From the Department of Pharmacology, University of Washington, Seattle, Washington 98195
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ABSTRACT |
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We report the cloning, expression, and characterization of a new family of cyclic nucleotide phosphodiesterase (PDE) that has unique kinetic and inhibitor specificities. A clone corresponding to the C terminus of this PDE was initially identified by a bioinformatic approach and used to isolate a cDNA that is likely full-length. This novel PDE, designated as MMPDE9A1, shows highest mRNA expression in kidney with lower levels in liver, lung, and brain. The mRNA size by Northern blot analysis is approximately 2.0 kilobases, and the cDNA encoding PDE9A1 is 1929 base pairs in length. The largest open reading frame predicts a protein of 534 amino acids with a molecular mass of 62,000 Da. When expressed in COS-7 cells, PDE9A1 activity was not inhibited well by either the nonselective inhibitor 3-isobutyl-1-methyl-xanthine or the new selective PDE5 inhibitor, sildenafil, but it is inhibited by the PDE1/5 inhibitor (+)-cis-5,6a,7,8,9 hyl] phenylmethyl]-5-methyl-cylopent[4,5]imidao[2,1-b]purin-49(3H)one (SCH51866) with an IC50 of 1.55 µM. This new phosphodiesterase is highly specific for cGMP. Its Km of ~0.07 µM for cGMP is the lowest yet reported for a PDE, being at least 40-170 times lower than that of PDE5 and PDE6, respectively.
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INTRODUCTION |
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The cyclic nucleotides cAMP and cGMP serve as second messengers for a wide variety of extracellular signals such as neurotransmitters, hormones, light, and odorants. The diverse cellular and behavioral responses to these second messengers are mediated by the action of cAMP and cGMP on their intracelluar targets, which include kinases, ion channels, transcriptional activators, and several isoforms of phosphodiesterases (PDEs).1 Accordingly, these responses are regulated by the rates of synthesis of cyclic nucleotides by cyclases and their degradation by PDEs to biologically inactive 5' monophosphate nucleosides.
Seven2 different gene
families of PDEs previously have been isolated based on their distinct
kinetic and substrate characteristics, inhibitor profiles, allosteric
activators and inhibitors, and amino acid sequence (1). Family 1 is
activated by Ca2+/calmodulin and hydrolyzes both cAMP and
cGMP; family 2 is stimulated by cGMP, and both cAMP and cGMP serve as
substrate; family 3 is distinguished by cAMP hydrolysis that is
inhibited by cGMP; family 4 is cAMP-specific; family 5 binds cGMP at a
noncatalytic site and specifically hydrolyzes cGMP; family 6 is the
retinal PDE that is inhibited by a subunit in the absence of
activated transducin and hydrolyzes cGMP; and family 7 is a very low
Km cAMP-specific PDE. Not only does each family of
PDE have specialized substrate and regulatory features, but each PDE
family and even members within a family also exhibit tissue-, cell-,
and subcell-specific expression patterns and therefore participate in
distinct signal transduction pathways. The precise cellular and
subcellular profile of PDE expression then will determine the cyclic
nucleotide phenotype of a cell and how it responds to first messengers.
Identifying and characterizing these functionally distinct PDEs is
therefore crucial for our understanding of the mechanisms by which
cyclic nucleotides moderate their biologic effects.
We report here the cloning, expression, and characterization of a previously unknown PDE designated as MMPDE9A1 (2). This PDE represents a new gene family because it shares less than 50% amino acid identity in the conserved catalytic domain with the other seven PDE families. A search of GenBankTM reveals that PDE9A1 has slightly higher sequence homology to a recently described Dictyostelium discoideum PDE referred to as RegA (3) rather than other mammalian PDEs. Additionally, expression of PDE9A1 in COS cells results in functional PDE activity that is unique kinetically from the other seven families in that it is cGMP-specific and has the lowest Km for cGMP reported so far for a PDE.
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MATERIALS AND METHODS |
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Data Base Searching for EST PDE Sequences-- The amino acid sequence of MMPDE1A2 was used as a query to search the data base of expressed sequence tags (4, 5). The program used was the Basic Local Alignment Search Tool (BLAST) (6) accessed from the data base search and analysis Search Launcher (7). This search resulted in many EST sequences with homology to the PDE1A2 query. Each of these EST sequences were then used as queries in a BLASTN search of GenBankTM to determine whether they represented different but known PDEs or whether the EST sequence represented a truly unknown PDE. EST clone identification number 404030 was isolated in this manner as a sequence that appeared to represent a novel PDE.
Sources of EST Clones-- Clone 404030 was ordered from the American Type Culture Collection. Clones 420451 was ordered from Genome Systems, Inc.
DNA Sequencing and Sequence Assembly-- Plasmid DNA was prepared using the SNAP kit (Invitrogen). Primers were designed using the program Amplify (freeware by William Engels, Genetics Department, University of Wisconsin, Madison, WI). Sequencing was done using ABI PRISM dye terminator cycle sequencing kit (Perkin-Elmer), and sequencing reactions were purified using Centri-sep columns (Princeton Separations). Sequences were assembled using the program Sequencher 3.0 (Gene Codes Corporation).
Primers Used-- The primers used were: Krace.S1, gcgatgggggccggctcctcaagctac; 4040AS2, gtttcaaacattgggatcaggacaaac; PDE9stp.AS2, ccacaattcatctgctctaactggtacagtc; PDE9stp.AS1, cctgggaagcctcgcttggctctgtccac; Gal4ActAS.pri, gaaattgagatggtgcacgatgcacag; AP1, ccatcctaatacgactcactatagggc AP2, actcactatagggctcgagcggc.
DNA Probe Synthesis and Northern Blotting--
DNA probes were
generated from EST clone 404030 and B-actin using Prime-It RmT random
primer labeling kit (Stratagene). [-32P]dCTP at 6000 Ci/mmol was used, and the reaction product was purified using
Centri-sep columns. Multiple tissue mRNA blot was purchased from
CLONTECH. Prehybridization, hybridization, and washing was done according to the manufacturer's guidelines.
Hybridization was done using 2 × 106 cpm/ml in a
final volume of 20 ml at 42 °C overnight.
Library Screening--
EST clone 404030 was used to screen
approximately 700,000 plaques from a mouse kidney gT10 cDNA
library (CLONTECH). Briefly, the library was
incubated with an overnight culture of c600Hfl Escherichia
coli and plated onto NZY plates with NZY plus top agarose and
incubated for 8 h. Plates were then stored overnight at 4 °C.
Nitrocellulose filters were placed on top of these plates for 3 min in
duplicate and denatured for 5 min in 1.5 M NaCl and 0.5 N NaOH followed by neutralization in 1.5 M NaCl
and 0.5 M Tris-HCl, pH 8, for 5 min again. Filters were
then rinsed in 2× SSC, air dried, and UV cross-linked (0.15 J/cm2). Filters were then prewetted in 2× SSC and then
incubated in hybridization solution (50 mM Tris-HCl, 1%
SDS, 10% dextran sulfate, 1 M NaCl) for 1 h at
65 °C. 5 × 106 cpm/ml of boiled probe was added,
and hybridization was continued overnight. Filters were then washed in
2× SSPE for 15 min twice at room temperature and then twice in 0.1×
SSPE, 0.5% SDS for 30 min at 65 °C. Filters were then exposed to
autoradiographic film overnight, and positive plaques were picked and
stored in 50 mM Tris-HCl, pH 7.5, 100 mM NaCl,
10 mM MgSO4, 2% gelatin until further rounds
of screening were done. This resulted in two independent clones, clones
2.1 and 6.1, which were identical and contained the sequence of EST
clone 404030 in addition to a polyadenine tail, representing the full
3' end of PDE9. Additionally, primers 4040AS2 and a primer against the
multiple cloning site, Gal4ActAS.pri, were used in a screen by
polymerase chain reaction of 6 × 106 colony forming
units of a rat brain pGAD10 library (CLONTECH) for
sequences 5'' to the 404030 sequence. This resulted in a clone that
contained additional 5' end sequence but still was missing the start
methionine. This rat sequence was then used to "walk" the EST data
base, looking for new EST clones that contained additional 5' end
sequence. This resulted in clone ID 420451, which contains the start
methionine but is 3' truncated at nucleotide 1,446 because of an
internal NotI site in PDE9.
5' RACE-- Marathon-adapted cDNA and Advantage polymerase PCR mix was purchased from CLONTECH. Reactions were set up as follows: 0.5 ng of adapted cDNA, 0.2 µM AP1 or AP2 primer, 0.2 µM gene-specific primer, 5 µl of 10× reaction buffer (supplied with Advantage polymerase), 0.2 mM dNTP, and 1 µl of Advantage KlenTaq Polymerase mix in a final volume of 50 µl. Reaction cycles were as follows: 94 °C for 1 min; 5 cycles of 94 °C for 30 s, 72 °C for 4 min; 5 cycles of 94 °C for 30 s, 70 °C for 4 min; 25 cycles of 94 °C for 30 s, 68 °C for 4 min.
Generation of Full-length MMPDE9A1-- A NotI/HindIII fragment from the mouse kidney clone 6.1 was subcloned into a NotI/HindIII digest of clone 420451 to insert the 3' end of PDE9A1 missing from this otherwise full-length clone because of an internal NotI site and the use of NotI in library construction for clone 420451. This generated a full-length PDE9A1 cDNA. This was subcloned into PCR3.1 (Invitrogen) by amplifying 0.5 µg of full-length PDE9A1 for 10 cycles using primers Krace.S1 and PDEstp.AS2. NotI digests were used to select for clones with PDE9A1 in the correct orientation. The cDNA as reported here has been confirmed to exist as a single mRNA species by PCR of kidney cDNA using primers Krace.S1 and PDE9.stp.AS1 (Fig. 1).
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Expression of PDE9A1--
PDE9A1 cDNA subcloned in PCR3.1
was purified using Qiagen tip-500 columns. 10 µg of DNA was
electroporated into 2 × 106 COS-7 cells/plate in a
0.4-cm electroporation cuvette at 250 microfarad and infinite
resistance using 300 V, 50 watts, and 50 mA. The same amount of
PCDNA-3 vector (Invitrogen) alone was also transfected under the
same conditions as a negative control for expression. Four plates for
each construct were transfected and plated onto 100-mm dishes and kept
in 5% CO2 for 24 h. After this period fresh medium
was added, and cells were placed in 10% CO2 for another
24-48 h. Each set of four plates was pooled and harvested in 4 ml of
homogenization buffer containing 40 mM Tris-HCl, pH 7.5; 15 mM benzamidine; 15 mM 2-mercaptoethanol; 1 µg/ml pepstatin A; 1 µg/ml leupeptin; and 5 mM EDTA.
This was homogenized in a Dounce homogenizer on ice with 25 strokes,
and then 1 volume of glycerol was added. This preparation was stored at
70 °C in aliquots and did not lose appreciable activity over 2 months.
Kinetics and Inhibitor Studies-- All PDE assays were done according to the method of Hansen and Beavo (8) in a buffer containing 40 mM MOPS, pH 7.5, 0.8 mM EGTA, 15.0 mM magnesium acetate, 0.2 mg/ml bovine serum albumin, and [3H]cGMP (25,000 cpm/reaction) in a final volume of 250 µl. All assays were done in triplicate, and reaction times and enzyme amounts were kept such that the lowest substrate concentration gave no more than 30% hydrolysis. PDE9 activity is defined as the total specific activity in PDE9-transfected COS cells minus the background specific activity of mock transfected COS cells. cGMP hydrolytic activity was approximately 8-16-fold higher in PDE9-transfected cells compared with mock transfected cells at 1 µM cGMP and approximately 50-fold higher at 0.06-0.1 µM cGMP. Thus the basal cGMP hydrolytic activity in COS-7 cells was not a significant contributor to the total activity in PDE9-transfected cells. Inhibitor studies were done without added unlabeled cGMP to keep the final substrate concentration low so that IC50 values would approximate the Ki value. IBMX, erythro-9-(2-hydroxy-3-nonyl)-adenine, and dipyridamole were obtained from Sigma. Zaprinast was a gift from May & Baker LTD, UK. Rolipram was obtained from BioMol (Plymoth Meeting, PA). SCH 51866 was a gift from Schering-Plow Research Institute, and sildenafil was a gift from Pfizer Central Research (Sandwich, UK). Enoximone was a gift from the Merrell Dow Research Institute.
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RESULTS AND DISCUSSION |
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Cloning, Tissue Distribution, and Predicted Sequence of PDE9A1-- We have cloned and characterized a cDNA encoding a new cyclic nucleotide phosphodiesterase that represents a previously unknown gene family. A partial cDNA representing this novel PDE, which had low but significant homology to the C terminus of calmodulin PDE MMPDE1A2, was initially isolated from the data base of expressed sequence tags. This sequence (mouse embryo EST clone 404030) did not match any GenBankTM PDE sequence with high homology but did match PDEs in general better than other GenBankTM sequences, suggesting that this sequence may represent a PDE of a new family. This EST was used to probe a mouse multiple tissue Northern blot to assess tissue distribution and mRNA size. (Fig. 2) Northern blot analysis indicates that mRNA representing this EST has a high level of expression in mouse kidney and lower levels of expression in liver, lung, and brain. This mRNA is estimated to be 2.0 kb in size.
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Comparison of the PDE9A1 Conserved Domain to Other Phosphodiesterases-- All mammalian PDEs characterized to date contain a conserved region of approximately 250 amino acid residues representing the catalytic domain of each of these enzymes (12). Many residues within this region are either absolutely or chemically preserved across species, from yeast to humans, and between PDE families. PDE9 is interesting in that three of these very highly conserved amino acids are divergent and all three cluster to the C-terminal end of the catalytic domain. The divergent amino acids identified here in mouse PDE9 are conserved in human PDE9 (11) also. Fig. 4B is an alignment of the C-terminal end of the catalytic domain from representatives of each of the eight families of PDEs. The first divergent substitution (1, Fig. 4B) is a conservative switch from a phenylalanine that is absolutely preserved among all other known PDEs to a tyrosine at this position in PDE9. Variation two (2, Fig. 4B) is less conserved. This alteration results in the replacement of a wholly conserved glycine found in all other mammalian PDEs to a serine at this position in PDE9. The third deviation found within PDE9 is a divergence from the chemically conserved hydrophobic residues isoleucine, leucine, and valine to the dissimilar and positively charged residue lysine. Although the exact function of these residues has not been determined to date, it is anticipated that some of these deviations from the highly conserved amino acids may participate in defining the unique kinetic characteristics of PDE9 (see kinetic and inhibitory studies). Ultimately, crystal structure solutions of several PDE catalytic domains will be needed to determine what functional roles these residues may play.
PDE9A1 Is a Low Km cGMP-specific Phosphodiesterase-- To demonstrate functional activity for PDE9 and to further characterize this novel PDE, we subcloned a cDNA constructed from overlapping clones for PDE9 and containing the full open reading frame reported here into the vector PCR3.1 for expression in mammalian cells. This construct was transfected into COS-7 cells, and PDE activity was assayed after 48-72 h. A low Km cGMP PDE activity that was significantly higher (6-18-fold higher, depending on transfection batch) in PDE9-transfected cells compared with PCDNA-3 alone-transfected cells was detected. This PDE9 activity does not appear to hydrolyze cAMP, and cGMP activity is not appreciably effected by cAMP, up to 100 µM (data not shown). This indicates that PDE9 is very specific for the hydrolysis of cGMP alone. Kinetic assays were done in triplicate on two different batches of COS-7 cell homogenates transfected with PDE9. Endogenous cGMP hydrolysis from vector only-transfected cells were subtracted from corresponding assays of PDE9-transfected cells. These assays demonstrate that this PDE has a Km of 0.07 µM ± 0.03 (average of five separate assays) (Fig. 5). This is the lowest Km for cGMP observed for a phosphodiesterase to date.
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Inhibitor Profile of PDE9A1-- Inhibitor studies were performed on COS-7 cell homogenates expressing PDE9 activity. PDE9 activity varied between transfections from 6- to 18-fold higher than COS-7 cGMP PDE endogenous activity. Any background endogenous cGMP PDE activity was subtracted, and inhibitor vehicle effects were likewise subtracted from the effects of inhibitor. These assays were done in triplicate on two separate transfection batches. No unlabeled cGMP was added to these reactions so that the final substrate concentrations were determined only by the addition of tracer [3H]cGMP to the reaction (11.3 nM). At these concentrations the IC50 should approach the Ki. Although PDE9 demonstrates cGMP-specific PDE activity, this PDE is not inhibited well by most of the PDE inhibitors, including the nonspecific inhibitor IBMX (IC50 greater than 200 µM) (Table I). It is important to note that this PDE is also not inhibited by the PDE5-specific inhibitor sildenafil (21) nor the PDE5 and PDE6 inhibitor dipyridamole at doses within ranges considered specific. Zaprinast, another PDE5 and PDE6 inhibitor, will inhibit PDE9 at moderately high concentrations (29 µM). Interestingly, although PDE9 is not inhibited well by IBMX or the PDE5 or PDE6 inhibitors, it is inhibited by the relatively new PDE 1 and PDE5 inhibitor SCH 51866 with an IC50 of 1.55 µM. This is only 15 times higher than the IC50 of SCH 51866 for both PDE1 and PDE5, both of which have an IC50 of approximately 0.1 µM (22).
Few studies examining the biologic effects of SCH 51866 have been done to date; however, one such study has found this drug to have antiplatelet, antiproliferative, and hemodynamic effects (22). In this study, SCH 51866 inhibited platelet aggregation with an IC50 of 10 µM. Additionally, this compound also inhibited platelet adhesion, and platelet accumulation of cGMP was noted at these inhibitor concentrations. In balloon angioplasty injury of rat carotid arteries, SCH 51866 inhibited neointima formation. Furthermore, SCH 51866 dose-dependently reduced blood pressure in spontaneously hypertensive rats. It is possible that some of these results may be attributable to inhibition of PDE9 in addition to inhibition of either PDE1 or 5. Inhibition of PDE9 by SCH 51866 may also point to possible starting compounds for future studies assaying for more selective and potent inhibitors of PDE9. SCH 51866 may be useful in future studies of PDE9 function in tissues or cells that have low or no endogenous PDE1 or PDE5 phosphodiesterase activity.PDE9A1 Is Most Homologous to RegA, a D. discoideum PDE-- A search of GenBankTM using PDE9 sequence as a query indicates this PDE is slightly more homologous to a recently cloned D. discoideum PDE, RegA (3), than to any of the known mammalian PDEs. Alignment of the conserved domain of RegA to that of PDE9 shows that 41% amino acid sequence identity exists between these two PDEs, which although higher than that for any of the mammalian PDEs (Fig. 3) is still low enough that these two PDEs cannot be considered to be in the same gene family. The significance of this higher degree of homology to RegA is unknown at this time; however, it appears that these two enzymes may not be functionally related because they do not share the same substrate specificity (23) and also do not share extended structural homology outside of the conserved domain. Previous work has noted that the N-terminal sequence of RegA is highly homologous to that of response regulator enzymes found in bacteria and lower eukaryotes, suggesting that this PDE may play a role in two-component signaling, possibly catalyzing histidine phosphotransfer to an aspartic acid to regulate PDE activity (3). It should be noted, however, that histidine phosphorylation has only been described in mammalian cells in a few cases (24, 25), and histidine phosphotransfer has only recently been documented to occur with Nm23 proteins (26, 27). The N terminus of PDE9 does not appear to share significant homology to Nm23 proteins and does not share homology to the region of RegA that is homologous to bacterial response regulators. The slightly higher degree of similarity these PDEs share may simply indicate that PDE9 evolved early in mammals and that RegA and PDE9 are closer in evolutionary distance to each other than more recently evolved mammalian PDEs. It is of interest to observe that of the three amino acid residues divergent within the conserved region of PDE9, two of these substitutions are also found in RegA (1 and 3, Fig. 4B), lending credence to the idea that these two PDEs are more closely related to each other than to any of the other mammalian PDEs. Additional functional characterization of both RegA and PDE9 will be necessary to determine whether this sequence homology extends to some unknown functional homology.
Conclusion and Possible Physiologic Roles for PDE9A1-- Although at this time it is unknown what physiologic roles PDE9 may regulate, the identification and initial description of this new family will aid in defining these roles in future studies. Because PDE9 is dedicated to the high affinity specific hydrolysis of cGMP, PDE9 must be involved in the regulation of signal transduction pathways that concern activation of guanylyl cyclase activity. For example, ANP receptors expressed in kidney regulate diuresis by ligand-activated receptor guanylyl cyclase activity (28-30). Because PDE9 is most highly expressed at the mRNA level in kidney, it is tempting to speculate that this novel PDE may participate in regulating this pathway by maintaining basal cGMP levels at low concentrations, by enlarging the ANP cGMP signal, and/or by lowering intracellular cGMP levels after ANP receptor activation, thus terminating ANP-mediated diuresis. Indeed, previous studies have shown that inhibition of cGMP hydrolysis by infusion of zaprinast (which at higher concentrations will inhibit PDE9; Table I) potentiates the effect of ANP on natriuresis without causing deleterious drops in blood pressure (31, 32). Because ANP receptors are known to be localized within the glomerulus and inner medullary collecting ducts (33), it will be interesting to determine the cellular localization of PDE9 enzyme in kidney.
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ACKNOWLEDGEMENTS |
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We thank Arlene Lamont-Cubitt for work on parts of this project as a rotation student and Todd Berard and Rejean L. Idzerda and the University of Washington Molecular Pharmacology DNA Core Facility for help in DNA sequencing. We also thank Douglas Fisher for sending us an advance copy of his manuscript, Pfizer Central Research (Sandwich, UK) for the gift of sildenafil, and the Schering-Plow Research Institute for the gift of SCH 51866.
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FOOTNOTES |
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* This work was supported by National Institutes of Health Grants DK21723 and GMO7750.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.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) AF031147.
To whom correspondence should be addressed: Dept. of Pharmacology,
Box 357280, University of Washington, Seattle, WA 98195. Tel.:
206-543-4006.
1 The abbreviations used are: PDE, phosphodiesterase; IBMX, 3-isobutyl-1-methyl-xanthine; RACE, rapid amplification of cDNA ends; EST, expressed sequence tag; kb, kilobase(s); SCH 51866, (+)-cis-5,6a,7,8,9 hyl] phenylmethyl]-5-methyl-cylopent[4,5]imidao[2,1-b]purin-49(3H)one; ANP, atrial natriuretic peptide; PCR, polymerase chain reaction; MOPS, 3-[N-morpholino]propanesulfonic acid.
2 Note that an eighth family of cyclic nucleotide phosphodiesterases has also been identified by our lab and by Fisher et al. Fisher, D. A., Smith, J. F., Pillar, J. S., St. Denis, S. H., and Cheng, J. B. (1998) Biochem. Biophys. Res. Commun. 246, in press.
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REFERENCES |
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