(Received for publication, March 30, 1995; and in revised form, October 12, 1995)
From the
cDNAs corresponding to two human calcium, calmodulin (CaM)-regulated 3`,5`-cyclic nucleotide phosphodiesterases (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 represented in human RNA from several tissues, including brain, kidney, testes, and heart. Two carboxyl-terminal splice variants for Hcam3 were isolated. One, Hcam3b (PDE1C1), encodes a protein 634 amino acids (72 kDa) in length. The other, Hcam3a (PDE1C3), diverges 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, including brain and heart, and that there may be additional 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 kinetic parameters from Hcam1, which has a higher affinity for cGMP than for cAMP. Both PDE1C enzymes were inhibited by isobutylmethylxanthine, 8-methoxymethyl isobutylmethylxanthine, zaprinast, and vinpocetine.
Cyclic nucleotides are involved in a large number of mammalian
signal transduction pathways. The intracellular concentrations of cAMP
and cGMP reflect their rate of synthesis, by adenylyl and guanylyl
cyclases, and their rate of degradation to 5`-monophosphate
nucleosides, by cyclic nucleotide phosphodiesterases (PDEs). ()A number of biochemically distinct PDEs have been
identified. They fall into seven families distinguished by their
allosteric regulation, substrate kinetics, amino acid sequence
homology, and interaction with specific
inhibitors(1, 2) . All known mammalian PDEs share a
conserved 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
binding of calmodulin in the presence of Ca (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 Ca
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 molecular weight, K values for
cyclic nucleotide substrates, affinities for calmodulin, and regulation
by
phosphorylation(5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16) .
The cDNAs for several of these type I PDEs have been isolated and
characterized(17, 18, 19, 20) . The
existence of type I PDEs with distinct sequences and properties
suggests a diversity of cellular functions for these enzymes. However,
this diversity complicates 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 counterparts 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 corresponding to each gene. Hcam3 shows alternative splicing that would generate phosphodiesterase proteins with different carboxyl termini; this is the first example of carboxyl-terminal splice variants for mammalian type I PDEs.
Figure 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.
Figure 2: A, map of Hcam3 cDNAs. The divergent 3`-ends of the cDNAs are represented by a hatched line (cDNAs He19a and He11a) or a jagged line (cDNA H3a). The two different open reading frames represented by these cDNAs are diagrammed. The portion of cDNA He19a represented as a dashed line has not been completely sequenced. B, nucleotide sequence and predicted protein sequence of Hcam3b (PDE1C1). The Hcam3a/3b divergence 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 nucleotide 611 and the protein expressed by the truncated Hcam3a(met150) construct begins at the methionine underlined at amino acid position 150. The positions of oligonucleotides used for RT-PCR are indicated above the sequence by numbered arrows. Nucleotide and amino acid residues are numbered on the right. C, nucleotide sequence and predicted protein sequence specific to Hcam3a (PDE1C3). The first nucleotide of this sequence corresponds to nucleotide 2067 of Panel B. Following this nucleotide (G) the two sequences diverge. The Hcam3a protein contains a glycine (G) rather than the aspartic acid (D) marked with a diamond in Panel B and extends another 79 amino acids before terminating. The cDNA insert 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. Nucleotide and amino acid residues are numbered on the right.
RNase protection conditions were based on published
methods(24) . The probe was resuspended in 80% formamide, 400
mM NaCl, 40 mM PIPES pH 7, and 1 mM EDTA.
Human total RNA (4-10 µg in 1.7-8.9 µl) was added
to 50-µl aliquots of the probe (5 10
dpm),
denatured at 90-95 °C for 5 min, and allowed to hybridize
overnight at 52 °C. Digestion buffer (350 µl of 50 mM NaCl, 10 mM Tris-HCl, pH 7.5, 5 mM EDTA)
containing 1 unit/µl RNase T1 (Sigma) was added to each sample and
incubated at room temperature for 45 min. The samples were
phenol/chloroform-extracted (200 µl of phenol, 400 µl of
chloroform, 10 µl of 20% SDS, and 1.5 µl 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 analyzed by denaturing acrylamide-urea gel
electrophoresis and autoradiography.
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 construct 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 (nucleotides 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.
YKS17 was generated from SX50-2D by deleting the endogenous PDE2 gene (32) (nucleotides 7-1564 of PDE2 open reading frame deleted) 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 sequencing 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.
The yeast cell pellets were lysed and phosphodiesterase
activity was assayed as described elsewhere (38) with the
following modifications. [P]cAMP and
[
P]cGMP were used (25 Ci/mmol, ICN Biochemical).
The
P
product was separated from the substrate
using charcoal(39, 40) . Two volumes of 25 mg/ml
activated charcoal in 0.1 M KH
PO
were
added to terminate the reaction. Following centrifugation, the
supernatant was removed and quantitated by Cerenkov counting. Extracts
prepared from YKS76 containing only the pBNY6n vector yielded
activities indistinguishable from assay background.
Kinetic and
inhibitor analyses were performed as described previously (38) . Cyclic nucleotide hydrolysis was measured at substrate
concentrations ranging from 0.03 to 100 µM. The specific
activity of the substrate was held constant (0.2 Ci/mmol). The kinetic
data were fitted to the Michaelis-Menton model using
TableCurve (Jandel Scientific). Inhibitor analyses
were performed at 0.1 µM cGMP. At this substrate
concentration the observed IC
should approximate the
apparent inhibitor constant (K
) 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 (TableCurve
) using a four-parameter or a
two-parameter logistic model(38) . The four-parameter model
derives values for the minimum PDE activity, the maximum PDE activity,
and the IC
, as well as a parameter that determines the
slope of the fitted curve at the IC
. The two-parameter
model derives values for the maximum PDE activity and the
IC
. The minimum PDE activity is set to 0, and the
parameter affecting the slope is set to 1. The two-parameter model was
used when the highest concentration of inhibitor was unable to inhibit
fully the enzymatic activity. All other data sets utilized 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 Triangle Park, NC);
and 8-methoxymethyl IBMX was a gift from Jack Wells, Vanderbilt
University (Nashville, TN). Cilostamide was synthesized using published
procedures(41) . Protein concentrations were assayed using a
protein assay kit (Bio-Rad) and published methods(42) .
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.
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 H2a contains a 47-bp insertion following nucleotide 74
and diverges from the Hcam1 composite sequence following nucleotide 807 (Fig. 1A). The 47-bp insertion is in the
5`-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 sequences 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. (
)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.
The sequence of the cDNA
insert in A2d contains 15 nucleotides at its 5`-end that
represent an inverted repeat of sequences found elsewhere in the cDNA
and which are presumably a cloning artifact. It also contains 4
additional nucleotides located just 5` to the poly(A) tail (AGCT
following nucleotide 1999 of Fig. 1B). These may
reflect the use of a second polyadenylation site. The composite Hcam1
sequence (Fig. 1B) has been submitted to GenBank
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 mammalian phosphodiesterases that corresponds to the phosphodiesterase 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-terminal 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).
RT-PCR using human heart cDNA was performed to determine if the Hcam3a 3`-end is associated with the Hcam3b 5`-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 5`-end and a Hcam3a 3`-end. This full-length Hcam3a would encode a 709-amino acid protein with a predicted molecular mass of 80,759. Although the sequence of Hcam3 shares certain characteristics with the human (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 number 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.
Figure 3: Hcam1 Northern blot analysis. The blot was purchased from Clontech. Each lane contains 2 µg of poly(A)-selected mRNA isolated from the tissue indicated. Positions of the RNA size markers are shown on the left.
We used RNase protection to determine if the specific mRNA structure predicted from the Hcam1 cDNAs was present in 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 3`-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 confirmed that both the 42-nucleotide insertion and the human type of 3`-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 5` 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.
Figure 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
(µg) 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. X174 (HaeIII) and Bluescript II
SK
(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 µg of tRNA and was processed in parallel with the
other samples. B, the diagram indicates the position of the
probe used in the protections.
Hcam1 mRNA expression was detected by RNase protection in RNA extracted from temporal cortex, hippocampus, brain stem, cerebellum, occipital cortex, heart ventricle, aorta, femoral 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 samples indicated that the RNA included molecules that were long enough to span the RNase protection probe, although the signals obtained may be underrepresented or absent.
The results obtained in the RNase protections and the Northern 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 differences reflect different expression levels in the different tissue donors, differing locations of the tissue within the organ, or some variation due to technical reasons is unknown.
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 hybridized to an approximately 10-kb mRNA present in the brain and heart, and to a lesser extent in the lung mRNA (Fig. 5B). 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 tissue mRNA. Hcam3 expression was also detected in the uterus and testes by RNase protection (data not shown).
Figure 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).
Figure 6: Comparison of Hcam1, bovine 63-kDa CaM PDE (18) , Hcam3a, and Hcam3b proteins. Boxed residues are identical in all four proteins. The sequence encoded by the Hcam3a cDNA begins 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-binding 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 K for cAMP 15-fold greater than its K
for cGMP (Fig. 7A, Table 2). The specific activity or maximal rate of hydrolysis for
cGMP and cAMP was determined for the yeast extract
(nmol
min
mg protein
).
This enzyme had a higher maximal rate of hydrolysis for cAMP than for
cGMP, although an accurate estimate for cAMP was not obtained because
of the high substrate concentrations required.
Figure 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
(-
) or cGMP (
-
)
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
(nmol
min
mg
) 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 2.
The data presented in Panel B are summarized in experiment 3
(Hcam3a(met150)) of Table 2.
The kinetic
parameters of Hcam3a(met150) differed significantly from those of
Hcam1(met141) (Fig. 7B, Table 2). Hcam3a(met150)
had a K of 0.6 µM for cGMP and 0.3
µM for cAMP, among the lowest ever reported for a
calmodulin-dependent PDE(4) . The specific activity or maximal
rate of hydrolysis for cGMP was approximately 1.2 times that for cAMP.
Eadie-Hoffstee plots (not shown) gave very similar results to those
reported in Table 2.
The effects of different PDE inhibitors on the truncated Hcam1(met141) and Hcam3a(met151) proteins were examined. IBMX, a relatively nonselective inhibitor, and 8-methoxymethyl 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 3). Cilostamide, 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 vinpocetine inhibits both recombinant enzymes, it shows greater potency for Hcam1(met141) than for Hcam3a(met150).
Figure 8:
Effect of selected inhibitors on the
hydrolysis of cGMP (0.1 µM) catalyzed by Hcam1(met141) (A) or Hcam3a(met150) (B). The data are expressed as
the percent of the total PDE activity measured in the absence of
inhibitor (Hcam1(met141): 10 ± 0.3
pmolmin
ml
;
Hcam3a(met150): 615 ± 19
pmol
min
ml
).
Hcam1(met141) was assayed for 45 min and Hcam3a(met150) was assayed for
15 min. The inhibitors tested were: IBMX (
), 8-methoxymethyl IBMX
(
), vinpocetine (
), rolipram (
), cilostamide
(
), and zaprinast (
).
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 3`-end of the open reading frame. The bovine 61-kDa CaM PDE is believed to undergo alternative splicing near the 5`-end of 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 transcripts 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), assuming it has the same 5`-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, interaction
with calmodulin, stability, or intracellular localization is not known.
Some of these possibilities can be tested by comparing the properties
of Hcam3a and Hcam3b when full-length, active proteins are expressed.
Preliminary results indicated that the K values
for full-length Hcam3a and Hcam3b are similar to the data for the
truncated Hcam3a reported here. (
)
A 19-amino acid region
that bound calmodulin was identified in the bovine 61-kDa CaM PDE
(amino acids 24-42) by peptide/calmodulin binding
studies(5) . This region is predicted to form an amphipathic
-helix, a feature often found in calmodulin 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 alternative 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
-helix, although 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 protein (49) . Phosphorylation of the serine at amino acid position 120 reduced the affinity of the 61-kDa CaM PDE for calmodulin. A serine is also found in this position in Hcam1 and Hcam3, although the context is slightly different in each protein. The serine at amino acid 138 is also phosphorylated in the bovine 61-kDa CaM PDE. A threonine is found in this position 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 compares 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 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 consistent with its
identification as the human counterpart of the bovine 61-kDa CaM PDE.
The K value for cGMP (3.5 µM) that is
much lower than the K
value for cAMP (51
µM), 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 affinity for both cAMP and cGMP have been reported. A CaM PDE with
a 1 µMK for cAMP and cGMP was
detected in immature rat testis (12) and in the germ cells of
male mice(15) . A 68-70-kDa CaM PDE was partially
purified from mouse testis(13) . This enzyme had a K
of 2 µM for both cAMP and cGMP. It
cross-reacted with polyclonal rabbit antisera raised against the bovine
brain CaM PDEs. In view of the size of the human Hcam3a protein (72
kDa), the presence of Hcam3 mRNA in the testis and the low K
for both cAMP and cGMP of Hcam3a(met150), one of
the splice variants of the Hcam3 gene may correspond to the rat testis
CaM PDE activity described. CaM PDEs with low K
values for cAMP and cGMP have also been reported in
heart(8, 9, 14) , pancreas(7) , and
in olfactory receptor neurons (10) and may also correspond to
Hcam3 gene products. The mouse and rat counterparts of Hcam3 have been
recently isolated and expression of the rat gene in olfactory neurons
has been detected(52) .
A 74-kDa bovine brain CaM PDE has also been identified(11) . Although Hcam3 and this 74-kDa protein are similar in size and are both expressed in the brain, their kinetic properties differ. Thus the 74-kDa protein is not likely to be Hcam3 and may represent a type I PDE for which cDNAs have not yet been isolated.
Although Hcam1(met141) and Hcam3a(met150) have distinctive
kinetic parameters, they show similar sensitivities to inhibitors. The
IC values for IBMX, 8-methoxymethyl-IBMX, rolipram, and
cilostamide differed less than 2-fold among these CaM PDEs. The
apparent affinity of Hcam1(met141) for zaprinast was about 3-fold lower
than that observed for Hcam3a(met150). The largest difference seen was
with vinpocetine where the IC
value for Hcam1(met141) (8.1
µM) was 6-fold lower than that observed for Hcam3a(met150)
(50 µM). The reported IC
values for
inhibition of CaM PDEs by vinpocetine vary from 20 to 95 µM 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 properties 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 IC 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.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBank(TM)/EMBL Data Bank with accession number(s) U40370 [GenBank](HSPDE1A3), U40371 [GenBank](HSPDE1C1), and U43072 [GenBank](HSPDE1C3).