THE JOURNAL OF BIOLOGICAL CHEMISTRY
796 –806, 1996
© 1996 by The American Society for Biochemistry and Molecular Biology, Inc.
Isolation and Characterization of cDNAs Corresponding to Two
Human Calcium, Calmodulin-regulated, 3*,5*-Cyclic Nucleotide
Phosphodiesterases*
(Received for publication, March 30, 1995, and in revised form, October 12, 1995)
Kate Loughney‡§, Timothy J. Martins‡, Edith A. S. Harris‡, Krishna Sadhu‡, James B. Hicks‡¶,
William K. Sonnenburgi, Joseph A. Beavoi, and Ken Ferguson‡
From the ‡Icos Corporation, Bothell, Washington 98021 and the iDepartment of Pharmacology, University of Washington,
School of Medicine, Seattle, Washington 98195
cDNAs corresponding to two human calcium, cal-
modulin (CaM)-regulated 3*,5*-cyclic nucleotide phos-
phodiesterases (PDEs) were isolated. One, Hcam1
(PDE1A3), corresponds to the bovine 61-kDa CaM PDE
(PDE1A2). The second, Hcam3 (PDE1C), represents a
novel phosphodiesterase gene. Hcam1 encodes a 535-
amino acid protein that differs most notably from the
bovine 61-kDa CaM PDE by the presence of a 14-amino
acid insertion and a divergent carboxyl terminus.
RNase protection studies indicated that Hcam1 is rep-
resented in human RNA from several tissues, including
brain, kidney, testes, and heart. Two carboxyl-termi-
nal splice variants for Hcam3 were isolated. One,
Hcam3b (PDE1C1), encodes a protein 634 amino acids
(72 kDa) in length. The other, Hcam3a (PDE1C3), di-
verges from Hcam3b 4 amino acids from the carboxyl
terminus of Hcam3b, and extends an additional 79
amino acids. All the cDNAs isolated for Hcam3a are
incomplete; they do not include the 5*-end of the open
reading frame. Northern analysis revealed that both
splice variants were expressed in several tissues, in-
cluding brain and heart, and that there may be addi-
tional splice variants. Amino-truncated recombinant
proteins were expressed in yeast and characterized
biochemically. Hcam3a has a high affinity for both
cAMP and cGMP and thus has distinctly different ki-
netic parameters from Hcam1, which has a higher af-
finity for cGMP than for cAMP. Both PDE1C enzymes
were inhibited by isobutylmethylxanthine, 8-me-
thoxymethyl isobutylmethylxanthine, zaprinast, and
vinpocetine.
Cyclic nucleotides are involved in a large number of mam-
malian signal transduction pathways. The intracellular con-
centrations of cAMP and cGMP reflect their rate of synthesis,
by adenylyl and guanylyl cyclases, and their rate of degrada-
tion to 59-monophosphate nucleosides, by cyclic nucleotide
* This work was supported in part by Grants HL44948 and DK21723
from the National Institutes of Health. The costs of publication of this
article were defrayed in part by the payment of page charges. This
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) U40370
(HSPDE1A3), U40371 (HSPDE1C1), and U43072 (HSPDE1C3).
§ To whom correspondence should be addressed: Icos Corporation,
22021 20th Ave. S.E., Bothell, WA 98021. Tel.: 206-485-1900; Fax:
206-486-0300.
¶ Present address: Hedral Corporation, Suite 1800, 222 S.W. Colum-
bia, Portland, OR 97201.
796
Vol. 271, No. 2, Issue of January 12, pp.
Printed in U.S.A.
phosphodiesterases (PDEs).1 A number of biochemically dis-
tinct PDEs have been identified. They fall into seven families
distinguished by their allosteric regulation, substrate kinetics,
amino acid sequence homology, and interaction with specific
Downloaded from http://www.jbc.org/ at Eli Lilly and Company on December 18, 2019
inhibitors (1, 2). All known mammalian PDEs share a con-
served region of approximately 250 amino acids that contains
the phosphodiesterase catalytic site (3). In certain PDEs the
regions amino-terminal to the catalytic domain are known to be
involved in the allosteric regulation caused by the binding of
calmodulin or cGMP (3).
Type I PDEs (PDE1, CaM PDEs) are activated by the bind-
ing of calmodulin in the presence of Ca21 (CaM). This binding
increases the hydrolysis of both cAMP and cGMP (4). The
regulation of cyclic nucleotide hydrolysis by changes in calcium
concentration may allow the type I enzymes to integrate the
Ca21 and cyclic nucleotide second messenger pathways within
a cell.
Biochemical characterizations have distinguished at least
five type I PDEs differing from each other in apparent molec-
ular weight, Km values for cyclic nucleotide substrates, affini-
ties for calmodulin, and regulation by phosphorylation (5–16).
The cDNAs for several of these type I PDEs have been isolated
and characterized (17–20). The existence of type I PDEs with
distinct sequences and properties suggests a diversity of cellu-
lar functions for these enzymes. However, this diversity com-
plicates the analysis of type I PDEs in tissues where more than
one biochemical form is present. Isolating each of the type I
PDE genes helps aid in understanding the role played by each
member of this complex family of enzymes.
We report here the isolation and characterization of cDNAs
corresponding to two human type I PDE genes. One, Hcam1,
corresponds to the bovine 61-kDa CaM PDE. The other,
Hcam3, is a novel type I PDE, whose mouse and rat counter-
parts have also been recently isolated (52). We examine the
tissue distribution of the mRNAs for Hcam1 and Hcam3 and
the biochemical properties of amino-truncated proteins corre-
sponding to each gene. Hcam3 shows alternative splicing that
would generate phosphodiesterase proteins with different car-
boxyl termini; this is the first example of carboxyl-terminal
splice variants for mammalian type I PDEs.
MATERIALS AND METHODS
Library Screening—A bovine 61-kDa CaM PDE cDNA fragment
was used as a probe to screen human cDNA libraries. Degenerate
oligonucleotides (corresponding to the bovine 61-kDa CaM PDE
1
PDE, phosphodiesterase; CaM, calmodulin; PCR, polymerase chain
reaction; RT, reverse transcription; PIPES, 1,4-piperazinediethanesul-
fonic acid; IBMX, isobutylmethylxanthine; bp, base pair(s); kb, kilobase
pair(s).
 Human Type I Phosphodiesterases
TABLE I
Yeast strains
Strains Genotype
SX50–1C Mata his3 leu2 trp1 ura3
YKS42 Mata his3 leu2 trp1 ura3 Dpde1::HIS3
SX50–2D Mata his3 leu2 trp1 ura3
YKS17 Mata his3 leu2 trp1 ura3 Dpde2::TRP1
KSX15 Diploid of YKS42 3 YKS17
YKS44 Mata his3 leu2 trp1 ura3 Dpde1::HIS3 Dpde2::TRP1 (haploid segregant of KSX15)
YKS45 Mata his3 leu2 trp1 ura3 Dpde1::HIS3 Dpde2::TRP1 (haploid segregant of KSX15)
5B a Mata leu2 trp1 ura3 pep4–3 prb1–1122 prc1–126
KSX24 Diploid of YKS44 3 5Ba
YKS76a Mata his3 leu2 trp1 ura3 Dpde1::HIS3 Dpde2::TRP1 pep4–3 prb1–1122? prc1–126?
(haploid segregant of KSX24)
a
YKS76 mates poorly with both Mata and Mata strains. It is assigned Mata because its growth is arrested near a Mata strain but not near a
Mata strain. The presence of pep4 –3, confirmed by DNA sequence analysis, rendered it difficult to determine the alleles at the prb1 and prc1 loci.
amino acid sequences KMGMMKKK and NMKGTTND) and bovine
heart cDNA were used in a PCR reaction to generate an 1108-bp
bovine cDNA fragment as previously described (17). Hybridization
probes were prepared by isolating DNA fragments from agarose gels
and labeling them with [32P]dCTP and [32P]dTTP (800 Ci/mmol,
DuPont) using a Boehringer Mannheim random priming kit. Library
screening and hybridization conditions were as described elsewhere
(21). Three positively hybridizing l phage (H2a, H3a, H6a) were
isolated from a human hippocampus cDNA library (Clontech). The
cDNA inserts from these phage were subsequently used as probes to
isolate additional l phage. An H6a probe (1.2-kb HindIII/EcoRV
fragment) was used to isolate l phage A2d from an aorta cDNA
library (Clontech). An H3a probe (2.4-kb HindIII/EcoRI fragment)
was used to isolate two additional l phage, He11a and He19a, from
the heart library (Stratagene).
Subcloning and DNA Sequencing—cDNA inserts from H6a, H2a,
H3a, and A2d were subcloned into Bluescript vectors (Stratagene).
Bluescript plasmids containing the cDNA inserts from He11a and
He19a were excised in vivo from the l ZAP vector (Stratagene) following
the manufacturer’s instructions. Plasmid DNA was extracted using kits
from Promega Biotech Inc. and QIAGEN Inc. Restriction and modifica-
tion enzymes were purchased from Boehringer Mannheim. Oligonucleo-
tides were synthesized using an ABI 394 DNA synthesizer. The cDNA
inserts from H6a, H3a, He11a, and A2d were completely sequenced on
both strands using a Sequenase kit (U. S. Biochemical Corp). Those
from He19a and H2a were partially sequenced. Additional molecular
biological techniques are as previously described (22).
RNA Isolation—Human tissue samples were obtained from the
National Disease Research Interchange and the Cooperative Human
Tissue Network. Tissue was frozen within 8 h of death or surgery except
for three of the four heart samples which were frozen within 30 h of
death. The tissues were pulverized under liquid nitrogen, and RNA was
extracted by the method of Chomczynski and Sacchi (23).
RT-PCR—First strand cDNA was reverse-transcribed from 2 mg of
human heart poly(A)1 mRNA using a Boehringer Mannheim cDNA
synthesis kit. 0.5 ml of the 20-ml final reaction volume was used per
subsequent PCR reaction, which also contained 10 mg/ml of each
primer, 10 mM Tris, pH 8.3, 1.5 mM MgCl2, 50 mM KCl, 0.2 mM each
dNTP (Boehringer Mannheim), 0.6 unit Taq polymerase (Boehringer
Mannheim) in 25 ml. Reaction conditions were 94 °C for 4 min, followed
by 30 cycles of 94 °C for 1 min, and 60 °C for 2 min and 72 °C for 4 min.
The polymerase was added after the reaction temperature reached
94 °C. Primary PCR reactions were diluted 1/100, and 1 ml was added to
a 25-ml secondary PCR reaction. Hcam1 primers are shown in Fig. 1B,
primers 1 and 4 were used in the primary reaction, and primers 2 and
3 in the secondary reaction. Hcam3 primers are shown in Fig. 2, B and
C; primers 1 and 4 were used in the first reaction and primers 2 and 3
in the secondary reaction. The PCR-derived DNA fragments were gel
purified and partially sequenced.
RNase Protection Assays—An antisense Hcam1 RNA probe (made
from a plasmid containing nucleotides 1252–1939; Fig. 1B) was pre-
pared by in vitro transcription using 20 ng of a linearized template. The
transcription reaction was performed at 37 °C for 30 min and contained
40 mM Tris-HCl, pH 8, 50 mM NaCl, 8 mM MgCl2, 2 mM spermidine, 0.25
mM each ATP, CTP, and GTP, 0.1 unit of RNase block I (Stratagene), 5
mM dithiothreitol, 8 mM UTP, 5 mM [a-32P]UTP (800 Ci/mmol, DuPont),
and 10 units of T7 RNA polymerase (Stratagene) in a reaction volume
of 5 ml. Escherichia coli tRNA (30 mg) and 92 ml of 40 mM Tris-HCl, pH
8, 6 mM MgCl2, and 10 mM NaCl were added prior to treatment with 10
units of RNase-free DNase (Boehringer Mannheim) for 15 min at 37 °C.
797
Source
Ivy et al. (27)
This laboratory
J. Ivy
This laboratory
This laboratory
This laboratory
This laboratory
J. B. Hicks
This laboratory
This laboratory
The reaction was extracted sequentially with phenol/chloroform and
chloroform and then precipitated (50 ml of 7.5 M ammonium acetate, 300
ml of ethanol).
RNase protection conditions were based on published methods (24).
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The probe was resuspended in 80% formamide, 400 mM NaCl, 40 mM
PIPES pH 7, and 1 mM EDTA. Human total RNA (4 –10 mg in 1.7– 8.9
ml) was added to 50-ml aliquots of the probe (5 3 104 dpm), denatured at
90 –95 °C for 5 min, and allowed to hybridize overnight at 52 °C. Diges-
tion buffer (350 ml of 50 mM NaCl, 10 mM Tris-HCl, pH 7.5, 5 mM EDTA)
containing 1 unit/ml RNase T1 (Sigma) was added to each sample and
incubated at room temperature for 45 min. The samples were phenol/
chloroform-extracted (200 ml of phenol, 400 ml of chloroform, 10 ml of
20% SDS, and 1.5 ml of 10 mg/ml tRNA), ethanol-precipitated, and
resuspended in sample loading buffer (95% formamide, 20 mM EDTA,
0.05% bromphenol blue, 0.05% xylene cylanol) prior to being ana-
lyzed by denaturing acrylamide-urea gel electrophoresis and
autoradiography.
Northern Analysis—A multiple tissue blot was purchased from Clon-
tech. The probes were prepared as described for the RNase protections
except that no unlabeled UTP was present in the reaction. Prehybrid-
izations were performed in 50% formamide, 50 mM sodium phosphate,
pH 6.8, 5 3 SSC, 1 mM EDTA, 2.5 3 Denhardt’s solution, 200 mg/ml
denatured salmon testes DNA, 0.5 mg/ml tRNA, and 20 units/ml hep-
arin at 65 °C; fresh solution containing the probe was added for the
hybridization overnight. 5 3 SSC and 2.5 3 Denhardt’s solutions are as
defined in Sambrook et al. (22). The filters were washed first at room
temperature and then at 65 °C in 0.1 3 SSC containing 0.1% SDS prior
to autoradiography. The filter was stripped between uses by immersion
in boiling water. The Hcam1 riboprobe was the same as that used in the
RNase protection analysis. The Hcam3a-specific probe extended from
nucleotides 2068 –2686 (Fig. 2C) and the Hcam3b-specific probe ex-
tended from nucleotides 2062–2652 (Fig. 2B). Fragments corresponding
to these regions were generated by PCR and subcloned prior to their use
in preparing an antisense riboprobe (as described above).
Expression Constructs—The cDNAs were cloned into the yeast ex-
pression vector pBNY6n and expressed in Saccharomyces cerevisiae.
This vector, which provided EcoRI and XhoI cloning sites, was con-
structed from pADNS (25) and Yeplac181 (26) as follows. The polylinker
of pADNS was replaced with an EcoRI/NotI/XhoI linker (59-AGCTC-
GAATTCTGCGGCCGCTCGAGA-39 and 59-AATTTCTCGAGCGGCCG-
CAGAATTCG-39). This linker was ligated into pADNS that had been
cut to completion with HindIII and partially digested with EcoRI. The
resultant plasmid was cleaved with BamHI and the fragment that
contained the ADHI promoter and terminator flanking the new
polylinker was inserted into the BamHI site of a YEplac181 derivative.
In this derivative the EcoRI site of YEplac181 had been removed by
digestion with HpaI and SmaI followed by ligation.
The plasmid Hcam3a(met150) was constructed by ligating the cDNA
insert of He11a (nucleotides 610-2067 of Fig. 2B followed by 2068 –2686
of Fig. 2C) into the EcoRI site of pBNY6n. New junctions in the con-
struct were verified by DNA sequencing. The first ATG in the cDNA
insert is nucleotide 624 of Hcam3a (Fig. 2B) which corresponds to amino
acid 150.
The plasmid Hcam1(met141) contains an EcoRI/XhoI fragment (nu-
cleotides 505-1692, Fig. 1B) inserted into pBNY6n. The first ATG in the
cDNA insert is nucleotide 505 of Hcam1 (Fig. 1B) which corresponds to
amino acid 141. DNA sequences generated by PCR and new junctions
were verified by DNA sequencing.
Yeast Strains—The genotypes of the yeast strains used in this work
are shown in Table I. YKS42 was generated from SX50 –1C by deleting
798 Human Type I Phosphodiesterases

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FIG. 1. A, map of Hcam1 cDNAs. The inverted triangle in cDNA H2a represents the 47-nucleotide insertion. The hatched portion of cDNA H2a
represents DNA sequences that differ from those found in the corresponding regions of cDNAs A2d and H6a. The star indicates the position of the
2 nucleotides missing in cDNA H6a. The open reading frame encoded by a composite of these cDNAs is diagrammed, and the putative CaM binding
region and the phosphodiesterase catalytic region are indicated. B, nucleotide sequence and predicted protein sequence of Hcam1 cDNAs
(PDE1A3). Nucleotide and amino acid residues are numbered on the right. The methionine at amino acid position 141 is underlined to indicate the
initiation point of the truncated protein Hcam1(met141). Also underlined is the insertion in the human Hcam1 relative to the bovine 61-kDa CaM
PDE (see Panel C). The exact boundaries of the insertion are uncertain as it is flanked by AG nucleotides only one pair of which is part of the
insertion. The positions of oligonucleotides used for RT-PCR are indicated above the sequence by numbered arrows. C, an alignment of the
predicted human Hcam1 amino acid sequence (top) and the bovine 61-kDa CaM PDE amino acid sequence (bottom). A dot in the bovine sequence
indicates that the amino acid in that position is identical to that found in the human sequence. The dashes in the bovine sequence indicate a region
present in the human sequence that is not present in the bovine sequence.

the endogenous PDE1 gene (28) (BamHI/HincII fragment containing
the open reading frame deleted) and replacing it with the HIS3 gene
(29) (Eco47III/BamHI fragment containing the open reading frame
inserted). Standard methods were used for gene replacements (30) and
yeast transformations (31).
YKS17 was generated from SX50-2D by deleting the endogenous
PDE2 gene (32) (nucleotides 7–1564 of PDE2 open reading frame de-
leted) and replacing it with the TRP1 gene (33) (1.4-kb EcoRI fragment
containing the open reading frame inserted).
The presence of the pep4-3 allele in YKS76 was confirmed by se-
quencing the region of the gene that contains the nonsense mutation
present in this allele (34). Tests (35, 36) for the presence of the prb1-
1122 and prc1-126 alleles gave ambiguous results because pep4-3 is
epistatic to their expression.
Biochemistry—YKS76 was transformed (37) with the plasmids
Hcam1(met141) and Hcam3a(met150) and the transformants were
grown at 30 °C in synthetic medium lacking leucine to 1–2 3 107
cells/ml, collected by centrifugation, washed once with water, and fro-
zen in a dry ice-ethanol bath.
The yeast cell pellets were lysed and phosphodiesterase activity was
assayed as described elsewhere (38) with the following modifications.
[32P]cAMP and [32P]cGMP were used (25 Ci/mmol, ICN Biochemical).
The 32Pi product was separated from the substrate using charcoal (39,
40). Two volumes of 25 mg/ml activated charcoal in 0.1 M KH2PO4 were
added to terminate the reaction. Following centrifugation, the super-
natant was removed and quantitated by Cerenkov counting. Extracts
prepared from YKS76 containing only the pBNY6n vector yielded ac-
tivities indistinguishable from assay background.
Kinetic and inhibitor analyses were performed as described previ-
ously (38). Cyclic nucleotide hydrolysis was measured at substrate
concentrations ranging from 0.03 to 100 mM. The specific activity of the
substrate was held constant (0.2 Ci/mmol). The kinetic data were fitted
to the Michaelis-Menton model using TableCurvey (Jandel Scientific).
Inhibitor analyses were performed at 0.1 mM cGMP. At this substrate
concentration the observed IC50 should approximate the apparent in-
hibitor constant (Ki) for both competitive and noncompetitive inhibitors.
The reaction time and amount of enzyme in the assay were adjusted
such that less than 25% of the substrate was consumed in the reaction.
Dose-response curves were fitted (TableCurvey) using a four-parameter
or a two-parameter logistic model (38). The four-parameter model de-
rives values for the minimum PDE activity, the maximum PDE activity,
and the IC50, as well as a parameter that determines the slope of the
fitted curve at the IC50. The two-parameter model derives values for the
maximum PDE activity and the IC50. The minimum PDE activity is set
to 0, and the parameter affecting the slope is set to 1. The two-param-
eter model was used when the highest concentration of inhibitor was
unable to inhibit fully the enzymatic activity. All other data sets uti-
lized the four-parameter model. Stocks of inhibitors were prepared in
dimethyl sulfoxide (Aldrich), and the final solvent concentrations in the
PDE assay never exceeded 2% (v/v). Vinpocetine was obtained from
Andard Mount Co.; IBMX from Sigma; rolipram and zaprinast were
gifts from Paul Feldman, Glaxo Inc. Research Institute (Research Tri-
angle Park, NC); and 8-methoxymethyl IBMX was a gift from Jack
Wells, Vanderbilt University (Nashville, TN). Cilostamide was synthe-
 Human Type I Phosphodiesterases 799

FIG. 2. A, map of Hcam3 cDNAs. The

divergent 39-ends of the cDNAs are repre-
sented by a hatched line (cDNAs He19a

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and He11a) or a jagged line (cDNA H3a).
The two different open reading frames
represented by these cDNAs are dia-
grammed. The portion of cDNA He19a
represented as a dashed line has not been
completely sequenced. B, nucleotide se-
quence and predicted protein sequence of
Hcam3b (PDE1C1). The Hcam3a/3b di-
vergence follows nucleotide 2067. The D
marked with a diamond is the first amino
acid that is specific to the Hcam3b splice
variant. cDNA He11a begins at nucleo-
tide 611 and the protein expressed by the
truncated Hcam3a(met150) construct be-
gins at the methionine underlined at
amino acid position 150. The positions of
oligonucleotides used for RT-PCR are in-
dicated above the sequence by numbered
arrows. Nucleotide and amino acid resi-
dues are numbered on the right. C, nucle-
otide sequence and predicted protein se-
quence specific to Hcam3a (PDE1C3). The
first nucleotide of this sequence corre-
sponds to nucleotide 2067 of Panel B. Fol-
lowing this nucleotide (G) the two se-
quences diverge. The Hcam3a protein
contains a glycine (G) rather than the as-
partic acid (D) marked with a diamond in
Panel B and extends another 79 amino
acids before terminating. The cDNA in-
sert from He19a contains a G at position
2246, whereas that from He11a contains
an A. This change does not affect the
amino acid sequence of the protein. Nu-
cleotide and amino acid residues are num-
bered on the right.
800 Human Type I Phosphodiesterases
FIG. 3. Hcam1 Northern blot analysis. The blot was purchased
from Clontech. Each lane contains 2 mg of poly(A)-selected mRNA
isolated from the tissue indicated. Positions of the RNA size markers
are shown on the left.
sized using published procedures (41). Protein concentrations were
assayed using a protein assay kit (Bio-Rad) and published methods (42).
RESULTS
Three phage were isolated from a human hippocampus
cDNA library screened with a cDNA fragment derived from the
bovine 61-kDa CaM PDE (17). DNA fragments derived from
these phage were used to screen heart and aorta human cDNA
libraries, and three additional phage were isolated. Subcloning
and sequencing revealed that these six cDNAs correspond to
two different genes, Hcam1 and Hcam3.
Hcam1—Three Hcam1 cDNAs (H2a and H6a from hip-
pocampus and A2d from aorta) provide a composite sequence of
2008 nucleotides (Fig. 1, A and B). This composite cDNA en-
codes a 535-amino acid protein with a predicted molecular
mass of 61,251 Da that is similar to the bovine 61-kDa CaM
PDE protein (5, 17) (Fig. 1C).
The three cDNAs used in building the composite (Fig. 1B)
differ from it as follows (Fig. 1A). The composite sequence is
identical to the sequence of cDNA H6a except that H6a is
lacking nucleotides 626 and 627. These nucleotides are present
in H2a and in the bovine 61-kDa CaM PDE. Deletion of them
alters the reading frame. We believe their absence in H6a is a
cloning artifact.
The cDNA insert in l H2a contains a 47-bp insertion follow-
ing nucleotide 74 and diverges from the Hcam1 composite
sequence following nucleotide 807 (Fig. 1A). The 47-bp inser-
tion is in the 59-untranslated region and does not alter the open
reading frame of the cDNA. It contains sequences that show a
good match to consensus splice donor and acceptor sequences
(44), which suggests it may be an intron that was not spliced
out of the mRNA from which the cDNA was made. It was not
included in the Hcam1 composite sequence (Fig. 1B). The se-
quences following nucleotide 807, which show a good match to
consensus splice donor sequences (44), may also represent an
intron. It is known from an analysis of genomic DNA sequences
that an intron is present at this position.2 These sequences
were also not included in the composite sequence (Fig. 1B).
RT-PCR was used to isolate a portion of Hcam1 from human
heart cDNA. A PCR-derived band extending from primer 2 to
primer 3 (Fig. 1B) was sequenced. The 2 bp (626 – 627) were
present, and the presumed intron following nucleotide 807 was
absent, thus confirming our interpretation of the cDNA
structures.
2
P. Snyder, unpublished observation.
The sequence of the cDNA insert in l A2d contains 15 nu-
cleotides at its 59-end that represent an inverted repeat of
sequences found elsewhere in the cDNA and which are presum-
ably a cloning artifact. It also contains 4 additional nucleotides
located just 59 to the poly(A) tail (AGCT following nucleotide
1999 of Fig. 1B). These may reflect the use of a second poly-
adenylation site. The composite Hcam1 sequence (Fig. 1B) has
been submitted to GenBankTM as HSPDE1A3.
The human Hcam1 protein sequence contains residues that
are identical to a calmodulin-binding region identified in the
bovine 61-kDa CaM PDE (amino acids 24 – 42, Fig. 1, A and B)
(5, 17). Hcam1 also contains a region conserved in all mamma-
lian phosphodiesterases that corresponds to the phosphodies-
terase catalytic domain (amino acids 194 – 447, Fig. 1, A and B)
(3). Although Hcam1 is most similar to the bovine 61-kDa CaM
PDE (PDE1A2), the human protein contains two regions that
are not found in the bovine protein. One region is a 14-amino
acid insertion in the human protein following amino acid 458.
The second region is the carboxyl terminus; the carboxyl-ter-
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minal 14 amino acids of the human protein have no homology
to the carboxyl-terminal 23 amino acids of the bovine protein.
When these two regions are excluded from comparison, the
human and bovine proteins share 94% amino acid identity
(Fig. 1C).
Hcam3—The sequences of three Hcam3 cDNAs (He11a and
He19a from heart and H3a from hippocampus) yield a compos-
ite that predicts two splice variants with different carboxyl
termini (Fig. 2A). One predicted splice variant, Hcam3b, is
represented by cDNA H3a. This splice variant is designated
HSPDE1C1 and was submitted to GenBankTM. The 2694-nu-
cleotide cDNA contains an open reading frame that encodes a
634-amino acid protein with a predicted molecular mass of
72,207 Da (Fig. 2B). The second splice variant, Hcam3a, is
represented by cDNAs He11a and He19a. This splice variant is
designated HSPDE1C3. Both cDNAs (He11a and He19a) en-
code a protein with a carboxyl terminus that diverges from that
found in Hcam3b, and both cDNAs are lacking the 59-end of the
open reading frame. He11a (nucleotides 611-2067 of Fig. 2B
followed by nucleotides 2068 –2686 of Fig. 2C) was submitted to
GenBankTM as HSPDE1C3.
RT-PCR using human heart cDNA was performed to deter-
mine if the Hcam3a 39-end is associated with the Hcam3b
59-end. A DNA fragment was amplified in two rounds of PCR
using first primers 1 and 4 (Fig. 2, B and C) and then primers
2 and 3 (Fig. 2, B and C). Sequence analysis revealed that the
the DNA fragment had a Hcam3b 59-end and a Hcam3a 39-end.
This full-length Hcam3a would encode a 709-amino acid pro-
tein with a predicted molecular mass of 80,759. Although the
sequence of Hcam3 shares certain characteristics with the hu-
man (this work) and bovine (5, 17) 61-kDa CaM PDEs and to
the human (19), bovine (6, 18), mouse (19, 20), and rat (19)
63-kDa CaM PDEs, it appears that Hcam3 represents a novel
CaM PDE gene.
The divergence of the human Hcam1 sequence from the
bovine 61-kDa CaM PDE sequence and the presence of two
different carboxyl-terminal sequences for Hcam3 raised a num-
ber of questions about the structures of these cDNAs. Northern
and RNase protection analyses were performed both to confirm
the cDNA structures and to examine the tissue distribution of
the CaM PDE transcripts.
Northern Analysis and RNase Protection Assay of
Hcam1—In Northern blots a probe for Hcam1 hybridized to
two different sized mRNAs in brain, heart, liver, skeletal mus-
cle, and kidney samples (Fig. 3). The larger mRNA is 4.8 kb.
Little of it is present in the liver and skeletal muscle samples.
The smaller mRNA is 2.4 kb in the brain sample and 2.6 kb in
 Human Type I Phosphodiesterases 801

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FIG. 4. RNase protection analysis using a Hcam1 probe and human RNA samples. A, the human RNA samples are described at the top
of the figure, and the amount of RNA (mg) included in each lane is indicated. One star indicates the sample was slightly degraded, and two stars
indicate it was significantly degraded as assessed by denaturing agarose gel electrophoresis. The integrity of the leftmost uterus sample was not
examined. fX174 (HaeIII) and Bluescript II SK1 (HinfI) markers are present on both the left and right sides of the figure. In the lanes labeled sense
RNA the antisense probe protected 714 nucleotides of a sense RNA transcript. This includes the 688 nucleotides of Hcam3 sequences and 26
nucleotides of flanking polylinker sequences. The lane labeled probe contains the radiolabeled probe. The lane labeled negative control contained
10 mg of tRNA and was processed in parallel with the other samples. B, the diagram indicates the position of the probe used in the protections.

the other samples. (The 2.4-kb/2.6-kb size difference was seen transcripts were most abundant in mRNA from the brain,
on two different blots (data not shown).) Lung and pancreas heart, kidney, and skeletal muscle.
mRNA samples contain a small amount of the 2.6-kb mRNA. We used RNase protection to determine if the specific mRNA
No hybridization signal was seen in the placenta sample. The structure predicted from the Hcam1 cDNAs was present in
802 Human Type I Phosphodiesterases
FIG. 5. Hcam3 Northern blot analysis. The positions of the RNA
markers are shown on the left. A, Hcam3a-specific probe (nucleotides
2068 –2686 of Fig. 2C). B, Hcam3b-specific probe (nucleotides 2062–
2652 of Fig. 2B).
human RNA samples. The RNase protection probe extended
across the two regions where the bovine and human sequences
differed from each other (42-nucleotide insertion in human
sequence and divergent 39-ends, Fig. 4B). Using this probe a
688-nucleotide band was seen (Fig. 4A). This band was the size
expected for protection of the entire Hcam1 probe. This con-
firmed that both the 42-nucleotide insertion and the human
type of 39-end were found in RNA from these tissues. A human
mRNA lacking the 42-nucleotide insertion would have given
rise to a 207–209-nucleotide band. No such band was detected.
The smaller bands included a prominent 397-nucleotide band
(Fig. 4A). This was the size expected for protection of the probe
59 to the human/bovine divergence point and suggested the
possibility that an additional form(s) of human Hcam1 mRNA
might exist. Isolation of additional cDNAs will be required to
test this suggestion.
Hcam1 mRNA expression was detected by RNase protection
in RNA extracted from temporal cortex, hippocampus, brain
stem, cerebellum, occipital cortex, heart ventricle, aorta, fem-
oral and renal arteries, psoas skeletal muscle, and uterus (Fig.
4A). No expression was detected in the heart atrium, liver, or
spleen.
Some of the RNA samples used in this RNase protection
(indicated by stars in Fig. 4A) were partially degraded, possibly
due to the length of time between donor death and tissue
acquisition. The presence of protected bands using these sam-
ples indicated that the RNA included molecules that were long
enough to span the RNase protection probe, although the sig-
nals obtained may be underrepresented or absent.
The results obtained in the RNase protections and the North-
ern blot agree reasonably well, although a few discrepancies
were seen. The liver RNA used in the RNase protection showed
no Hcam1 signal, while a different liver sample used in the
Northern blot was positive. The skeletal muscle signal was also
weaker in the RNase protection than might have been expected
from the Northern blot results (again, a different sample). In
the RNase protections, different samples of the heart and the
testes gave qualitatively different results. Whether these dif-
ferences reflect different expression levels in the different tis-
sue donors, differing locations of the tissue within the organ, or
some variation due to technical reasons is unknown.
Northern Analyses of Hcam3—For Hcam3 the cDNAs had
predicted two distinct transcripts, one corresponding to
Hcam3a and one corresponding to Hcam3b. Hybridization
probes specific for each of these were prepared (see “Materials
and Methods”) and used on Northern blots.
The Hcam3a-specific probe hybridized to a 5.6-kb mRNA
present in the heart and brain samples (Fig. 5A). A weaker
hybridization signal was detected in the lung, liver, kidney,
and skeletal muscle samples. The Hcam3b-specific probe hy-
bridized to an approximately 10-kb mRNA present in the brain
and heart, and to a lesser extent in the lung mRNA (Fig. 5B).
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Additional faint bands were also detected with this probe. A
Hcam3 catalytic region probe, which should hybridize to both
transcripts, hybridized to both a 10- and a 5.6-kb transcript as
well as additional smaller transcripts in the heart sample (data
not shown). These results confirmed that both Hcam3a and
Hcam3b represent transcripts that are present in human tis-
sue mRNA. Hcam3 expression was also detected in the uterus
and testes by RNase protection (data not shown).
Sequence Comparison of CaM PDEs—Human Hcam1, bo-
vine 63-kDa CaM PDE (18), and human Hcam3 show 59%
identity of amino acids when the gaps and the divergent car-
boxyl termini are excluded from the comparison (Fig. 6). Com-
pared pairwise, Hcam1 and bovine 63-kDa CaM PDE are 65%
identical as are bovine 63-kDa CaM PDE and Hcam3. Hcam1
and Hcam3 are 77% identical. A partial sequence for the hu-
man counterpart of the bovine 63-kDa CaM PDE has been
reported (19), and it is 97% identical to the bovine sequence.
Thus the greater identity of human Hcam1 and Hcam3 to each
other than to the bovine 63-kDa CaM PDE is probably not a
species difference. Hcam3 differs from Hcam1 and the 63-kDa
CaM PDE by the presence of a 9-amino acid insertion in the
putative calmodulin binding domain. Hcam3a and 3b are also
larger than Hcam1 and the 63-kDa CaM PDE because of the
longer carboxyl termini found in the Hcam3 proteins.
Biochemical Characterization of Hcam1 and Hcam3 Proteins
Expressed in S. cerevisiae—The proteins encoded by Hcam1,
Hcam3a, and Hcam3b were expressed in a strain of yeast that
lacked endogenous PDE activity (YKS76). Expression of full-
length proteins resulted in very low PDE activity that was not
stimulated by 10 mM calmodulin (data not shown). This cannot
be explained by the simple absence of enzyme, because full-
length Hcam1, Hcam3b, and Hcam3a (with the Hcam3b amino
terminus) proteins were detected in these yeast extracts by
Western analysis (data not shown). This result is also not a
simple consequence of the yeast expression system because
full-length human Hcam2 (a human counterpart of the bovine
63-kDa CaM PDE) can be expressed in yeast3 and the activity
of the expressed protein is stimulated by calcium and calmod-
ulin. In contrast to the full-length proteins, amino-truncated
proteins beginning at methionine 141 of Hcam1 (Fig. 1B) or
methionine 150 of Hcam3a (Fig. 2, B and C) gave measurable of
PDE activity and hydrolyzed both cAMP and cGMP (truncated
Hcam3b was not expressed).
3
J. Yu, A. B. Frazier, V. A. Florio, T. J. Martins, S. L. Wolda, E. A. S.
Harris, K. N. McCaw, C. Farrell, L. S. Cousens, B. H. Steiner, J. K.
Bentley, J. A. Beavo, K. Ferguson, R. E. Gelinas, manuscript in
preparation.
 Human Type I Phosphodiesterases
FIG. 6. Comparison of Hcam1, bo-
vine 63-kDa CaM PDE (18), Hcam3a,
and Hcam3b proteins. Boxed residues
are identical in all four proteins. The se-
quence encoded by the Hcam3a cDNA be-
gins at amino acid 146, from that position
through amino acid 630 Hcam3a and
Hcam3b are identical and are shown as
one line of sequence. The unique regions
of Hcam3a and Hcam3b are indicated
separately. Amino acids are numbered on
the right. The putative calmodulin-bind-
ing regions and the phosphodiesterase
catalytic domains are indicated.
The effect of substrate concentration on the initial velocity of
cyclic nucleotide hydrolysis was examined. Hcam1(met141)
had a Km for cAMP 15-fold greater than its Km for cGMP (Fig.
7A, Table II). The specific activity or maximal rate of hydrolysis
for cGMP and cAMP was determined for the yeast extract
(nmolzmin21zmg protein21). This enzyme had a higher maximal
rate of hydrolysis for cAMP than for cGMP, although an accu-
rate estimate for cAMP was not obtained because of the high
substrate concentrations required.
The kinetic parameters of Hcam3a(met150) differed signifi-
cantly from those of Hcam1(met141) (Fig. 7B, Table II).
Hcam3a(met150) had a Km of 0.6 mM for cGMP and 0.3 mM for
cAMP, among the lowest ever reported for a calmodulin-de-
pendent PDE (4). The specific activity or maximal rate of hy-
drolysis for cGMP was approximately 1.2 times that for cAMP.
Eadie-Hoffstee plots (not shown) gave very similar results to
those reported in Table II.
The effects of different PDE inhibitors on the truncated
Hcam1(met141) and Hcam3a(met151) proteins were exam-
ined. IBMX, a relatively nonselective inhibitor, and 8-me-
thoxymethyl IBMX, an inhibitor reported to be more selective
for type I PDEs (45), were equipotent inhibitors of both
Hcam1(met141) and Hcam3a(met150) (Fig. 8, Table III). Cilos-
803
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tamide, a type III PDE inhibitor, and rolipram, a type IV PDE
inhibitor, were relatively poor inhibitors of these recombinant
enzymes. Zaprinast (a type V inhibitor with activity against
type I PDE) and vinpocetine (a type I PDE inhibitor) (46, 47)
inhibit both recombinant enzymes. These data are consistent
with the behavior expected of a type I PDE. Although vinpoc-
etine inhibits both recombinant enzymes, it shows greater po-
tency for Hcam1(met141) than for Hcam3a(met150).
DISCUSSION
We report here the isolation and characterization of cDNAs
encoding two human type I PDEs: Hcam1 and Hcam3. Hcam1
corresponds to the bovine 61-kDa CaM PDE (5, 17). The 535-
amino acid human protein differs from the bovine protein in
two regions (a 14-amino acid insertion and a divergent carboxyl
terminus). RNase protection studies show that the nucleotide
sequences predicted by the human cDNAs are present in RNA
from human tissues, thus these differences are not likely to be
cloning artifacts. The 42-nucleotide insertion is also found in a
rat cDNA (data not shown). The RNase protection studies also
raised the possibility of an additional splice variant near the
39-end of the open reading frame. The bovine 61-kDa CaM PDE
is believed to undergo alternative splicing near the 59-end of
804 Human Type I Phosphodiesterases
the open reading frame since the bovine 59-kDa CaM PDE
differs from the 61-kDa CaM PDE at the amino terminus but is
otherwise identical to it (6). Two different human mRNA tran-
scripts were observed on Northern blots. It is not known
whether these correspond to different splice variants. This
question can be addressed by the isolation of additional cDNAs.
The second human type I PDE reported here is Hcam3. Two
splice variants, which encode proteins that diverge from each
other at the carboxyl terminus, were observed. Hcam3a is
predicted to be 709 amino acids in length (80,759 Da), assum-
ing it has the same 59-end as Hcam3b (see above). Hcam3b is
634 amino acids in length (72,207 Da). The Hcam3b protein
extends only 4 amino acids beyond the Hcam3a/3b divergence
point before terminating, whereas the Hcam3a protein extends
an additional 79 amino acids beyond the divergence point.
The carboxyl-terminal region specific to Hcam3a is very
basic (43). Whether this unique carboxyl terminus of Hcam3a
has any consequences for the enzyme’s catalytic activity, inter-
action with calmodulin, stability, or intracellular localization is

FIG. 7. Hydrolysis of cAMP and cGMP by Hcam1(met141)
(Panel A) and Hcam3a(met150) (Panel B). The graphs in both
panels show enzyme activity as a function of cAMP (EOOE) or cGMP
(●OO●) concentration. For each substrate concentration tested, the
rate of hydrolysis was measured at several protein concentrations. The
rate of hydrolysis as a function of protein concentration was fit by linear
regression. The values (nmolzmin21zmg21) from these linear regressions
and their standard errors (shown by error bars) are plotted in the figure.
When the standard error is small the symbols obscure the error bars.
The data presented in Panel A are summarized in experiment 4
(Hcam1(met141)) of Table II. The data presented in Panel B are sum-
marized in experiment 3 (Hcam3a(met150)) of Table II.
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not known. Some of these possibilities can be tested by com-
paring the properties of Hcam3a and Hcam3b when full-length,
active proteins are expressed. Preliminary results indicated
that the Km values for full-length Hcam3a and Hcam3b are
similar to the data for the truncated Hcam3a reported here.4
A 19-amino acid region that bound calmodulin was identified
in the bovine 61-kDa CaM PDE (amino acids 24 – 42) by pep-
tide/calmodulin binding studies (5). This region is predicted to
form an amphipathic a-helix, a feature often found in calmod-
ulin binding domains (48). The human Hcam1 protein also
contains this region (Fig. 1C). However the human Hcam3
protein has a different amino acid sequence (6/19 amino acids
differ) and contains a nine amino acid insertion (Fig. 6). This
insertion is found at the same relative position as the alterna-
tive splicing that yields the bovine 61- and 59-kDa CaM PDEs.
The Hcam3 protein, both with and without the 9-amino acid
insertion, is predicted to form an amphipathic a-helix, al-
though the insertion alters the amino acids that would be
included. Characterizing the binding of CaM to Hcam3 will
help determine the effect of the amino acid insertion.
Two sites of phosphorylation by cAMP-dependent protein
kinase A have been identified in the bovine 61-kDa CaM PDE
4
V. Florio, L. Uher, P. Snyder, K. Loughney, and K. Ferguson, un-
published observations.

TABLE II
Biochemical characterization of Hcam1 and Hcam3a
cAMP cGMP
Enzyme Exp.a Specific Specific
b b
Km Km
activityb,c activityb,c
mM mM
Hcam1(met141) 1 40 (1) 3.0 (0.2) 0.311 (0.005)
2 53 (1) 3.6 (0.3) 0.376 (0.007)
3 27 (2) 3.4 (0.1) 0.446 (0.005)
4 71 (3) 3.9 (0.1) 0.695 (0.002)
5 73 (2) 4.1 (0.6) 0.66 (0.03)
6 38 (1) 3.2 (0.4) 0.40 (0.01)
Mean 51 (19)d 3.5 (0.4)d 0.5 (0.2)d

Hcam3a(met150) 1 0.31 (0.03) 2.61 (0.05) 0.56 (0.06) 3.14 (0.06)

2 0.40 (0.06) 1.96 (0.05) 0.46 (0.05) 2.34 (0.05)
3 0.29 (0.05) 2.23 (0.06) 0.70 (0.1) 2.97 (0.09)
Mean 0.33 (0.06)d 2.3 (0.3)d 0.57 (0.12)d 2.8 (0.4)d
a
These data were derived from six independent yeast cultures grown from a single transformant expressing Hcam1 and three independent
cultures grown from a single transformant expressing Hcam3. Experiment numbers correspond to particular cultures.
b
Values were determined as described under “Materials and Methods” and the legend for Fig. 7. The value in parentheses is the standard error.
c
nmol z min21 z mg protein21, measured in the yeast extract, accurate values for the specific activity or maximal rate of hydrolysis were not
obtained for Hcam1(met141) and cAMP because of the high substrate concentrations required.
d
The standard deviation of the mean is in parentheses.
 Human Type I Phosphodiesterases 805
in Hcam1 and Hcam3. It is not known whether either the
serine, threonine, or any additional sites are phosphorylated in
the human proteins.
Excluding gaps and divergent termini, Hcam1 and Hcam3
proteins are 77% identical, whereas Hcam1 and the bovine
61-kDa CaM PDE proteins are 94% identical. When one com-
pares the DNA sequences over the corresponding regions of the
cDNAs, they show 76 and 93% identity, respectively. Thus the
analogous genes in two different mammals show higher levels
of identity than do two different type I CaM PDEs in the same
organism. Presumably, this reflects the conservation of both
the regulatory interactions and the specific functions that these
PDEs fill in mammalian cells.
Hcam1 expression was seen in brain, testes, and kidney
samples. Lower levels of expression were seen in heart and
uterus. The pattern of human Hcam1 expression resembled
that of the bovine 59- and 61-kDa CaM PDEs (17). Hcam3 also
showed a similar pattern of expression and was most readily
detected in brain, heart, and testes (data not shown) but was

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also found in lung, uterus (data not shown), and kidney. Hcam1
and Hcam3 were coexpressed in human tissues. Whether this
coexpression extends to the cellular level is unknown.
The biochemical characterization of Hcam1(met141) is con-
sistent with its identification as the human counterpart of the
bovine 61-kDa CaM PDE. The Km value for cGMP (3.5 mM) that
is much lower than the Km value for cAMP (51 mM), and the
response of Hcam1 to inhibitors, are in general agreement
with the literature values for the bovine 61-kDa CaM PDE
(4, 50, 51).
The biochemical properties of Hcam3a(met150) are distinct
from those of Hcam1 and the bovine 63-kDa CaM PDE. Of
these proteins, Hcam3a(met150) has the highest affinities for
both cAMP and cGMP. A variety of CaM PDEs with high
FIG. 8. Effect of selected inhibitors on the hydrolysis of cGMP affinity for both cAMP and cGMP have been reported. A CaM
(0.1 mM) catalyzed by Hcam1(met141) (A) or Hcam3a(met150) (B). PDE with a 1 mM Km for cAMP and cGMP was detected in
The data are expressed as the percent of the total PDE activity meas-
ured in the absence of inhibitor (Hcam1(met141): 10 6 0.3 immature rat testis (12) and in the germ cells of male mice (15).
pmolzmin21zml21; Hcam3a(met150): 615 6 19 pmolzmin21zml21). A 68 –70-kDa CaM PDE was partially purified from mouse
Hcam1(met141) was assayed for 45 min and Hcam3a(met150) was testis (13). This enzyme had a Km of 2 mM for both cAMP and
assayed for 15 min. The inhibitors tested were: IBMX (●), 8-methoxy- cGMP. It cross-reacted with polyclonal rabbit antisera raised
methyl IBMX (E), vinpocetine (f), rolipram (M), cilostamide (å), and
zaprinast (Ç). against the bovine brain CaM PDEs. In view of the size of the
human Hcam3a protein (72 kDa), the presence of Hcam3
TABLE III mRNA in the testis and the low Km for both cAMP and cGMP
Inhibitor effects on Hcam1 and Hcam3a of Hcam3a(met150), one of the splice variants of the Hcam3
IC50a
gene may correspond to the rat testis CaM PDE activity de-
Compound scribed. CaM PDEs with low Km values for cAMP and cGMP
Hcam1(met141) Hcam3a(met150)
have also been reported in heart (8, 9, 14), pancreas (7), and in
mM olfactory receptor neurons (10) and may also correspond to
cGMP 3.4 (0.1)b 0.52 (0.03) Hcam3 gene products. The mouse and rat counterparts of
cAMP 54 (6) 0.36 (0.02)
IBMX 3.0 (0.2) 5.1 (0.5) Hcam3 have been recently isolated and expression of the rat
8-Methoxymethyl IBMX 2.2 (0.2) 4.9 (0.5) gene in olfactory neurons has been detected (52).
Vinpocetine 8.1 (0.9) 50 (8)c A 74-kDa bovine brain CaM PDE has also been identified
Rolipram 180 (29)c 160 (32)c (11). Although Hcam3 and this 74-kDa protein are similar in
Cilostamide 112 (9)c 200 (48)c
Zaprinast 10.1 (1.2) 3.5 (0.6) size and are both expressed in the brain, their kinetic proper-
ties differ. Thus the 74-kDa protein is not likely to be Hcam3
a
Determined using 0.1 mM cGMP as substrate (see “Materials and
Methods”).
and may represent a type I PDE for which cDNAs have not yet
b
The IC50 and its standard error were obtained using a four-param- been isolated.
eter model, except as noted in footnote c. The parameter that deter- Although Hcam1(met141) and Hcam3a(met150) have dis-
mines the slope of the fitted curve at the IC50 ranged from 0.8 to 1. tinctive kinetic parameters, they show similar sensitivities to
c
These IC50 values were obtained using a two-parameter model.
inhibitors. The IC50 values for IBMX, 8-methoxymethyl-IBMX,
rolipram, and cilostamide differed less than 2-fold among these
protein (49). Phosphorylation of the serine at amino acid posi- CaM PDEs. The apparent affinity of Hcam1(met141) for zap-
tion 120 reduced the affinity of the 61-kDa CaM PDE for rinast was about 3-fold lower than that observed for
calmodulin. A serine is also found in this position in Hcam1 and Hcam3a(met150). The largest difference seen was with vinpo-
Hcam3, although the context is slightly different in each pro- cetine where the IC50 value for Hcam1(met141) (8.1 mM) was
tein. The serine at amino acid 138 is also phosphorylated in the 6-fold lower than that observed for Hcam3a(met150) (50 mM).
bovine 61-kDa CaM PDE. A threonine is found in this position The reported IC50 values for inhibition of CaM PDEs by vinpo-
806 Human Type I Phosphodiesterases
cetine vary from 20 to 95 mM among enzymes isolated from
bovine aorta, brain, coronary artery, kidney, and rabbit aorta
(46, 47). This range of values may reflect the particular mixture
of CaM PDEs present in each tissue. The inhibition of the
truncated Hcam1 and Hcam3 PDEs, which do not contain
calmodulin binding regions, is consistent with the hypothesis
that vinpocetine is a catalytic region inhibitor (46).
In contrast to the full-length proteins, amino-truncated
Hcam1 and Hcam3a were active when expressed in yeast. It
has been reported that amino-truncation by proteolysis leads to
activation of bovine type I PDEs (5, 50, 51). The kinetic prop-
erties of the proteolyzed bovine enzymes were similar to the
calmodulin-activated full-length enzymes (50, 51). The amino
termini of the proteolyzed bovine 61-kDa CaM PDEs (5) are
similar in position to the amino terminus of Hcam1(met141),
suggesting that the recombinant truncated Hcam1 is activated
as well. Hcam3a(met150) is truncated at the corresponding
methionine and thus may also be activated.
The presence in tissues of proteins derived from more than
one CaM PDE gene and the observation that different splice
variants of the same gene can generate proteins that appear to
be regulated differently has complicated the interpretation of
studies using a mixture of CaM PDEs isolated from a tissue. An
additional layer of complexity is indicated by the results of the
inhibitor analysis presented here in which some, but not all, of
the PDE inhibitors show slightly different IC50 values with two
human type I PDEs. Continued characterization of each gene
and its products should aid in understanding the cellular roles
of CaM PDEs.
Acknowledgments—We thank Liz Hunter, Carmen Hertel, and Bart
Steiner for excellent technical assistance; Mike Cicirelli for developing
the charcoal-based PDE assay; Kerry Fowler for the synthesis of cilos-
tamide; Ernie Tolentino, Dina Leviten, and Christi Wood for oligonu-
cleotide synthesis; Peter Snyder for the human heart cDNA; and Vince
Florio and Bryan Jones for critically reading the manuscript.
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 Isolation and Characterization of cDNAs Corresponding to Two Human Calcium,
Calmodulin-regulated, 3 ′,5′-Cyclic Nucleotide Phosphodiesterases
Kate Loughney, Timothy J. Martins, Edith A. S. Harris, Krishna Sadhu, James B. Hicks,
William K. Sonnenburg, Joseph A. Beavo and Ken Ferguson
J. Biol. Chem. 1996, 271:796-806.
doi: 10.1074/jbc.271.2.796

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