©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
An Essential Aspartic Acid at Each of Two Allosteric cGMP-binding Sites of a cGMP-specific Phosphodiesterase (*)

(Received for publication, February 1, 1995; and in revised form, September 28, 1995)

Linda M. McAllister-Lucas (1) Tamara L. Haik (1) Janet L. Colbran (1) William K. Sonnenburg (2) Dalia Seger (2) Illarion V. Turko (1) Joe A. Beavo (2) Sharron H. Francis (1) Jackie D. Corbin (1)(§)

From the  (1)Department of Molecular Physiology and Biophysics, Vanderbilt University School of Medicine, Nashville, Tennessee 37232-0165 and and (2)Department of Pharmacology, University of Washington, Seattle, Washington 98195

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
MATERIALS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

The amino acid sequences of all known cGMP-binding phosphodiesterases (PDEs) contain internally homologous repeats (a and b) that are 80-90 residues in length and are arranged in tandem within the putative cGMP-binding domains. In the bovine lung cGMP-binding, cGMP-specific PDE (cGB-PDE or PDE5A), these repeats span residues 228-311 (a) and 410-500 (b). An aspartic acid (residue 289 or 478) that is invariant in repeats a and b of all known cGMP-binding PDEs was changed to alanine by site-directed mutagenesis of cGB-PDE, and wild type (WT) and mutant cGB-PDEs were expressed in COS-7 cells. Purified bovine lung cGB-PDE (native) and WT cGB-PDE displayed identical cGMP-binding kinetics, with 1.8 µM cGMP required for half-maximal saturation. The D289A mutant showed decreased affinity for cGMP (K > 10 µM) and the D478A mutant showed increased affinity for cGMP (Kapprox 0.5 µM) as compared to WT and native cGB-PDE. WT and native cGB-PDE displayed an identical curvilinear profile of cGMP dissociation which was consistent with the presence of distinct slowly dissociating (k = 0.26 h) and rapidly dissociating (k = 1.00 h) sites of cGMP binding. In contrast, the D289A mutant displayed a single k = 1.24 h, which was similar to the calculated k for the fast site of WT and native cGB-PDE, and the D478A mutant displayed a single k = 0.29 h, which was similar to that calculated for the slow site of WT and native cGB-PDE. These results were consistent with the loss of a slow cGMP-binding site in repeat a of the D289A mutant cGB-PDE, and the loss of a fast site in repeat b of the D478A mutant, suggesting that cGB-PDE possesses two distinct cGMP-binding sites located at repeats a and b, with the invariant aspartic acid being crucial for interaction with cGMP at each site.


INTRODUCTION

Cyclic nucleotide phosphodiesterases (PDEs) (^1)constitute a complex family of enzymes which catalyze the hydrolysis of 3`:5`-cyclic nucleotides to the corresponding nucleoside 5`-monophosphates. The multiple PDEs differ in their substrate specificities, sensitivities to inhibitors, modes of regulation, and tissue distributions. Most PDEs are chimeric multidomain proteins, possessing distinct catalytic and regulatory domains(1) . A 250-amino acid segment of sequence, which is conserved among all mammalian PDEs and is located in the more carboxyl-terminal portions of the PDE molecules, contains the catalytic site of these enzymes(1, 2, 3, 4) . Domains of the PDEs which interact with allosteric/regulatory factors are thought to be located within the more amino-terminal regions(1, 5, 6) .

The cGMP-binding PDEs comprise a heterogeneous subgroup of PDEs, all of which exhibit allosteric cGMP-binding sites that are distinct from the sites of cyclic nucleotide hydrolysis. This group consists of at least three classes of PDEs: the cGMP-stimulated PDEs (cGS-PDEs, or PDE2s)(^2)(7) , the photoreceptor PDEs (rod outer segment PDE (ROS-PDE; PDE6A/B) (8) and cone PDE (PDE6C)(9) ), and the cGMP-binding, cGMP-specific PDE (cGB-PDE; PDE5A)(10) . The stimulatory effect of cGMP binding to the allosteric sites of cGS-PDE upon cyclic nucleotide hydrolysis at the catalytic site is well documented(7) , but the functional role of the allosteric sites in the photoreceptor PDEs or cGB-PDE is not well understood. However, there is evidence that cGMP binding at allosteric sites of frog ROS-PDE regulates the interaction of its catalytic subunits with inhibitory subunits and with the G-protein, transducin(11) , and that binding of cGMP to the allosteric sites of bovine lung cGB-PDE allows for the phosphorylation of the enzyme by cGMP-dependent or cAMP-dependent protein kinase(12) .

cDNAs encoding each of these cGMP-binding PDEs have now been isolated(13, 14, 15, 16, 17) , and, as predicted(5) , the deduced amino acid sequence of each of these enzymes contains a conserved segment of approximately 340 residues located amino-terminal to the catalytic site. This conserved segment is not present in any PDEs other than the cGMP-binding PDEs, and is proposed to constitute an allosteric cGMP-binding region(5) . Limited proteolysis and photoaffinity-labeling studies of cGB-PDE and cGS-PDE have provided direct evidence to support this proposal(10, 13, 18) . Within their conserved cGMP-binding regions, all known cGMP-binding PDEs contain two internally homologous sequence repeats of 90 amino acids each (a and b), that are arranged in tandem, and share 15-45% sequence identity with the a and b repeats in other cGMP-binding PDEs(13, 14, 15, 16, 17) . cAMP-dependent protein kinase and cGMP-dependent protein kinase, which do not share significant sequence homology with the cGMP-binding PDEs, also contain a tandem repeat pattern of sequence. In these kinases, the repeated sequences, approximately 120 amino acid residues in length, are known to comprise two distinct cyclic nucleotide-binding sites (19) . By analogy, the repeats a and b present in the cGMP-binding region of the cGMP-binding PDEs may also represent two distinct cGMP-binding sites. In support of this proposal, kinetic analyses of cGMP binding to cGS-PDE(18) , ROS-PDE(20, 21) , and cGB-PDE (10) suggest the presence of two classes of allosteric cGMP-binding sites in these enzymes.

If the homologous sequence repeats in the cGMP-binding PDEs represent two distinct cGMP-binding sites, amino acids essential for interaction with cGMP should be conserved among all repeats. Seven amino acid residues are invariant in all repeats in all known cGMP-binding PDEs, and three of these seven residues possess charged side groups (Lys, Asp, Glu) which could be important for interaction with cGMP(13) . In this report, the invariant Asp in repeats a or b of cGB-PDE has been modified by site-directed mutagenesis (see Fig. 1). Analysis of the effect of these mutations on cGMP-binding activity has been utilized to determine if the repeats a and b represent two distinct cGMP-binding sites, and if the conserved Asp is essential for binding at both sites.


Figure 1: A, point mutations within the proposed domain organization of a cGB-PDE monomer. The cGMP-binding region (residues 142-526) is shown as a box with diagonal lines. Internally homologous repeats a(228-311) and b(410-500), within the cGMP-binding region, are shown as black boxes. Single point mutations introduced into repeats a or b are indicated above the diagram. The catalytic region(578-812) is shown as a light gray box, and extends through the putative cyclic nucleotide-binding component of the the catalytic region c(760-812), which is shown as a dark gray box. B, alignment of internally homologous repeats a and b and the putative cyclic nucleotide-binding component of the catalytic region (c) of cGB-PDE. Residues which are identical in each repeat a and b from all known cGMP-binding PDEs are enclosed in boxes. Arrows represent positions in which all residues are chemically conserved among each a and b repeat from all known cGMP-binding PDEs. Residues identical in repeats a and b in cGB-PDE are shown in boldface. The conserved Asp which has been mutated in repeat a of the D289A mutant and in repeat b of the D478A mutant is enclosed in a black box. Residues that are conserved in each region c (putative cyclic nucleotide-binding component of the catalytic region) of all known mammalian PDEs are indicated by stars. Vertical lines illustrate conserved residues among segments a, b, and c. Residues comprising each segment are described in the legend to Panel A.




EXPERIMENTAL PROCEDURES

Methods

Purification of Bovine Lung cGB-PDE

cGB-PDE was purified to apparent homogeneity according to previously described methods(10, 13) . This purified cGB-PDE will be referred to as native cGB-PDE.

Sequence Analysis

The methods used to identify regions of amino acid homology among PDEs have been described previously(13) .

Construction of Expression Plasmids and Site-directed Mutagenesis

The isolation of the cDNA encoding a full-length bovine lung cGB-PDE, designated cGB-8, has been described (13) . (^3)Subcloning and mutagenesis were performed using standard methods(22) . Specifically, the cGB-8-pBSSK(-) construct (13) was cut with BsaI, followed by Klenow treatment and subsequent digestion with XbaI. A resulting 3553-base pair (bp) fragment which included bp 6-3559 and therefore contained the entire coding sequence of cGB-8 (bp 99-2724), was isolated. The pCDNA1/Amp mammalian expression vector (Invitrogen) was prepared for subcloning manipulations as follows. The HindIII site within the polylinker was removed by HindIII digestion, Klenow treatment, and religation, producing a modified vector designated pCDNA1/Amp(-)H3. This step was necessary for efficient subcloning of cDNAs encoding mutant cGB-PDEs, as will be discussed below. The pCDNA1/Amp(-)H3 plasmid was cut with BamHI, followed by Klenow treatment and subsequent digestion with XbaI. The 3553-bp cGB-8 fragment was ligated into the BamHI/XbaI-digested pCDNA1/Amp(-)H3, producing the expression plasmid pCDNA-cGB-PDE(WT).

Oligonucleotide-directed mutagenesis was performed using standard techniques(22) . Specifically, a HindIII fragment of cGB-8, spanning bp 712-1546 (encoding the entire cGMP-binding region), was ligated into the HindIII cloning site of the pBluescript SK(-) subcloning vector, producing the phagemid construct cGB-8-H3f-pBSSK(-). Uracil-containing single-stranded cGB-8-H3f-pBSSK(-) DNA was prepared for use as a template by transformation of Escherichia coli CJ236 (dut, ung) with the phagemid and subsequent infection with M13K07 helper virus. The following mutagenic oligonucleotides were synthesized on a Cyclone Plus DNA synthesizer (Millipore): 1) cGBD289A: 5`-GGG ACA TTC ACT GAA AAA GCC GAA AAG GAC TTT GCT GCT-3` (encoded amino acid sequence: GTFTEKAEKDFAA), and 2) cGBD478A: 5`-CT TTC AAC CGC AAC GCT GAA CAG TTT CTG GA-3` (amino acid sequence: FNRNAEQFL). The altered bases and amino acid residues are underlined. Mutagenic oligonucleotides (100 pmol) were phosphorylated with T4 polynucleotide kinase(22) , and 5 pmol of phosphorylated oligonucleotide were annealed to 0.5-1 pmol of uracil-containing single-stranded DNA in 50 mM Tris (pH 7.5), 20 mM MgCl(2), and 50 mM NaCl (final volume, 10 µl) by incubating the mixture in a 500-ml beaker filled with 65 °C H(2)O, and allowing the temperature to cool to 22 °C over 30-60 min. After cooling, 2 µl of 10 times mutagenesis buffer (100 mM Tris (pH 7.5), 50 mM MgCl(2), 10 mM ATP, 5 mM dATP, 5 mM dCTP, 5 mM dGTP, 5 mM dTTP, and 20 mM dithiothreitol), 3 units of T4 DNA polymerase and 200 units of T4 DNA ligase were added to a final volume of 20 µl. The reactions were incubated for 5 min at 4 °C, followed by incubations for 5 min at 22 °C and 90 min at 37 °C. One µl of the resulting mutagenesis reaction products was used to transform E. coli XL1-blue (dut, ung). Transformants were screened for the presence of the appropriate mutation by sequencing miniprepped DNA (23) using Sequenase® Version 2.0 according to the manufacturer's protocol (U. S. Biochemical Corp.).

In order to create plasmid constructs for expression of mutant cGB-PDEs, the WT HindIII fragment of cGB-8 (bp 712-1546) was removed from the pCDNA-cGB-PDE(WT) plasmid, and replaced with HindIII fragments of cGB-8 containing the desired mutations, creating the mutant expression constructs pCDNA-cGB-PDE(D289A) and pCDNA-cGB-PDE(D478A). E. coli XL1-blue cells were used for all transformations, and DNA was purified from large scale plasmid preparations using QIAGEN Plasmid Mega kits according to the manufacturer's protocol (QIAGEN Inc.). All DNA segments subject to mutagenesis reactions (bp 640-1640 of the cGB-8 sequence in pCDNA- cGB-PDE(D289A) and bp 625-1625 in pCDNA-cGB-PDE(D478A) were sequenced (23) to ensure the presence of the desired mutation, the absence of any spurious mutations, and proper in-frame subcloning at the HindIII sites.

Transient Transfection and Expression in COS-7 Cells

COS-7 cells were cultured in Dulbecco's modified Eagle's medium containing 10% fetal bovine serum, 50 µg/ml penicillin, and 50 µg/ml streptomycin at 37 °C in a humidified CO(2) atmosphere. Transfections were performed using the DEAE-dextran method as described elsewhere (13) with 10 µg of either pCDNA-cGB-PDE(WT), pCDNA-cGB-PDE(D289A), or pCDNA-cGB-PDE(D478A) plasmid DNA or no DNA (mock transfection) as indicated. 48 h after transfection, cells were suspended in 0.45 ml of ice-cold homogenization buffer (40 mM Tris-HCl (pH 7.5), 15 mM benzamidine, 15 mM beta-mercaptoethanol, 0.7 µg/ml pepstatin A, 0.5 µg/ml leupeptin, and 5 mM EDTA) per 100-mm dish of cells and disrupted using a Dounce homogenizer prechilled in an ice bath. The resulting whole cell extracts were stored at -70 °C for 1 week to several months. Immediately prior to assaying cGMP-phosphodiesterase or cGMP binding activity, or performing immunoprecipitation and Western blot, the whole cell extract was thawed and subjected to centrifugation at 10,000 times g for 15 min at 4 °C, and the resulting supernatant was used in these procedures. Phosphodiesterase activity and cGMP binding activity were stable through this single round of freezing and thawing, with 80-100% of the activities of freshly harvested whole-cell extract remaining in the supernatant after centrifugation.

cGMP Saturation Binding

The cGMP-binding assay, modified from the previously described assay(10, 13) , was conducted in a total volume of 20 µl; 10 µl of the soluble extract of transfected COS-7 cells were combined with 10 µl of a binding mixture such that the final concentration of the components of the reaction were 5 µM cAMP, 10 µM 8-bromo-cGMP, 0.2 mM 3-isobutyl-1-methylxanthine, 1 mM EDTA, 25 mM beta-mercaptoethanol, 5 mM Na(2)HPO(4), 5 mM NaH(2)PO(4), and varying concentrations of [P]cGMP (10-10cpm/µmol) as indicated. For assays of native enzyme, purified bovine lung cGB-PDE was added to the soluble fraction of extract from mock transfected COS-7 cells to a final concentration of approximately 0.5-1.5 µg of cGB-PDE/mg of total protein (the approximate concentration of recombinant cGB-PDE in the cytosolic extracts of transfected cells, based on enzyme specific activity), and then combined with the binding mixture. This procedure was utilized in an attempt to simulate the conditions used for assaying expressed WT and mutant cGB-PDEs. The cAMP and 8-bromo-cGMP were added to block [P]cGMP binding attributable to either cAMP-dependent protein kinase or cGMP-dependent protein kinase, respectively. The concentration of [P]cGMP added to the reaction was determined by measurement of absorbence at 252 nm. The reactions were initiated by the addition of the COS-7 cell extract containing the cGB-PDE, followed by incubation for 3 h at 22 °C. This incubation period was sufficient for native, WT, and mutant cGB-PDE cGMP binding (at the lowest concentration measured (0.5 µM)) to attain equilibrium. Following this incubation, 1 ml of ice-cold KPE buffer (10 mM potassium phosphate (pH 6.8) and 1 mM EDTA) was added to the reaction tube, and the contents were immediately applied to a premoistened Millipore HAWP filter (pore size, 0.45 µm). The tube was then washed with 1 ml of additional cold KPE buffer, and this wash was applied to the filter, which was then rinsed 2 times with 6 ml each of cold KPE buffer, dried, placed in 4 ml of nonaqueous scintillation mixture, and counted on a Beckman LS 1801 scintillation counter. The data were corrected by subtraction of nonspecific binding, which was defined as either the [P]cGMP bound per mg of total protein in the soluble fraction of extract from mock-transfected COS-7 cells, or the [P]cGMP bound per mg of total protein in the presence of 100-fold excess of unlabeled cGMP. Identical results were obtained with each method. No significant breakdown of the [P]cGMP occurred during the incubations. The cGMP-binding assay is linear with respect to volume.

cGMP Dissociation

Exchange of unlabeled cGMP for [P]cGMP was used to measure rates of cGMP dissociation (k) from native, WT, and mutant cGB-PDEs, and therefore, binding sites remained occupied throughout the assay. The same binding mixture was used for the cGMP-dissociation and cGMP-saturation studies. 225 µl of soluble extract of transfected COS-7 cells were combined with 225 µl of binding mixture. The final concentration of [P]cGMP in the reaction tubes was 6 µM for assays of extracts containing native and WT cGB-PDE and 4 µM for assays of D478A mutant cGB-PDE. At these [P]cGMP concentrations, the cGMP-binding sites were saturated under our assay conditions (see Fig. 3). Native cGB-PDE was diluted into soluble extracts of mock-transfected COS-7 cells (as described under ``cGMP Saturation Binding''), prior to addition of the binding mixture. Assays of extract containing D289A mutant cGB-PDE were conducted at subsaturating [P]cGMP (8 µM) since full occupation of the cGMP-binding sites of this mutant could not be achieved under the assay conditions used in this study (see Fig. 3). Reactions were incubated for 3 h at 22 °C to allow binding equilibrium to be established and were then transferred to a 4 °C water bath. Following a 10-min incubation at 4 °C, which was sufficient for temperature equilibration, five 20-µl aliquots were withdrawn for Millipore filtration (as described under ``cGMP Saturation Binding''), in order to assess initial bound [P]cGMP (B(o)). To initiate the exchange, 100-fold excess unlabeled cGMP was added to the reaction, and equivalent aliquots were withdrawn for Millipore filtration at the indicated time points (B(t)). Immediately prior to addition of unlabeled cGMP, 90 µl of the reaction mixture were transferred to a new tube prechilled to 4 °C. 20-µl aliquots from this new tube were removed and filtered at 2-h intervals for WT, native, and D478A mutant cGB-PDEs, and at 30-min intervals for D289A mutant cGB-PDE, in order to verify the stability of the [P]cGMP binding during the course of the dissociation assay. This [P]cGMP binding activity, in the absence of unlabeled cGMP, was stable throughout the duration of the cGMP-dissociation assays. Data were corrected by subtraction of nonspecific binding.


Figure 3: Effect of [P]cGMP concentration on binding to native, WT, and mutant cGB-PDEs. Soluble extracts of COS-7 cells transfected with cDNAs encoding WT or D289A or D478A mutant cGB-PDE were prepared and cGMP-binding assays were performed using increasing concentrations of [P]cGMP as indicated. Purified bovine lung (native) cGB-PDE was added to soluble extract from mock transfected COS-7 cells prior to initiation of the assay. (See ``Experimental Procedures'' for details.) For Panels A and B, results are given as percent of maximum ([P]cGMP binding/mg of total protein). All results shown are the averages of at least three separate determinations. Error bars represent standard deviations. All assays were performed in duplicate. Panel A, [P]cGMP binding to extract containing WT cGB-PDE (bullet) and native cGB-PDE (circle) was compared. Maximum binding was achieved at 8 µM [P]cGMP. Panel B, [P]cGMP binding to extract containing WT (bullet), D289A mutant (), or D478A mutant () cGB-PDE was compared. Maximum binding was achieved at 8 µM [P]cGMP for WT cGB-PDE and 4 µM [P]cGMP for D478A mutant cGB-PDE. Since the concentration of [P]cGMP required for maximum binding to the D289A mutant cGB-PDE could not be determined (see ``Results''), the [P]cGMP binding observed at 24 µM was arbitrarily designated as maximum binding. Panel C, Scatchard analysis of [P]cGMP binding to WT (bullet) and D478A mutant () cGB-PDE. Units for ``bound'' and ``free'' are picomoles of [P]cGMP/mg of total protein and µM [P]cGMP, respectively. Data shown are representative of at least three separate experiments.



Rates of cGMP dissociation from theoretical slow and fast sites on native and WT cGB-PDE were calculated according to the 2-site model of Doskeland and Corbin, which was utilized in characterizing the two cyclic nucleotide-binding sites in cAMP-dependent protein kinase (24) and cGMP-dependent protein kinase(25) . Linear regression of the data points from 3 to 7 h was used to estimate the slope of the slow component of the dissociation curves, which represented the rate of cGMP dissociation (k) from the theoretical slow (higher affinity) cGMP-binding site. The line predicted by this analysis (y = -0.256, x - 0.733) had a y intercept of -0.733, which was consistent with the slow component representing the dissociation of 48% of the total [P]cGMP bound.

The rate of [P]cGMP dissociation from the theoretical fast (lower affinity) cGMP-binding site was calculated as follows. First, the contribution of cGMP binding to the slow site during time points 0-3 h was estimated. The natural log of the fraction of [P]cGMP remaining bound to the slow site at each time point (t), (ln(B(t)/B(o))), was calculated by inserting each early time point (x) into the equation of the line representing dissociation from the slow site (see above). The antilog of ln(B(t)/B(o)) represented the fraction of [P]cGMP remaining bound to the slow site at each time point. Second, the contribution of cGMP binding to the slow site, (B(t)/B(o)), was subtracted from total measured (B(t)/B(o)) at each time point from 0-3 h in order to calculate the fraction of [P]cGMP remaining bound to the fast site: ((B(t)/B(o)) - (B(t)/B(o)) = (B(t)/B(o))). Third, the natural log of (B(t)/B(o)) for each time point was calculated, and linear regression of the corrected fast site data was performed in order to estimate the slope of the fast component of the cGMP-dissociation curves. This slope represented the rate of cGMP dissociation (k) from the theoretical fast site. The line predicted by this analysis (y = -1.00x - 0.665) had a y intercept of -0.665, which was consistent with the fast component representing the dissociation of 50% of the total [P]cGMP bound.

cGMP-Phosphodiesterase Activity and Protein Concentration

Phosphodiesterase activity using [^3H]cGMP as the substrate was measured as described previously(13) . In all studies, [^3H]cGMP hydrolysis was measured at more than one time point in order to be certain that these measurements represented linear initial rates, and less than 15% of the total [^3H]cGMP was hydrolyzed during the reaction. The phosphodiesterase activity assay was linear with respect to volume. Protein concentrations of the soluble fraction of COS-7 cell extracts were determined by the method of Bradford (26) using bovine serum albumin as the standard.

Production of Anti-cGB-PDE Polyclonal Antisera

Aliquots containing 50-100 µg of cGB-PDE purified from bovine lung were subjected to SDS-polyacrylamide gel electrophoresis (10% acrylamide) in order to ensure separation of cGB-PDE from potentially antigenic contaminants. The cGB-PDE band was identified by Coomassie staining and excised. The gel slice was combined with Freund's complete adjuvant and injected subcutaneously into each of two laboratory rabbits at 3-week intervals. Approximately 15 ml of serum per rabbit were collected 1 week following each injection. Animal care, injection, and serum collection were performed by Bethyl Laboratories, Inc. (Montgomery, TX).

Immunoprecipitation and Western Blot Analysis

250-500 µl of soluble extracts of transfected COS-7 cells were combined with 10 µl of anti-cGB-PDE antisera and 100 µl of a 10% slurry of protein A-Sepharose CL-4B beads in 50 mM Hepes (pH 7.2) and 0.05% sodium azide. For analysis of native enzyme, purified cGB-PDE was diluted into soluble extract of mock-transfected COS-7 cells (as described under ``cGMP Saturation Binding'') or into PBS buffer (8.1 mM sodium phosphate (pH 7.5), 1.1 mM potassium phosphate, 2.7 mM KCl, and 137 mM NaCl) prior to addition of protein A-Sepharose. The mixtures were incubated overnight at 4 °C while shaking. Immunoprecipitates were collected by centrifugation (2 min, 10,000 times g, 4 °C), washed three times in 500 µl of 50 mM Tris-HCl (pH 7.5), 150 mM NaCl, 0.1% Triton X-100, and 10% glycerol, and then suspended in 60 µl of sample buffer (25 mM Tris-HCl (pH 6.8), 10% glycerol, 2% SDS, 0.1 M dithiothreitol, and 0.01% bromphenol blue) and boiled for 5 min. Samples were then subjected to centrifugation (2 min, 10,000 times g, 4 °C), and the resulting supernatant was loaded onto a 10% SDS-polyacrylamide gel. Following electrophoresis, proteins were transferred to nitrocellulose membrane using a Milliblot-SDE system semi-dry blotter according to the manufacturer's protocol (Millipore). The membrane was blocked in TBS-T (50 mM Tris (pH 7.5), 150 mM NaCl and 0.05% Tween 20) plus 5% nonfat dry milk for 1 h at 22 °C, followed by incubation in a 1500-fold dilution of anti-cGB-PDE antisera in TBS-T for 2 h at 22 °C. Membranes were washed at least twice for 5 min in TBS-T, followed by incubation in a 20,000-fold dilution of protein A-conjugated to horseradish peroxidase in TBS-T for 1 h at 22 °C. Membranes were washed four times for 10 min in TBS-T. Western blots were developed using the ECL kit (Amersham Corp.) according to the manufacturer's protocol.


MATERIALS

[P]cGMP (1500-3000 Ci/mmol) was purchased from ICN. [^3H]cGMP (19.2 Ci/mmol) was purchased from Amersham Corp. pCDNA1/Amp expression vector, E. coli CJ236, and helper phage M13K07 were purchased from Invitrogen. Horseradish peroxidase-protein A was purchased from Zymed. 3-Isobutyl-1-methylxanthine, dimethyl sulfoxide, benzamidine, pepstatin A, leupeptin, Crotalus atrox snake venom, cAMP, cGMP, 8-bromo-cGMP, and protein A-Sepharose CL 4B were purchased from Sigma. Klenow, T4 DNA polymerase, and T4 DNA ligase were purchased from Promega. E. coli XL1-blue and pBluescript SK(-) vector were purchased from Stratagene Cloning Systems. T4 polynucleotide kinase, 1-kilobase pair DNA ladder, Dulbecco's modified Eagle's medium, and fetal bovine serum were purchased from Life Technologies, Inc. Bradford reagent, Coomassie Brilliant Blue stain, and protein molecular weight markers were purchased from Bio-Rad. DEAE-dextran was purchased from Pharmacia Biotech Inc., Sequenase Version 2.0 from U. S. Biochemical Corp., Nitropure nitrocellulose membrane from Micron Separations Inc., Nu Serum from Collaborative Biomedical Products, DNA purification columns from QIAGEN Inc., Ready Safe aqueous scintillation mixture from Beckman Industries Inc., and Ecoscint-O nonaqueous scintillation mixture from National Diagnostics. All restriction enzymes were purchased from Promega and Life Technologies, Inc. COS-7 cells were obtained from the American Type Culture Collection.


RESULTS

WT and Mutant cGB-PDE Domain Structure and Sequence

The proposed domain structure of the cGB-PDE monomer is shown in Fig. 1A. The sequences of the conserved internally homologous repeats within the cGMP-binding region of cGB-PDE (labeled a and b in Fig. 1A) are shown in Fig. 1B. Segment c (see Fig. 1A), which occurs in the catalytic domain, shares limited homology with repeats a and b (see Fig. 1B) and will be addressed under ``Discussion.'' Oligonucleotide-directed mutagenesis was used to replace either Asp-289 with Ala (D289A mutant) or Asp-478 with Ala (D478A mutant). Asp-289 and Asp-478 are located at homologous positions within the conserved sequences of repeats a and b, respectively, of cGB-PDE, and these positions are conserved within a and b repeats of all known cGMP-binding PDEs (Fig. 1B).

Expression of WT and Mutant cGB-PDEs in COS-7 Cells

In order to express recombinant cGB-PDEs in COS-7 cells, cDNAs encoding WT and mutant cGB-PDEs were subcloned into the pCDNA1/Amp mammalian expression vector (see ``Experimental Procedures''). Previous studies showed that recombinant WT cGB-PDE, as expressed in COS-7 cells, displayed biochemical properties characteristic of native bovine lung cGB-PDE including cGMP-specific phosphodiesterase activity that was inhibited by Zaprinast and dipyridamole(10, 13) , cGMP-specific binding activity that was enhanced by 3-isobutyl-1-methylxanthine(10, 13) , and a dimeric quaternary structure (not shown). (^4)Expression of WT and mutant cGB-PDEs was monitored by measurement of cGMP-specific phosphodiesterase activity per total protein in cytosolic extracts of transfected COS-7 cells (Fig. 2). Extracts of cells transfected with cDNA encoding WT cGB-PDE (construct pCDNA-cGB-PDE(WT) described under ``Experimental Procedures'') exhibited approximately 30-fold higher cGMP phosphodiesterase activity than did extracts of mock-transfected cells. Extracts of cells transfected with cDNAs encoding the D289A (construct pCDNA- cGB-PDE(D289A)) and D478A (construct pCDNA-cGB-PDE(D478A)) mutant cGB-PDEs consistently (in 10 separate transfections) exhibited 40 and 50%, respectively, of the cGMP-phosphodiesterase activity per total protein measured in extracts of cells transfected with cDNA encoding WT cGB-PDE (Fig. 2).


Figure 2: Expression of WT and mutant cGB-PDEs in COS-7 cells. COS-7 cells were subjected to mock transfection or were transfected with cDNAs encoding WT or D289A or D478A mutant cGB-PDEs. After 48 h, the cells were harvested, and the soluble extracts were assayed for phosphodiesterase activity using 20 µM [^3H]cGMP as the substrate. (See ``Experimental Procedures '' for details.) Picomoles of cGMP hydrolyzed/(min)(mg total protein) was calculated for each extract, and results are given as the percentage of phosphodiesterase activity measured in extracts of cells transfected with cDNA encoding WT cGB-PDE during the same transfection. The results shown are the averages of five separate transfections. All assays were performed in duplicate. Error bars represent S.D.



cGMP Saturation of WT and Mutant cGB-PDEs

cGMP binding to cytosolic extracts of COS-7 cells transfected with cDNA encoding WT, D289A mutant, or D478A mutant cGB-PDEs was measured at various concentrations of [P]cGMP (Fig. 3). As a control, [P]cGMP binding to native cGB-PDE that was diluted into cytosolic extract from mock-transfected COS-7 cells was also measured (Fig. 3A) (as described under ``Experimental Procedures''). Under these conditions, native and WT cGB-PDE displayed identical patterns of cGMP saturation, with 1.8 µM cGMP required for half-maximal saturation (Fig. 3A). This value is approximately 10-fold higher than the value previously reported for purified bovine lung cGB-PDE (0.2 µM cGMP required for half-maximal binding)(10) . This difference in apparent affinity is most likely due to the fact that histone VIII-s, which enhances cGMP binding to cGB-PDE via an unknown mechanism(27) , had to be omitted from the assays described in this report, due to precipitation in the presence of COS-7 cell extract. Transformation of the WT (Fig. 3C) and native enzyme (data not shown) cGMP-saturation data produced an upward concave Scatchard plot, which was consistent with the presence of multiple cGMP-binding sites.

D478A mutant cGB-PDE displayed slightly higher affinity for cGMP as compared to WT and native enzyme (K(d) approx 0.5 µM) (Fig. 3B). A Scatchard plot of the data was consistent with the presence of a single class of cGMP-binding site (Fig. 3C). The D289A mutant cGB-PDE displayed significantly decreased affinity for cGMP (K(d) > 10 µM) (Fig. 3B). Binding to the D289A mutant cGB-PDE was not saturated at up to 24 µM [P]cGMP, and measurements of [P]cGMP binding at concentrations greater than 24 µM were not reliable due to high nonspecific background binding and low specific radioactivity of cGMP. Therefore, the range of [P]cGMP concentrations at which binding to the D289A mutant cGB-PDE could be measured was insufficient for K(d) determinations and/or Scatchard analyses.

cGMP Dissociation from WT and Mutant cGB-PDEs

The patterns of [P]cGMP dissociation from native, WT, and mutant cGB-PDEs were compared. The curvilinear patterns of [P]cGMP dissociation from recombinant WT and native cGB-PDE were identical with t approx 1.2 h at 4 °C (Fig. 4A). This curvilinearity suggested the presence of multiple cGMP-binding sites on WT and native cGB-PDE. Assuming that the curvilinearity of the [P]cGMP dissociation was the result of the presence of two distinct cGMP-binding sites, each exhibiting a distinct dissociation rate (``fast site'' and ``slow site''), the individual rates of dissociation from the two theoretical sites were calculated according to a two-site model as explained under ``Experimental Procedures'' (Table 1). These methods and calculations have been used successfully for studies of the two cyclic nucleotide-binding sites of cAMP-dependent (24) and cGMP-dependent (25) protein kinase. The predicted patterns of cGMP dissociation from the theoretical sites of the cGB-PDE are illustrated in Fig. 4A.


Figure 4: Dissociation of [P]cGMP from native, WT, and mutant cGB-PDEs. Soluble extracts of COS-7 cells transfected with cDNAs encoding WT or D478A mutant cGB-PDEs or extracts of mock-transfected cells containing native cGB-PDE were incubated under saturating conditions for [P]cGMP binding. Since the concentration of [P]cGMP required to saturate the cGMP-binding sites in extracts of cells containing the D289A mutant cGB-PDE could not be determined, these extracts were incubated with subsaturating concentrations of [P]cGMP (8 µM) (see ``Results'' for the explanation). Binding reactions were equilibrated to 0 °C and 100-fold excess unlabeled cGMP was added to initiate exchange. Aliquots were removed at the indicated time points in order to measure [P]cGMP binding (described under ``Experimental Procedures''). Data are plotted as ln(B/B) versus time where B/B represents the ratio of [P]cGMP binding at any time t to the P binding measured at time = 0 (before addition of unlabeled cGMP). Each data point represents the natural log of the average of at least three separate determinations of B/B. Parabolic regression of the data was performed to generate the binding curves. Panel A, comparison of [P]cGMP dissociation from extracts containing WT (bullet) and native (circle) cGB-PDE. The range of ln(B/B) was <0.25 for all time points, with the exception of time points 5 h (native) and 2 and 6 h (WT) for which the range was 0.5. Calculation of the rates of the slow and fast components of cGMP dissociation is explained in the text under ``Experimental Procedures.'' In order to facilitate visual comparison of the rates of cGMP dissociation from theoretical fast and slow sites of WT/native cGB-PDE and mutant cGB-PDEs (Panel B), the plots of cGMP dissociation from the theoretical sites have been corrected such that B/B at time = 0.0 min is equal to 1.0, and ln(B/B) = 0.0. The true values for ln(B/B) at time = 0.0 min for the slow and fast component are -0.733 and -0.655, respectively (explained under ``Experimental Procedures''). Thus, at time = 0.0, 48% of the total bound [P]cGMP is bound to the theoretical slow site, and 50% is bound to the theoretical fast site. Panel B, comparison of [P]cGMP dissociation from extracts containing WT (bullet), D289A mutant (), and D478A () mutant cGB-PDEs. For D478A (), the range of ln(B/B) was <0.20 for all time points, with the exception of time points 4, 5.5, and 7 h for which the ranges were 0.6, 0.4, and 0.5, respectively. For the D289A () the range of ln(B/B) was <0.4 for all time points.





[P]cGMP dissociated from the D478A mutant cGB-PDE at a single rate of k = 0.29 h (Fig. 4B), which was similar to the calculated rate of dissociation of cGMP from the theoretical slow site of WT and native cGB-PDEs (Table 1), whereas [P]cGMP dissociated from the D289A mutant cGB-PDE at a single rate of k = 1.24 h, which was similar to the calculated rate of cGMP dissociation from the theoretical fast site of WT and native cGB-PDE (Table 1). Similar results were obtained when [P]cGMP dissociation from WT and mutant cGB-PDEs was measured at 22 °C (not shown); a curvilinear pattern of cGMP dissociation from WT was observed, whereas dissociation from the D478A and D289A mutant cGB-PDEs occurred at single rates which were similar to the calculated rates of cGMP dissociation from the theoretical slow and fast sites, respectively, of WT cGB-PDE.

Comparison of Catalytic Activity of WT and Mutant cGB-PDEs

Since extracts of COS-7 cells containing D289A mutant or D478A mutant cGB-PDEs consistently exhibited lower levels of cGMP-phosphodiesterase activity than did extracts containing WT cGB-PDE (Fig. 2), the possibility that the D289A and D478A mutations had affected the catalytic activity of cGB-PDE was investigated. In order to estimate if either mutation might have a measurable effect on the K(m) of the enzyme, the ratio of cGMP-phosphodiesterase activity at 50 µM substrate concentration to the activity at 0.5 µM substrate concentration in extracts containing native, WT, and mutant cGB-PDEs was compared. The K(m) of cGMP hydrolysis by purified bovine lung cGB-PDE has been reported to be 5 µM cGMP (10) . At 50 µM cGMP, the rate of hydrolysis would be predicted to be 90% of V(max) based on simple Michaelis-Menten enzyme kinetics, and the rate of hydrolysis at 0.5 µM cGMP would be predicted to be 9% of V(max). Therefore, the ratio of the catalytic activity of cGB-PDE at 50 µM cGMP to the catalytic activity at 0.5 µM cGMP would be predicted to be 10, based on a reported K(m) of 5 µM(10) . The ratios determined for native, WT, and mutant cGB-PDEs ranged from 12 to 16 (native = 11.4, WT = 16.1, D289A mutant = 15.9, and D478A mutant = 11.9) (average of two separate determinations). These results suggested that, under the assay conditions described in this report, native, WT, and mutant cGB-PDEs each displayed cGMP-hydrolytic activity with a K(m) that was similar, if not identical, to the value previously reported for native enzyme. Therefore, neither the D289A nor the D478A mutation markedly affected the affinity of the catalytic site for cGMP.

Immunoblot Analysis of WT and Mutant cGB-PDEs

Immunoblot analysis was performed to assess the sizes of native, WT, and mutant cGB-PDEs (Fig. 5) (see ``Experimental Procedures''). The anti-cGB-PDE antisera recognized two prominent protein species (140 and 400 kDa) other than cGB-PDE that were present in equivalent amounts in extract of mock-transfected COS-7 cells (lane 6) and in extracts of cells transfected with cDNAs encoding WT and mutant cGB-PDEs (lanes 3-5). The native cGB-PDE appeared as a single 98-kDa band when the protein was diluted into either soluble extract of mock-transfected COS-7 cells (lane 2) or PBS buffer prior to immunoprecipitation (lane 7). The WT (lane 3) and mutant cGB-PDEs (lanes 4 and 5) appeared as identical 99/90-kDa doublets. The densities of each of the bands appeared to be similar in extracts of cells transfected with cDNAs encoding WT, D289A mutant, and D478A mutant cGB-PDEs, suggesting that WT and mutant cGB-PDEs were expressed at similar levels per mg of total protein, with the relative proportions of the high and low molecular mass species also being similar. Therefore, the observed differences in the cGMP-binding properties could not be attributed to a difference in the sizes of WT and mutant cGB-PDEs. The higher molecular mass protein band of this doublet migrated more slowly on SDS-polyacrylamide gel electrophoresis than did the native cGB-PDE protein band (compare lanes 2 and 3). This difference in the size of the native and recombinant cGB-PDE was probably due to the addition of 10 amino acid residues on the carboxyl-terminal end of the recombinant cGB-PDE.^3


Figure 5: Immunoblot analysis of native, WT, and mutant cGB-PDEs. Samples (625 µg of total protein) of soluble extract from COS-7 cells were analyzed by immunoprecipitation and Western blot detection as described under ``Experimental Procedures.'' COS-7 cells were transfected with cDNA encoding WT cGB-PDE (lane 3), D289A mutant cGB-PDE (lane 4), D478A mutant cGB-PDE (lane 5), or no DNA (mock transfection) (lane 6). Lanes 2 and 7 show approximately 700 ng of native cGB-PDE diluted into soluble extract of mock-transfected COS-7 cells (625 µg of total protein) and 250 µl of PBS buffer, respectively. Lane 1 shows extract from COS-7 cells transfected with WT cGB-PDE which was subjected to immunoprecipitation with preimmune sera in the place of anti-cGB-PDE antisera as a control. All extracts used in this experiment were from a single transfection experiment. This experiment has been repeated with four different sets of extracts (from four separate transfections), and the same results were obtained with each experiment.



The lower molecular mass species of the 99/90-kDa doublet may have been generated by proteolysis of full-length cGB-PDE. However, increasing the number and concentration of protease inhibitors in the buffer in which the COS-7 cells were homogenized did not affect the appearance of the 90-kDa band, nor did inclusion of 0.01% SDS in the homogenization buffer (not shown). It is also possible that the 90-kDa species was produced as a result of initiation of translation of the cGB-PDE mRNA at an alternative AUG start site which is 126 bp downstream from the AUG identified as the start site of translation of the 99-kDa cGB-PDE (nucleotide position 99-101)(13) .

It is unlikely that the presence of the 90-kDa species affected the cGMP-binding properties of WT and mutant cGB-PDEs. The loss of 10 kDa on either the amino-terminal or carboxyl-terminal end of cGB-PDE would leave the cGMP-binding region intact (see Fig. 1). Previous studies showed that the curvilinear pattern of [^3H]cGMP dissociation from a 30-45-kDa fragment of cGB-PDE containing the cGMP-binding region was identical to the dissociation pattern observed with intact cGB-PDE(28) , suggesting that the cGMP-dissociation pattern was an intrinsic property of the cGMP-binding region, and that it was not altered by the loss of outlying segments of cGB-PDE. The data presented in this report showed that WT and native cGB-PDEs displayed identical cGMP-saturation and cGMP-dissociation kinetics ( Fig. 3and 4) despite the fact that the native cGB-PDE appeared as a single band on Western blot (Fig. 5). As a control, native cGB-PDE, diluted into soluble extract of mock-transfected COS-7 cells, was incubated under the exact conditions used in the cGMP-saturation or cGMP-dissociation assay, followed by immunoprecipitation and Western blot detection. The native cGB-PDE still appeared as a single band in both cases (data not shown), removing the possibility that native cGB-PDE was degraded into a 99/90-kDa doublet during the course of the cGMP-binding assay. Therefore, the presence of two species (99/90 kDa) in the WT cGB-PDE was not responsible for producing cGMP-saturation and dissociation patterns consistent with the presence of two distinct cGMP-binding sites, as the identical patterns were obtained when measuring cGMP binding to native cGB-PDE, which remained as a single species throughout these assays.


DISCUSSION

Two internally homologous repeats of sequence (a and b) are conserved within the cGMP-binding region of all cGMP-binding PDEs, and have been proposed to form two distinct cGMP-binding sites in these PDEs(13) . The studies described in this report were designed to test this hypothesis using site-directed mutagenesis of bovine lung cGB-PDE, and the results of the cGMP-saturation and cGMP-dissociation analyses of native, WT, and mutant cGB-PDEs strongly support this hypothesis. WT and native cGB-PDE displayed a higher cGMP-binding affinity than that of the D289A mutant cGB-PDE and a lower cGMP-binding affinity than that of the D478A mutant. Scatchard analysis of cGMP binding to native and WT cGB-PDE produced an upward concave plot which is consistent with the presence of two classes of cGMP-binding sites, whereas Scatchard analysis of the D478A mutant cGB-PDE produced a linear plot, which suggests the presence of a single class of cGMP-binding site. The observed curvilinearity of cGMP dissociation from native and WT cGB-PDE is consistent with the presence of two distinct cGMP-binding sites from which cGMP dissociates at two distinct rates, and the single rates of cGMP dissociation from the D289A and D478A mutant cGB-PDEs were very similar to those calculated for the theoretical fast and slow sites, respectively, of WT and native cGB-PDE (Table 1).

These findings provide compelling evidence for a model of cGB-PDE containing a higher affinity (slow) cGMP-binding site at repeat a and a lower affinity (fast) site at repeat b, with the Asp residue, which is invariant among all repeats a and b of all cGMP-binding PDEs, being crucial to the interaction of cGMP with each site. According to this model, replacement of the Asp residue in site a with Ala (as in the D289A mutant cGB-PDE) diminishes or ablates binding to this site, thereby producing a cGB-PDE which binds to cGMP primarily at lower affinity site b. Therefore, this mutant displays lower cGMP-binding affinity than does WT or native cGB-PDE, and dissociation of cGMP from this mutant occurs at a single rate, which is similar to the predicted rate of dissociation from the fast site of WT and native cGB-PDE. Conversely, the model predicts that the D478A mutant cGB-PDE binds to cGMP primarily at higher affinity site a. Therefore, this mutant displays higher affinity for cGMP binding than does WT or native cGB-PDE, and dissociation of cGMP from this mutant occurs at a single rate which is similar to the predicted rate of dissociation from the slow site of WT and native cGB-PDE. It seems unlikely that the cGMP saturation binding and dissociation kinetics of each of the mutant cGB-PDEs would coincidentally resemble the predicted saturation binding and dissociation kinetics of the respective theoretical sites in WT and native cGB-PDE.

Although the current results provide strong evidence for the existence of two distinct cGMP-binding sites at repeats a and b on cGB-PDE, it is not yet known if these binding sites are formed by the interaction of two identical repeats residing on separate subunits of the homodimer, resulting in a stoichiometry of 2 mol of cGMP per mol of homodimer, or if each repeat on each individual subunit forms a separate binding site, resulting in a stoichiometry of 4 mol of cGMP per mol of homodimer. The stoichiometry of cGMP binding to purified bovine lung cGB-PDE has been reported to be 1.9 mol of cGMP per mol of homodimer(10) , and the reported stoichiometry of cGMP binding to any of the cGMP-binding PDEs has not exceeded 2 mol of cGMP per mol of holoenzyme(9, 18, 21) . Limited proteolysis studies have shown that the dimerization domain of cGB-PDE (10) and cGS-PDE (4) is within or near the cGMP-binding region of the enzyme. Such an arrangement would allow for the formation of cGMP-binding sites through the interaction of two identical repeats residing on separate subunits.

The invariant Asp present in repeats a and b is the first amino acid residue to be identified as a critical component of the cGMP-binding sites of the cGMP-binding PDEs. A general conformational deterioration due to mutation of the Asp seems unlikely, since cGMP binding to the unmodified site is not affected by mutation of either Asp. The structural features of the binding sites in two other families of guanine nucleotide-binding proteins, the E. coli catabolite activator protein (CAP) family (CAP, cAMP-dependent protein kinase, cGMP-dependent protein kinase, and the cyclic nucleotide-gated channels), and the GTP-binding protein family, have now been well characterized, and may serve as useful models in the analysis of the role of individual amino acid residues of the cGMP-binding sites of the cGMP-binding PDEs. In the cyclic nucleotide-binding domains of all of the members of the CAP family, an invariant Glu forms a hydrogen bond with the 2`OH of the nucleotide(19) . Although the cGMP-binding region of the cGMP-binding PDEs shows no sequence homology to the CAP family, it is possible that this invariant Asp may play a similar role to that known for the Glu. In support of this hypothesis, cyclic nucleotide analog studies of cGB-PDE suggest the existence of a hydrogen bond between the allosteric cGMP-binding sites and the 1-, C^6-ketone-, and 2`OH-positions of the cGMP molecule(29) . Another possible role of the Asp could be similar to that played by an invariant Asp in GTP-binding proteins(30) , which forms hydrogen bonds with both the 1-NH and the 2-NH(2) groups of guanosine in GTP. It is of interest that the conserved sequence of the guanosine-binding component of G proteins is Asn-Lys-X-Asp, which resembles the Asn-Lys-(X)(n)-Asp of the proposed cGMP-binding sites of cGMP-binding PDEs (Fig. 1), suggesting that the cGMP-binding domain of the cGMP-binding PDEs and the GTP-binding domain of G proteins may interact with the guanosine moiety through a common mechanism. If so, then the conserved Asn of cGB-PDE could form a hydrogen bond with the 7-N of guanine as is the case for G proteins(30) .

Since the conserved catalytic domain of PDEs and the conserved cGMP-binding region of the cGMP-binding PDEs share a common function, i.e. the ability to bind cyclic nucleotides, the catalytic and allosteric binding sites may have evolved by duplication of an ancestral cyclic nucleotide-binding domain(31) . In support of this proposal, a segment of sequence located in the carboxyl-terminal portion of the conserved catalytic domain of PDEs (labeled c in Fig. 1B) shares limited homology with repeats a and b of all cGMP-binding PDEs. All published amino acid sequences of mammalian PDEs (as of the listing compiled in April 1994 for the American Society of Pharmacology and Experimental Therapeutics, Colloquium and Symposium on Multiple PDEs(32) ) share the common sequence K/R(X)F(X)(4)D(X)E within segment c. (^5)Alignment of region c with repeats a and b of the cGMP-binding PDEs reveals that the spatial arrangement of these conserved residues within region c is similar, but not identical, to that of the corresponding residues in repeats a and b (K(X)F(X)(3)DE) (Fig. 1B). Segment c may represent a cyclic nucleotide-binding component of the catalytic domain. If so, it could act in concert with another segment(s), such as the zinc-binding domain (33) , to elicit catalysis.

The functional role of cGMP binding to sites a and b of cGB-PDE is still unclear. Comparison of the cGMP-phosphodiesterase activities of the WT and mutant cGB-PDEs suggests that the loss of cGMP binding at site a or b does not profoundly affect the K(m) of catalysis. However, the phosphodiesterase activity measured at 20 µM cGMP was consistently lower in extracts of cells transfected with the mutant cGB-PDEs as compared to WT cGB-PDE (Fig. 2). A method for more precisely quantitating the concentration of cGB-PDE in COS-7 cell extracts is needed to determine if this small difference in the cGMP-phosphodiesterase activity is due to a decreased catalytic V(max) of the mutant cGB-PDEs as compared to WT, or to a consistently lower level of expression of mutant cGB-PDEs as compared to WT cGB-PDE.

The studies described in this report provide the first biochemical evidence for the existence of two allosteric cGMP-binding sites located at repeats a and b of a cGMP-binding PDE, and the first evidence for a role of an individual amino acid residue in interacting with the cGMP molecule at the allosteric sites in these enzymes. These findings represent an important first step in characterizing the structural elements that contribute to the function of the allosteric cGMP-binding sites in cGB-PDE and other cGMP-binding PDEs.


FOOTNOTES

*
This work was supported in part by National Institutes of Health Research Grants DK21723 (to W. K. S., D. S., and J. A. B.), HL 44948 (to W. K. S. and J. A. B.), GM 41269 (to L. M. M.-L., T. L. H., J. L. C., S. H. F., and J. D. C.), the Medical Scientist Training Program Grant GM07347 (to L. M. M.-L.), and a Drug Discovery Grant from the ICOS Corporation. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed. Tel.: 615-322-4384; Fax: 615-322-7236.

(^1)
The abbreviations used are: PDE, 3`5`-cyclic nucleotide phosphodiesterase; cGS-PDE, cGMP-stimulated phosphodiesterase; ROS-PDE, rod outer segment phosphodiesterase; cGB-PDE, cGMP-binding, cGMP-specific phosphodiesterase; bp, base pair; PBS, phosphate-buffered saline; WT, wild type; CAP, catabolite activator protein.

(^2)
The newest PDE nomenclature presented at the American Society for Pharmacology and Experimental Therapeutics Colloquium on Multiple Phosphodiesterases (April 22-23, 1994) is shown in italics(32) .

(^3)
After the completion of the studies described in this report, we recognized an error in the sequence of the cDNA encoding bovine lung cGB-PDE which is an artifact of cDNA library construction. As a result of this artifact, the recombinant protein product encoded by the cDNA is lacking 2 amino acid residues of cGB-PDE sequence on its carboxyl-terminal end, and approximately 12 residues which do not represent cGB-PDE sequence have been attached to the carboxyl terminus. This error has had no detectable effect on the biological activity of the recombinant cGB-PDE, since it displays cGMP-phosphodiesterase and cGMP binding activities characteristic of native cGB-PDE purified from bovine lung. Our laboratories are currently working on correcting this problem.

(^4)
Although the levels of recombinant cGB-PDE expressed in COS-7 cells are insufficient for studies of phosphorylation, we have shown that recombinant WT cGB-PDE, as expressed in an sf9/baculovirus system, is phosphorylated by cGMP-dependent protein kinase and by the catalytic subunit of cAMP-dependent protein kinase in a cGMP-dependent manner. For this recombinant enzyme, mutation of Asp-289 to Ala causes loss of the stimulatory effect of low concentrations of cGMP on phosphorylation by cAMP-dependent protein kinase. Slight stimulation may occur at >1 mM cGMP. In contrast, phosphorylation of WT cGB-PDE is stimulated by µM levels of cGMP.

(^5)
The published sequence of rat PDE2 appeared to be one exception to this rule (a His residue was originally thought to be present at the position corresponding to the conserved Asp)(34) . However, the sequence of segment c of rat PDE2 was recently reexamined and the residue was found to be Asp (M. Conti, personal communication).


ACKNOWLEDGEMENTS

We thank Alfreda Beasley-Leach for technical assistance, Dr. David Poon for helpful advice on mutagenesis techniques, and Drs. Peter C. Lucas and Deborah V. Horstman for expert assistance with immunoblot analysis.


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