.5 (black-square) and 8 nM of total Pgamma -1-83BC (bullet ) was determined at varying concentrations of cGMP. The curves (black-triangle, Km = 90 ± 10 µM; black-square, Km = 215 ± 23 µM, Vmax = 95%; and bullet , Km = 480 ± 76 µM, Vmax = 80%) fit the Michaelis-Menten equation with r values of 0.99, 0.99, and 0.98, respectively.
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Binding of Pgamma -1-83BC to Palpha beta

Addition of tPDE to Pgamma -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 Pgamma -1-83BC was detected upon the addition of holoPDE or tPDE reconstituted with Pgamma . Furthermore, addition of Pgamma to the Pgamma -1-83BC-Palpha beta complex readily reduced fluorescence to a basal F0 level, suggesting that the fluorescent increase reflects a specific interaction between Palpha beta and Pgamma -1-83BC (not shown). Fluorescence of Pgamma BC was not notably affected in the presence of tPDE. It appears that in the Pgamma BC-Palpha beta complex the probe is oriented away from the Palpha beta subunits, whereas in the Pgamma -1-83BC-Palpha beta complex the probe occupies the pocket for binding of the Pgamma C terminus.

Effects of Zaprinast on the Interaction between Pgamma -1-83BC and Palpha beta

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 Pgamma -1-83BC and Palpha beta . Zaprinast had no effect on the basal fluorescence of Pgamma -1-83BC. Addition of increasing concentrations of zaprinast resulted in a complete reversal of the fluorescent enhancement of Pgamma -1-83BC bound to Palpha beta (IC50 of 0.9 µM) (Fig. 3). Zaprinast was effective in blocking the Pgamma -1-83BC-Palpha beta 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 Pgamma -24-45BC-Palpha beta interaction (see below) and cannot completely displace Pgamma -1-83BC from Palpha beta . 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 Pgamma -1-83BC binding to Palpha beta , the binding curves were obtained in the presence of different concentrations of zaprinast (Fig. 4). Increasing concentrations of zaprinast reduced the fluorescent enhancement of Pgamma -1-83BC by Palpha beta and increased the apparent Kd of the Pgamma -1-83BC binding to Palpha beta . 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 Pgamma -1-83BC-Palpha beta complex and the increase in apparent Kd values suggest that zaprinast competitively displaces the fluorescently labeled Pgamma -1-83BC C terminus from the binding pocket on Palpha beta . 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.

Effects of Zaprinast on the Interaction between Pgamma -24-45BC and Palpha beta

To investigate if zaprinast can compete for binding between the polycationic region of Pgamma , Pgamma -24-45, and Palpha beta , we utilized an assay of interaction between a synthetic peptide, Pgamma -24-45Cys, labeled with BC and tPDE (13). Zaprinast at concentrations that completely reversed the fluorescent enhancement of the Pgamma -1-83BC-Palpha beta complex had no effect on the fluorescent increase of Pgamma -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 Pgamma -24-45BC-Palpha beta complex by only ~15% without affecting the Kd . The Kd values for Pgamma -24-45BC binding to Palpha beta 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 Pgamma -1-83BC binding to Palpha beta at higher concentrations of zaprinast (Fig. 4) approached the Kd for the Pgamma -24-45BC-Palpha beta complex. This supports the notion that zaprinast competes with the Pgamma -1-83BC C terminus for binding to Palpha beta and does not affect the interaction of the polycationic region, Pgamma -24-45, with Palpha beta .

Effects of cGMP and Its Analogs on the Interaction between Pgamma -1-83BC and Palpha beta

The relative potency of zaprinast in competition with the Pgamma -1-83BC C terminus for binding to Palpha beta indicated that cGMP and its analogs might be effective as well. Effects of cGMP on the Pgamma -1-83BC-Palpha beta 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 Pgamma -1-83BC bound to Palpha beta 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 Pgamma -1-83BC C terminus for binding to Palpha beta (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).


DISCUSSION

Interaction between the inhibitory gamma  subunits of PDE and the catalytic Palpha beta 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 Pgamma subunits bind to Palpha beta with very high affinity (Kd< 100 pM) (22). The high affinity of the Pgamma -Palpha beta interaction is provided by two major binding sites on Pgamma , the central polycationic region, Pgamma -24-45, and the C-terminal 5-7 amino acid residues (5-9). The main role of the Pgamma -24-45 region is to enhance the affinity of Pgamma interaction with Palpha beta . The C terminus of Pgamma is critical for PDE inhibition. Truncations of the Pgamma C-terminal residues lead to a loss of the Pgamma inhibitory function (5, 8, 9). Peptides corresponding to the C-terminal region of Pgamma can fully inhibit PDE activity (6, 7, 9). Recently, we have shown that the C-terminal region of Pgamma binds within the catalytic domain of PDE (10). This finding raised the possibility that Pgamma may inhibit PDE activity by physically blocking the binding site for cGMP. An alternative mechanism of PDE inhibition by Pgamma 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 Pgamma binds to Palpha beta 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 Pgamma from Palpha beta (>10 min) (8, 22) may not allow cGMP to compete with Pgamma bound to Palpha beta . It has been shown previously that the addition of Pgamma 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 Pgamma .

To study the mechanism of PDE inhibition by Pgamma , we developed an assay that reports binding of the Pgamma C terminus to Palpha beta . The assay utilizes a Pgamma mutant with the C-terminal amino acid residues replaced with a fluorescent probe, BC. The fluorescently labeled mutant, Pgamma -1-83BC, was a more potent inhibitor of PDE activity than the unlabeled mutant, Pgamma -1-83Cys, suggesting that the probe interacts with the inhibitory pocket on Palpha beta . Addition of Pgamma -1-83BC to Palpha beta led to a dose-dependent increase of the apparent Km values for cGMP hydrolysis. Binding of Pgamma -1-83BC to Palpha beta produced a large ~8-fold increase in the probe fluorescence. Zaprinast, a specific competitive inhibitor of photoreceptor PDEs, effectively competed for the interaction between Pgamma -1-83BC and Palpha beta , but had no effect on binding of the polycationic region, Pgamma -24-45, to Palpha beta . Perhaps the fact that Pgamma -1-83BC binds to Palpha beta (Kd of 4 nM) less tightly than Pgamma has helped zaprinast to compete for the Pgamma -1-83BC-Palpha beta interaction. cGMP and its analogs, 8-Br-cGMP and 2'-butyryl-cGMP, were also effective in blocking the interaction between the Pgamma -1-83BC C terminus and Palpha beta using the fluorescent assay. Effects of cGMP and its analogs on the Pgamma -1-83BC binding to Palpha beta 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 Pgamma inhibits PDE activity by physically blocking access of the substrate, cGMP, to the PDE catalytic site. The region of Palpha , Palpha -751-763, that interacts with the C terminus of Pgamma (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.


FOOTNOTES

*   This work was supported by National Eye Institute Grant EY-10843.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.
Dagger    To whom correspondence and reprint requests should be addressed: Dept. of Physiology and Biophysics, University of Iowa College of Medicine, 5-660 Bowen Science Bldg., Iowa City, IA 52242. Tel.: 319-335-7864; Fax: 319-335-7330; E-mail: Nikolai-Artemyev{at}uiowa.edu.
1   The abbreviations used are: PDE, rod outer segment cGMP phosphodiesterase; tPDE, phosphodiesterase activated with limited trypsin digestion to remove gamma  subunits; ROS, rod outer segment; BC, 3-(bromoacetyl)-7-diethylaminocoumarin; HPLC, high performance liquid chromatography.

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