From the
cDNAs for two distinct Type III cGMP-inhibited (cGI) cyclic
nucleotide phosphodiesterases (PDE), designated cGIP1 and cGIP2, were
previously cloned from rat adipose and human cardiac cDNA libraries,
respectively. In this study, another cDNA (
Cyclic nucleotide phosphodiesterases (PDEs)
Activities of cGI-PDEs that are regulated by insulin (2, 3, 4, 5) have been extensively
characterized using rat adipocytes(6, 7, 8) , in
which insulin decreases intracellular cAMP and inhibits
lipolysis(9, 10, 11) . cGI-PDEs purified from
human platelets, rat adipose tissue, bovine fat, bovine heart, and
bovine aorta(12, 13, 14, 15, 16, 17, 18) exhibited
similar kinetic properties, substrate affinities, and inhibitor
specificities. Activation of cGI-PDE by insulin involves intracellular
protein kinase(s) but not direct participation of the tyrosine-specific
protein kinase of the insulin
receptor(19, 20, 21, 22) .
Recently
cDNAs encoding a variety of PDEs have been cloned, contributing to our
understanding of PDEs at the molecular level (23-29). Among all
known mammalian PDE cDNAs, a conserved domain consisting of
We previously reported
the presence of insulin-sensitive cGI-PDE in human
placenta(33, 34) . The placental cGI-PDE was purified by
affinity chromatography on Sepharose coupled to a derivative of a
cGI-PDE specific inhibitor, cilostamide. The purified placental cGI-PDE
preparations contained multiple proteins with apparent molecular
weights of 138,000, 83,000, 67,000, 63,000, and 44,000, all of which
reacted with antiserum against platelet cGI-PDE. Ten peptides from
endoproteinase Lys-C digests of the purified placental cGI-PDE were
isolated and sequenced. Sequences of eight peptides were identical to
the deduced amino acid sequences in the C-terminal half of the human
cardiac cGI-PDE(34) . Kinetic properties and inhibitor
specificity of the purified placental cGI-PDE indicated subtle
differences between placental and other cGI-PDEs. To determine at which
level, transcriptional, translational, or post-translational, these
different properties arise, we have carried out molecular cloning of
placental cGI-PDE. In this article, we present evidence consistent with
the existence of an alternative promoter in the human cardiac Type III
PDE gene (cGIP2), from which a 4.4-kb mRNA is transcribed in human
placenta.
For expression of the
When kinetic properties of 80 and 125 kDa
recombinant cGI-PDEs for cGMP were compared, significant differences
were observed in the K
Analysis of human cGIP2 genomic clones
identified the presence of an intron/exon 3 boundary at nt
1291-1292 of the cardiac cGIP2 cDNA, and the exon 3/exon 4
junction at nt 1444-1445 (Fig. 8).
cGI-PDEs are susceptible to proteolysis, and most preparations of
cGI-PDEs contain immunologically related proteins in the
60-135-kDa range (despite the presence of protease inhibitors
during preparation). For instance,
A number of other
studies have identified N-terminal domains as being important in
subcellular localization. Shakur et al.(45) have
reported that deletion of the N terminus prevents association of a
recombinant Type IV cAMP PDE with COS cell membranes. In yeast, two
different sizes of transcripts are produced from SUC2 locus
which encodes invertase(46, 47) . The transcription
start sites for each type are
Alternative transcription and splicing
mechanisms have been reported to generate different protein products
from other PDE mRNAs. The Drosophiladunce
In
summary, we have found that a 4.4-kb transcript for a cGIP2 (Type III
PDE) is significantly expressed in human placenta and HeLa cells. The
transcript shares its sequence with the 7.6-kb transcript but differs
in size. The 4.4- and 7.6-kb transcripts are transcribed from different
transcription start sites of the same gene in a tissue-specific manner
and code for 80- and 125-kDa cGI-PDEs, respectively. These two cGIP2s
differ in the subcellular localization and at least one enzymatic
characteristic, the K
We appreciate the indispensable contribution of Dr.
Vincent C. Manganiello of the NIH to the execution of this work, which
included providing us with recombinant viruses as well as valuable
discussions and sharing unpublished data. We thank Kathy Barbrow and
Belinda Lew for their excellent technical assistance, Drs. Stefano
Giannini, Elisabetta Meacci, and Masato Taira for their contribution to
the early phase of this work, Dr. Linda Iverson for valuable comments,
Dr. John Termini for analysis of secondary structure of mRNA, Dr.
Lu-Hua Wang for supplying the recombinant pVL1393-cGIP2 (
Addendum-After primary submission of this manuscript, Pillai et al.(65) reported biochemical characterization of N
terminus deletion cGI-PDE mutants expressed in yeast. They showed that
significant differences in V
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
4.0 kilobase (kb))
encoding a cGI-PDE of 74 kDa (658 amino acids) was isolated from a
human placental cDNA library. The nucleotide sequence of its open
reading frame was virtually identical to a corresponding region in the
3` portion of the cardiac cGIP2 cDNA (
7.6 kb) which encoded a
125-kDa cGI-PDE (1141 amino acid). Northern blots and RNase
protection assays revealed a prominent 4.4-kb transcript and a 7.6-kb
transcript in human placenta. The transcription start site of the
4.4-kb transcript was assigned to cardiac cDNA nucleotide 1292, the
putative beginning of exon 3 of the human cGIP2 gene, with a potential
translation initiation site 183 bases downstream, as determined by
RNase protection assay. The 5`-flanking region of the 4.4-kb transcript
exhibited promoter activity in HeLa cells which expressed the 4.4-kb
transcript, and contained a TATAA sequence 35 base pairs upstream from
the tentative transcription start site. Recombinant cGI-PDEs, expressed
in Sf9 cells from the 7.6- and 4.0-kb cDNA, exhibited differences in
their subcellular localization and K
for
cGMP. Thus, in human tissues, alternative transcription may contribute
to generating at least two cGIP2 isoforms, cytosolic and
membrane-associated cGI-PDEs with different K
values for cGMP.
(
)catalyze hydrolysis of intracellular second
messengers, cAMP and cGMP, which play important roles in a variety of
signal transduction processes. At least seven distinct mammalian PDE
families have been identified on the basis of molecular cloning and
enzymatic characterization. Type III cGI-PDEs
(
)exhibit a high affinity (``low K
'') for cAMP and are specifically
inhibited by cGMP, cilostamide (OPC 3689), and a number of
pharmacologic agents which increase myocardial contractility, inhibit
platelet aggregation, and increase smooth muscle relaxation(1) .
800
bases in the 3` region apparently serves as the catalytic core of PDEs,
whereas variations found in the 5` region appear to provide specific
functions for PDEs belonging to different families. To date, cDNAs
encoding two different cGI-PDEs, an adipocyte type (cGIP1) and a
cardiac type (cGIP2), have been cloned(30, 31) . The
cDNA encoding cGIP2 (
7.6 kb cDNA containing an open reading frame
of
3.5 kb) was first cloned from a human cardiac cDNA
library(30) . The predicted molecular mass of 125 kDa was in
good agreement with that of cGI-PDE purified from bovine cardiac muscle (17) or immunoprecipitated from solubilized cardiac sarcoplasmic
reticulum fractions after phosphorylation by cAMP-dependent protein
kinase(32) . cDNAs encoding two cGI-PDEs were also cloned from a
rat adipose tissue cDNA library, one partial cDNA similar to the human
cardiac cGI-PDE and a second cGI-PDE cDNA that was different from the
cardiac type, designated as cGIP2 and cGIP1, respectively(31) .
cGIP1 and cGIP2 cDNAs share high homology in their C-terminal regions
(84%) and low homology in their N-terminal regions. cGIP1 mRNA is
expressed in adipocytes but not in heart, while cGIP2 mRNA is expressed
in heart and adipose tissues. The expression of cGIP1 mRNA is induced
dramatically during differentiation of mouse 3T3-L1 fibroblasts to
adipocytes(31) , and correlated with the previously reported
increase in hormone-sensitive cGI-PDE activity in differentiated
adipocytes(1) . The human homolog of the adipocyte type cGI-PDE
(cGIP1) has been cloned from a human genomic and omental cDNA
libraries.
(
)These results indicated the
existence of at least two types of cGI-PDE, adipocyte type (cGIP1) and
cardiac type (cGIP2), in both rat and human.
cDNA Library Screening
An oligonucleotide probe
(58-mer PILA; corresponding to 2029-2086, in Ref. 30; see also Fig. 1) was designed according to the amino acid sequence of the
purified human placental cGI-PDE (34) and human cardiac cGIP2
cDNA sequence(30) . The oligonucleotide (PILA) was 5`-end
labeled with T4 kinase and [-
P]ATP and
purified by DEAE-Sephacel chromatography. A human placental
ZAP II
cDNA library (Stratagene, La Jolla, CA) was screened by plaque
hybridization. Duplicate filters were hybridized with 10
cpm/ml of
P-labeled oligonucleotide (PILA) in
hybridization buffer (10 mM Tris-HCl, pH 7.5, 5
SSC,
10
Denhardt's, 0.1% SDS, 10% dextran sulfate, 0.1 mg/ml
denatured salmon sperm DNA) at 37 °C for 17 h and washed in 6
SSC at 37 °C. A total of 7 positive phagemids from
screening 3
10
plaques were isolated by four rounds
of plaque hybridization screening. The inserts of positive phagemids
were subcloned into pBluescript by in vivo excision in the
presence of Escherichia coli XL1-Blue and R408 helper phage,
and sequenced by a dideoxy chain termination method (35) using
Sequenase version 2.0 (U. S. Biochemical Corp., Cleveland, OH).
Figure 1:
Schematic illustration of
two transcripts for the human cardiac Type III PDE gene (HcGIP2).
Translated regions of two transcripts are shown. The 7.6-kb transcript
contains a membrane-associated region (shaded box) and a
conserved catalytic region (solid box). The 5`-ends of the
placenta cGIP2 cDNA clone 8-1 and PCR products produced from 5`-RACE
are indicated by and
, respectively. The ¦
shows putative exon/intron boundary. The antisense cRNA probe
used in the RNase protection assay, the PILA probe used for the cDNA
library screening, and the 3` primer, ALA-3`, used for the 5`-RACE are
also presented.
Northern Blot Analysis
Total RNA was extracted
from human placenta and HeLa cells with guanidine thiocyanate as
described(36) . mRNA was prepared from the isolated total RNA (1
mg) using Oligotex(dT) (Qiagen, Chatsworth, CA) according to the
manufacturer's instructions, separated in 1% agarose, 2.2 M formaldehyde gels and transferred onto nylon membranes. Following
cross-linking, the membranes were prehybridized for 5 h at 37 °C in
5 SSPE, 2
Denhardt's, 0.1% SDS, 0.1 mg/ml
denatured salmon sperm DNA, 50% formamide, hybridized for 24 h at 50
°C with the placental cGIP2 cDNA (labeled with
[
-
P]dCTP using a random priming kit
(Amersham), washed 5 times with 0.1
SSC, 0.1% SDS at 65 °C
for 30 min each, and exposed to a X-Omat AR film (Kodak, Rochester, NY)
with two intensifying screens for 2 weeks at -75 °C.
5`-RACE (Rapid Amplification of cDNA 5`-End) to Determine
a 5`-End of the Placental cGI-PDE
5`-RACE was performed
according to the manufacturer's protocol (Life Technologies,
Inc., Gaithersburg, MD) with some modifications. Briefly, a
first-strand cDNA was generated with 2 pmol of an oligonucleotide
(37-mer, ALA, see Fig. 1), which corresponds to 109-145
bases downstream from the 5`-end of the placental cGIP2 cDNA, 200 units
of SuperScript reverse transcriptase (Life Technologies, Inc.), and 1
µg of denatured total placental RNA. After 30 min incubation at 42
°C, the template RNA was digested by 2 units of RNase H at 55
°C. The synthesized first strand cDNA was purified using 10 µl
of Qiatex (Qiagen) and the 3`-end of the purified cDNA was tailed with
dCTP using 10 units of terminal deoxynucleotidyltransferase (Life
Technologies, Inc.) and 4 nmol of dCTP. After inactivation of terminal
deoxynucleotidyltransferase, polymerase chain reaction (PCR) was
carried out to amplify the cDNA in the presence of both 5` primer
(5`-GGCCACGCGTCGACTAGTACGGGIIGGGIIGGGIIG-3`) and 3` nested primer
(5`-ACCTGGTCGACTTTGCTTT-3`). The sizes of PCR products were identified
by Southern blotting using an internal probe, or cloned into dideoxy
T-tailed and/or blunt-ended pBluescript (Stratagene) for further
sequencing.
RNase Protection
Assay
HindIII-PstI fragment (corresponding to
nt 1275-1428(30) ) of the cardiac cGIP2 cDNA was cloned
into pBluescript at PstI and HindIII sites. The
plasmid was linearized by digesting with HindIII. Antisense P-labeled cRNA probes (213 bases) were produced by T3 RNA
polymerase (Promega, Madison, WI) in the presence of
[
-
P]CTP (800 Ci/mmol, DuPont NEN) using 0.5
µg of the linearized plasmid DNA as a template. The cRNA probe was
purified by 5% acrylamide, 7 M urea gel electrophoresis. The
purified cRNA probe (1
10
cpm) was hybridized with
20, 2.5, and 11 µg of total RNA preparations from placental
tissues, cardiac tissues, and HeLa cells, respectively. After digestion
with RNase A and RNase T1, protected
P-labeled cRNA was
analyzed by 5% acrylamide, 7 M urea gel electrophoresis.
CAT Assay
Genomic DNAs containing putative
5`-flanking regions of both 4.4- and 7.6-kb transcripts were isolated
by screening a human genomic library (Stratagene) using the cardiac
cGIP2 cDNA as a probe,(
)and designated as P1 and
P2, respectively. The P1 (
1700 bp) and P2 (
2000 bp) fragments
were subcloned into the CAT-basic vector (Promega, Madison, WI) at XbaI and PstI sites, respectively. Orientation of the
inserts was confirmed by restriction enzyme analysis and Southern blot
analysis. HeLa cells were plated at a density of 1
10
cells/cm
in tissue culture dishes (6 cm diameter),
and after 24 h, the cells were washed with serum-free media
(OPTI-reduced media, Life Technologies, Inc.). Varying amounts of the
reporter plasmids were transfected into the cells in the presence of 20
µg of Lipofectin (Life Technologies, Inc.) for 5-10 h. The
cells were washed and harvested at 72 h post-transfection and lysed in
0.25 M Tris-HCl buffer, pH 7.8, by repeating freezing and
thawing 4 times. Cell lysates were incubated at 60 °C to inactivate
endogenous deacetylase and centrifuged at 20,000
g for
2 min. The supernatants were assayed for CAT activity in the presence
of 3 µl of [
C]chloramphenicol (at 0.05
mCi/ml, Amersham), and 5 µl of n-butyryl coenzyme A (at 5
mg/ml, Sigma) in a total volume of 125 µl. The reaction was
terminated by adding 300 µl of mixed xylenes and followed by
centrifugation at 20,000
g for 5 min. The xylene phase
was transferred to a new tube, and back-extracted with 100 µl of
0.25 M Tris-HCl buffer, pH 7.8. The amount of acetylated
[
C]chloramphenicol in the organic phase was
either measured by liquid scintillation counting or visualized by thin
layer chromatography and autoradiography.
Expression of the Two cGIP2 cDNAs in Sf9 Insect
Cells
A 2.0-kb fragment of the coding region of the placenta
cGIP2 cDNA was amplified by PCR in order to create BamHI sites
at both 3`- and 5`-ends of the cDNA. The amplified 2.0-kb fragment,
after its DNA sequence was confirmed, was inserted into pBlueBacIII
transfer vector (Invitrogen, San Diego, CA). Orientation of the insert
was confirmed by restriction enzyme analysis. Sf9 cells were
co-transfected with the transfer vector containing the 2.0 kb of the
coding region of placental cGIP2 cDNA and the genomic DNA of Autographa california nuclear polyhediosis virus (kindly
provided by Dr. Max Summers, Texas A & M University). Recombinant
viruses were screened by monitoring blue color produced from the lacZ gene of recombinant DNA and Western blotting using rabbit
polyclonal antibody against human platelet cGI-PDE(19) . After
three rounds of screening, three independent recombinant viruses were
purified. The recombinant viruses were amplified to a volume of
100 ml, and aliquots were kept at 4 °C for short periods
storage or at -75 °C for long periods storage. The amplified
viruses were used for infection of Sf9 cells.
3.5 kb coding region (
125-kDa protein) of the cardiac cGIP2
cDNA, the
4.0-kb EcoRI fragment (30) was inserted
into the baculovirus transfer vector pVL 1393 (Pharmingen, San Diego,
CA). Transfer to A. california nuclear polyhedriosis virus was
accomplished by homologous recombination of the vector into the
resident polyhedrin gene after calcium phosphate co-transfection into
Sf9 cells with wild type linearized A. california nuclear polyhedriosis virus DNA (a modified baculoviral DNA
containing a lethal mutation that precludes viral propagation)
(Baculogold Transfection kit, Pharmingen, San Diego, CA).
Insect Cell (Sf9) Culture
Sf9 cells were cultured
in Grace's insect media, pH 6.0, supplemented with 0.33% of
Yeastolate, and Lactalbumin Hydrolysate (Difco Laboratories, Detroit,
MI), and 10% fetal calf serum in 25-cm flasks at 27 °C.
For a large scale (50
150 ml) cultures, spinner bottles (Belco,
Vineland, NJ) were used.
Partial Purification of Placental cGI-PDE Expressed in
Insect Cells
Sf9 cells (1
10
cells)
were infected with recombinant viruses at a multiplicity of infection
of 20. On day three post-infection, Sf9 cells were harvested, washed
three times with phosphate-buffered saline, and homogenized in 20 ml of
50 mM Tris-HCl buffer, pH 7.5, containing 154 mM NaCl, 10 µg/ml each of leupeptin, aprotinin, and pepstatin, 20
mMN,O-benzoyl-L-arginine ethyl ester, and
10 mM phenylmethylsulfonyl fluoride. Crude homogenates were
centrifuged at 100,000
g for 90 min. The supernatants
were applied onto a DEAE-cellulose (DE-52) column equilibrated with 50
mM Tris-HCl buffer, pH 7.5, and eluted with a linear gradient
of 0-0.5 M NaCl. The fractions that contained low K
cAMP PDE activity were used to examine
inhibitor sensitivity. Sf9 cells expressing
125-kDa cardiac
cGI-PDE were solubilized in 50 mM Tris-HCl buffer, pH 7.4,
containing 0.5 M NaBr, 5 mM MgCl
, 1%
C
E
, 1 mM EDTA, and 1 mM EGTA. Crude lysates were used for measurements of K
for cAMP and cGMP.
Other Methods
SDS-polyacrylamide slab gel (7.5%)
electrophoresis was carried out according to the method of Laemmli
(37). Western blot analysis was carried out as described previously
(34) with modification using the ECL reagent (Amersham).
Low-K cAMP PDE activity and inhibitor
specificity were determined as described
previously(33, 34) . In order to obtain kinetic
parameters for cGMP of both 125- and 80-kDa cGI-PDEs, two methods were
used. One set was carried out as described (4) using an equal
amount of [
H]cGMP for all the substrate
concentrations and relatively a small amount of
[
H]cGMP (
20,000 cpm). The other assays were
performed as described previously (33, 34) except that
the constant ratio of [
H]cGMP/cold cGMP and
relatively a large amount of [
H]cGMP was used
(
6,000 cpm at the lowest to 500,000 cpm at the highest substrate
concentration).
Molecular Cloning of Human Placental cGIP2
cDNA
Seven clones were isolated by screening a human placental
cDNA library using an oligonucleotide PILA (58-mer) as a probe. By
restriction enzyme mapping and Southern blot analyses, 3 of the 7
clones (8-1, 12-1, and 12-3) were identical and
contained the longest insert of 4.0 kb. Inserts (0.8-1.2 kb) from
other clones hybridized with the PILA probe, but not with the probes
corresponding to the sequences within the catalytic domain. DNA
sequences of both 5` and 3` regions of these inserts did not show
homology to any PDEs. The 4.0-kb insert of clone 8-1 consisted of
a 5` 2.5-kb EcoRI fragment which hybridized with the PILA
probe and a 3` 1.5-kb EcoRI fragment. As depicted in Fig. 1, DNA sequencing of the 2.5-kb fragment revealed that this
clone contained a 2.0-kb open reading frame (with a predicted mass of
74 kDa, 658 amino acids) that shared the same DNA sequence (except one
base at the third position of Ile-730, CT), and thus the same
deduced protein sequence as
two-thirds of 3` coding region of the
cardiac cGIP2 cDNA (corresponding to nt 1456-3448 (30)). The far
upstream region of placental cGIP2 mRNA was determined by the 5`-RACE
method using a 37-mer oligonucleotide, ALA, as a 3` primer,
corresponding to 109-145 bases downstream from the 5`-end of
clone 8-1 (Fig. 1). Of 18 independent clones isolated from the
PCR products, the longest 6 inserts (none >
200 bp) were
sequenced. All of the inserts contained the same sequence as that of
the corresponding part of the cardiac cGIP2 cDNA but were of different
sizes. The 5`-ends of these inserts are shown in Fig. 1. Since
stable secondary structures of far upstream mRNA which could cause an
incomplete extension of a first strand cDNA are not predicted by the
analysis using the program ``mfold 2.2''(38) , it is
not likely that the results from the 5`-RACE reflected artificial
termination points of reverse transcriptase due to the stable secondary
structure.
Northern Blot Analysis
In order to determine the
size of placental cGIP2 mRNA, Northern blot analysis was carried out
using P-labeled 4.0-kb insert of clone 8-1 as a probe.
Although a
7.6-kb transcript has been reported to be present in
human heart (30), only a 4.4-kb transcript was detected in 20 µg of
placental mRNA (Fig. 2). The apparent absence of the 7.6-kb
transcript is probably due to the sensitivity of this method since the
content of the 4.4 kb in placental mRNA was hardly detected. In HeLa
cells, however, an additional
7.6-kb transcript was faintly
detected along with the 4.4-kb transcript that was the major form (Fig. 2).
Figure 2:
Northern blot analysis. Twenty and 2
µg of mRNA prepared from HeLa cells (lanes 1 and 2) and human placental tissue (lanes 4 and 5), and 20 µg of total human placental RNA (lane
3) were probed with P-labeled 4-kb insert of clone
8-1. The
7.6- and
4.4-kb transcripts are indicated by 1 and 2, respectively.
RNase Protection Assay
The results of cDNA cloning
and 5`-RACE indicated that the sequence of the 4.4-kb transcript was
virtually identical to part of the 7.6-kb cGIP2 transcript. Although
the results of 5`-RACE suggested that the 5`-end of the 4.4-kb
transcript was located in the coding region of 7.6-kb transcript, the
precise transcription start site of the 4.4-kb transcript was not
determined. Analysis of genomic DNA fragments of the cGIP2 gene, which
have been isolated by screening a human genomic DNA library, indicated
the presence of a putative intron/exon 3 boundary at nt 1291-1292
of the cardiac cGIP2 cDNA sequence (30) (Fig. 3A). Thus, we hypothesized
that the beginning of exon 3 could be the 5`-end of the 4.4-kb
transcript. In an effort to examine this possibility, RNase protection
assays were undertaken using an antisense cRNA probe containing the
putative 5`-end of the 4.4-kb transcript (Fig. 3A). The
cRNA probe is 213 bases long, consisting of 154 and 59 bases derived
from the cardiac cGIP2 cDNA sequence and the Bluescript sequence,
respectively. As illustrated in Fig. 3A, if transcripts
of
7.6- and 4.4-kb mRNA were present and if the putative
intron/exon 3 boundary is the transcription start site for the 4.4-kb
transcript, two protected cRNAs should be detected, one a completely
protected cRNA of 154 bases and a partially protected cRNA of 137 bases
corresponding to the 4.4-kb transcript. As shown in Fig. 3B, the two predicted bands with different
intensities were detected in all RNA samples examined. Their sizes were
estimated from three different sizes of standard cRNAs. The difference
in the two sizes was in good agreement with the distance from the
5`-end of the cRNA probe and an intron/exon 3 boundary corresponding to
nt 1291-1292 in the cardiac cGIP2 cDNA. Thus, the 137-base
fragment reflected a mRNA which was truncated at a position
corresponding to nt 1292, the putative intron/exon 3 boundary for the
cardiac cGIP2 cDNA, due to either alternative transcription (with the
beginning of exon 3, nt 1292, representing the transcription start site
of the 4.4 kb transcript) or alternative splicing (existence of an
unidentified exon which is spliced out in generation of the 7.6-kb
transcript). In placenta, the 137-base fragment was strongly detected
whereas the 154-base fragment was faintly detected (Fig. 3B,
lane 3). The presence of a protected 154-bp fragment suggested
existence of a longer transcript than the 4.4-kb transcript, presumably
the 7.6 kb in human placenta, which was not detected by Northern blot
probably due to low sensitivity. Conversely, the shorter transcript,
which has not been reported, was also detected in human heart (Fig. 3B, lane 1). In HeLa cells, both transcripts were
significantly expressed although the shorter transcript was the
predominant form (Fig. 3B, lane 2), which is consistent
with the Northern blot result (Fig. 2).
Figure 3:
RNase protection assay. A, cDNA
sequence surrounding the putative 5`-end of the 4.4-kb transcript and
schematic illustration of the cRNA probe used in RNase protection
assays. B, the cRNA probe (1 10
cpm) was
hybridized with 2.5, 20, and 11 µg of total RNA from human heart
and placenta tissues, and HeLa cells, respectively. The protected cRNA
fragments were separated by 5% acrylamide, 7 M urea gel
electrophoresis as described under ``Experimental
Procedures.'' The size of unprotected cRNA probe is 213 bases,
while those of protected cRNA probes are 154 and 137 bases for the 7.6-
and 4.4-kb transcripts, respectively.
Promoter Activity (CAT Assay)
Since the RNase
protection assays indicated that the 5`-end of the 4.4-kb transcript
could be located at the beginning of the exon 3 of cardiac cGIP2 gene,
to determine the possibility of alternative transcription mechanisms
for the 4.4-kb transcript, promoter activities in the 3` portion of the
intron/exon 3 boundary region (P2 fragment) as well as in 5`-flanking
region of exon 1 (P1 fragment) were examined (Fig. 4A).
The P1 fragment (1700 bp) was excised from the genomic clone
containing the putative 5`-end of the 7.6-kb transcript by digesting
with XbaI. The 5`-end of 7.6-kb transcript was tentatively
assigned at the 5`-end of the 7.6-kb cGIP2 cDNA cloned from a human
cardiac cDNA library(30) . As illustrated in Fig. 4A, the P1 fragment contained both
900 bp of
5`-untranslated region and
800 bp of 5`-flanking DNA fragment of
putative 5`-end of the 7.6-kb mRNA. The genomic P2 fragment (
2000
bp) from the intron/exon 3 region was excised from the genomic clone by
digestion with PstI; it consisted of
1,900 bp of the
intron and 137 bases of the 5` portion of exon 3. Using HeLa cells in
which both mRNA species are expressed with the 4.4-kb transcript as a
predominant form, promoter activity was only found in the P2 fragment
inserted into the CAT vector with a correct orientation, P2(+)CAT,
whereas no significant promoter activity was detected from other
constructs including the P2 fragment with an opposite orientation,
P2(-)CAT, and both orientations of the P1 fragment, P1(+)CAT
and P1(-)CAT (Fig. 4B). Fig. 4B shows an autoradiogram in which two forms of acetylated
[
C]chloramphenicol were detected with P2 with a
correct orientation but not with the others. When quantitated by
scintillation counting, CAT activities of HeLa cells transfected with
the CAT, P1(+)CAT, P2(+)CAT, P1(-)CAT, and
P2(-)CAT were 1.46 ± 0.54 (n = 7), 1.12
± 1.07 (n = 10), 8.20 ± 2.13 (n = 5), 1.67 ± 0.28 (n = 4), and 1.87
± 0.42 (n = 4) pmol/3
10
cells, respectively. P2 promoter activity was therefore 5.6-fold
over the control (CAT). The reason that P1 promoter activity was not
detected in HeLa cells could be due to either a low abundance of the
7.6-kb transcript in HeLa cells or the use of an insufficient length of
the 5`-flanking fragment in the P1 fragment.
Figure 4:
CAT assay. A, schematic
illustration of the 5`-flanking regions of 7.6- and 4.4-kb transcripts
used for CAT assays. Genomic DNA fragments, P1-1.7 kb and
P2-2 kb, have been isolated and cloned into the CAT-basic vector
as described under ``Experimental Procedures.'' Intron and
exon boundaries have been compared with the cardiac cGIP2 cDNA sequence
and established consensus sequences for splicing sites, and exons 1 and
3 tentatively assigned. B, HeLa cells were transfected with 1
µg of a CAT-reporter plasmid containing either P1 or P2 fragment
with both orientations, as well as a CAT-reporter plasmid without a
potential promoter region as a negative control. Promoter activity was
measured as described under ``Experimental Procedures.''
Diacetylated and mono-acetylated
[C]chloramphenicol separated by thin layer
chromatography are indicated by 1 and 2, respectively. (+) and
(-) indicate CAT-reporter plasmids with correct and opposite
orientations, respectively.
Characterization of Recombinantly Expressed
cGI-PDEs
The human placental cGIP2 cDNA, clone 8-1, was
expressed in Sf9 insect cells. Three recombinant viruses, 5-4, 5-3, and
13-4, isolated by screening three times using Western blotting with a
polyclonal antibody against human platelet cGI-PDE, produced a high
level of expression of 80-kDa cGI-PDE, in good agreement with the
predicted molecular mass of 74 kDa (Fig. 5). No band was detected
on Western blots of uninfected Sf9 cells or Sf9 cells overexpressing
insulin receptor kinase domain (Ref. 39, data not shown). Specific and
total activities of the expressed placental cGI-PDE in crude extracts
were 2.5 nmol/min/mg protein and 84 nmol/min per 100 ml of Sf9 cell
culture (
2
10
cells), respectively. The
specific and total activities of the expressed placental cGI-PDE in
insect cells were
160 and 150 times higher, respectively, than
those of placental cGI-PDE expressed in E. coli.
Figure 5:
Western blot analysis of the recombinantly
expressed 80-kDa cGI-PDE. Whole cell lysates of Sf9 cells (1
10
cells) uninfected (A, lane 2, and B, lane
1) or infected with the HcGIP2 recombinant virus 5-4 (A, lane
3, and B, lane 2) were separated by SDS-polyacrylamide
gel electrophoresis and immunoblotted by anti-cGI-PDE antibody as
described under ``Experimental
Procedures.''
The
recombinant 80 kDa and purified authentic placental (34) cGI-PDEs exhibited similar K
values for cAMP, 0.50 µM and 0.57
µM, respectively. The recombinant placental cGI-PDE was
sensitive to several PDE inhibitors, such as cGMP, and cilostamide, but
not to other inhibitors such as theophylline and Ro 20-1724, with
ED
values of 0.4, 0.02, >1000, and 300 µM,
respectively. These results were generally in good agreement with the
properties of purified placental cGI-PDE which had inhibitor
sensitivity with ED
values of 0.14, 0.22, and 120
µM for cGMP, cilostamide, and Ro 20-1724,
respectively(34) .
values of the two
forms. K
for cGMP of 125 and 80 kDa
cGI-PDE were 0.46 ± 0.22 µM (n = 8)
and 3.27 ± 1.75 µM (n = 9), which
indicated an apparent
7-fold difference in the K
values. Kinetic experiments comparing
both 125- and 80-kDa cGI-PDEs are shown in Fig. 6.
Figure 6:
Kinetic analysis of the recombinant
cGI-PDEs. Whole lysates from approximately 3 10
and
1
10
cells of Sf9 cells expressing 125-kDa cardiac
cGI-PDE and 80-kDa placental cGI-PDE, respectively, were used for each
assay point. Shown are Lineweaver-Burk plots of one of eight (125 kDa,
) or nine (80 kDa,
) representative experiments. The lines were constructed using the least squares method. The K values of 80-kDa cGI-PDE ranged from 1.3 to 6.8 µM in nine individual preparations. An average and S.D. of eight and
nine preparations were presented in the inset. The difference
in the K values of 125 and 80 kDa was statistically
significant (p < 0.0013). Note that V
values cannot be compared between the two cGI-PDE preparations
since the concentrations of cGI-PDE were not
determined.
Approximately 60% of the 80-kDa cGI-PDE activity was recovered in
the cytosolic fraction when Sf9 cells were disrupted in 50 mM Tris-HCl buffer, pH 7.4, containing 0.145 M NaCl (Fig. 7). In contrast, the recombinant 125-kDa cGI-PDE was barely
solubilized in the same buffer (Fig. 7, less than 5% of total
activity) as determined by both enzyme activity and immunoblotting.
Approximately 20-30% of total activity derived from the 125-kDa
cGI-PDE was, however, solubilized in 50 mM Tris-HCl buffer, pH
7.4, containing 0.5 M NaBr, 5 mM MgCl, 1%
C
E
, 1 mM EDTA, and 1 mM EGTA which is the buffer used for solubilizing the membrane bound
form of rat adipocyte cGI-PDE(19) . In addition, immunostaining
of the insect cells expressing the 125-kDa cGI-PDE showed a peripheral
staining pattern whereas cells expressing 80-kDa cGI-PDE were stained
diffusely (data not shown).
Figure 7:
Subcellular localization of the
recombinant cardiac 125-kDa and placental 80-kDa cGI-PDEs in Sf9 cells. A, whole lysates (H.) and supernatants after 100,000
g
1 h centrifugation (C.) were
subjected to immunoblotting as described under ``Experimental
Procedures.'' Cell extracts equivalent to approximately 1
10
cells were applied to each lane. B, cGI-PDE
activity in cytosolic fractions (100,000
g, 1 h) is
presented as the percent of cGI-PDE activity in whole
lysates.
There Are Two Distinct Sizes of Transcripts for
HcGIP2
Northern blot hybridization with the placental cGIP2 cDNA
and RNase protection assays identified a 4.4-kb transcript in
placenta, HeLa cells, and cardiac tissues, in addition to a previously
described 7.6-kb transcript(30) . The 4.4-kb transcript has been
found in human erythroleukemia (HEL) cells (40) and T84 human
colon carcinoma cells(30) , but has not been previously
characterized. A comparison of the nucleotide sequence of cardiac and
placental cGIP2 cDNAs indicate that the
7.6- and 4.4-kb
transcripts share the same sequence but differ in length, i.e. the
4.4-kb transcript lacks a portion (
1290 bases) of
the 5` region of the 7.6-kb transcript. The 4.0-kb placental and 7.6-kb
cardiac cGIP2 cDNAs contained
2- and 3.3-kb 3`-untranslated
regions, respectively. Analyses of these regions have not been
completed, but restriction mapping indicates possible alternative
splicing in these regions.
Results of
RNase protection assays and 5`-RACE (which did not detect sequences
other than the those of the 7.6-kb cGIP2 cDNA in the extended upstream
region of the 4.0-kb cDNA) suggest that the transcription of
4.4-kb mRNA is initiated at nt 1292, i.e. the beginning
of exon 3. The 5`-end of the 4.4-kb transcript is
183 bp upstream
from the first ATG (in exon 4 of the cGIP2 gene) at which translation
was apparently initiated in Sf9 cells.
Figure 8:
Proposed alternative transcription for
HcGIP2 transcription variants. The 7.6- and 4.4-kb transcripts are
transcribed from the beginning of exon 1 and exon 3, respectively, and
contain 3.3 and 2.0 kb of 3`-untranslated region, respectively. Solid and open boxes indicate translated region and
untranslated region, respectively. Exons 1-4 have been
tentatively assigned. Since exons downstream from exon 4 have not been
analyzed, they are not shown. A first ATG for the 7.6-kb transcript,
located in exon 1, is a putative translation start site for the 125-kDa
cGI-PDE. The ATG for the 80-kDa cGI-PDE is located 25 bases downstream
from the beginning of exon 4, and thus exon 3 and the 5` portion (25
bases) of exon 4 serve as untranslated regions in the 4.4-kb
transcript.
The P2 genomic fragment from
the intron/exon 3 boundary region exhibited significant promoter
activity in HeLa cells, and may very well correspond to the 5`-flanking
promoter region of the 4.4-kb transcript. A TATAA sequence was
found in the P2 fragment 35 base pairs upstream from the tentative
transcription start site. The TATA motif is a component of most
promoters utilized by RNA polymerase II, and factors that bind to the
TATA motif have been found in HeLa cells(41) . Thus, all our
results are consistent with the idea that the
7.6- and 4.4-kb
transcripts are derived from a single gene (Fig. 8), but that the
4.4-kb transcript has a different transcription initiation site from
the
7.6-kb transcript; i.e. perhaps at the intron/exon 3
boundary. In this scheme, exons 1 and 2 are not transcribed in the
4.4-kb transcript. Exon 3 and 25 bp of exon 4, which are part of the
coding region of the 7.6-kb transcript, serve as the untranslated
region for the 4.4-kb transcript (Fig. 8). Since RNase protection
assays do not, however, completely rule out the possible existence of
another unidentified exon, further studies will be required to confirm
the transcription initiation site of the 4.4-kb mRNAs.
Two Mechanisms for Production of Truncated
cGI-PDEs
With nt 1292 at the beginning of exon 3 as the
tentatively assigned 5`-end of the 4.4-kb transcript, a first ATG is
located 183 bases downstream of the 5`-end, which is in exon 4 (Fig. 8). Although the nucleotide context surrounding this ATG
does not confirm to Kozak's rules(42, 43) , this
ATG was used as a translation initiation site in insect cells as judged
by the molecular size (80 kDa) of the expressed protein, which is
in good agreement with the predicted size of 658 amino acid (with a
theoretical mass of
74 kDa) encoded by the placental cGIP2 cDNA.
60-kDa proteins were the
predominant cGI-PDE forms first purified from human
platelets(13) . In later studies, however, rapid immunoisolation
of
P-labeled cGI-PDE (44) and one-step purification
by cilostamide-agarose affinity chromatography (12) indicated that a
105/110-kDa polypeptide (
5% of the purified protein) might
represent the intact platelet cGI-PDE, and that several smaller
immunologically related cGI-PDE fragments (79, 62, and 55/53 kDa forms)
resulted from proteolysis(12) . Similarly, in purified placental
cGI-PDE preparations, we found immunologically related proteins of 135,
83, 67, 63, and 44 kDa, the 83 kDa being the predominant
form(34) . The presence of an apparently intact 135-kDa form is
consistent with the RNase protection assays which suggested the
presence of a
7.6-kb transcript in human placenta. Although the
placental 83-kDa cGI-PDE was the predominant form, we initially
considered it to arise from proteolysis of the 135-kDa material.
Results of the present study, however, suggest that at least some of
the 83-kDa material isolated from placenta represents an intact
placental cGI-PDE translated from the 4.4-kb transcript. While cGI-PDEs
are readily proteolyzed, resulting in recovery of catalytic core
domains of
60-80 kDa, our study suggests an alternative
mechanism for producing truncated cGI-PDEs.
The Recombinant Placental cGI-PDE Exhibits Similar
Characteristics to cGI-PDE Purified from Placenta
Both the
recombinant placental 80 kDa cGI-PDE and authentic purified placenta
cGI-PDE exhibited similar inhibitor specificities and substrate
affinities, including K values for cGMP
higher than those for other purified cGI-PDEs (3-20 versus 0.2-0.3 µM). Furthermore, both recombinant
80-kDa and placental cGI-PDE activities were predominantly recovered in
cytosol fractions. In contrast, a recombinant
125-kDa cardiac
cGI-PDE, also expressed in Sf9 cells, exhibited a low K
for cGMP (
0.4 µM) and
was found predominantly in association with particulate fractions.
Detailed characterization of this recombinant cGI-PDE will be presented
elsewhere.
The deduced sequences of
125-kDa human
cardiac cGIP2 (30) and
122-kDa rat adipocyte cGIP1 predict
hydrophobic domains in the N-terminal region. Studies with N-terminal
deletion and truncated recombinant cardiac cGIP2 and adipocyte cGIP1
indicate that this hydrophobic domain is critical for membrane
association.
Affinity for cGMP as substrate seems to
decrease with removal of the N-terminal region from 125-kDa cardiac
cGI-PDE. Since the catalytic core of cGI-PDE is located in the
C-terminal half, it is probable that the N-terminal regulatory domain
influences cGMP binding to the catalytic domain.
100 bases apart and the shorter mRNA
is transcribed downstream of the first ATG. The enzyme derived from the
longer mRNA contains a signal peptide and is secreted from the cell,
while the enzyme from the shorter mRNA is retained intracellularly.
Transcripts from LEU4 (encoding
-isopropylmalate
synthase; 48), FUM1 (fumarase; 49), TRM1 (tRNA
modification enzyme; 50), and HTS1 (histidine-tRNA synthase;
51, 52) consist of multiple mRNAs differing in the 5`-end, in which the
longer forms produce proteins containing an amino acid sequence
necessary for transporting these enzymes to mitochondria.
Multiple Promoters and/or Alternative Splicing Generate
Multiple Transcripts
A number of reports have described multiple
transcripts produced by use of multiple promoters and/or alternative
splicing. Although in most cases multiple transcripts produced
heterogeneous untranslated regions, in several cases similar to ours,
proteins with different N-terminal sequences were generated. They can
be classified into two types. Human c-Myc(53) , human fibroblast
growth factor(54) , and many virus proteins (55) are
examples of translational control in which two proteins can be
generated from different translation start sites of a single mRNA. For
the second type, including our findings, alternative transcription
start sites generate mRNAs with different coding potentials, with two
transcripts differing only in the length of their 5` terminus; examples
include genes encoding bovine
14-galactosyltranferase(56) , human gelsolin(57) ,
human porphobilinogen deaminase(58) , and human progesterone
receptor(59) .
gene encodes Type IV cAMP PDE whose mutants are known to cause
memory/learning dysfunction in fruit flies. Multiple transcription and
splicing mechanisms result in generation of at least six transcripts in
a tissue-specific manner(60) . So far, five distinct promoter
regions have been proposed, and in combination with alternative
splicing, five PDEs, differing in their N terminus regions, exhibit
altered functions in terms of specific activity and their potential
roles in initial learning or female fertility. Multiple transcripts of
mammalian homologs of the dunce
cAMP PDE, i.e. two and three transcripts for rat Type IVb and Type IVd
PDE, respectively, have also been identified(61) . Obernolte et al. (62) have demonstrated that the expression of
two transcripts for human lymphocyte Type IVa PDE is differently
regulated, i.e. one transcript (4.6 kb) but not the other
(
3.0 kb) is induced by the treatment of lymphocytic 43D cells with
dibutyryl-cAMP. Furthermore, based on RNase protection assays,
Sonnenburg et al. (63, 64) suggested the
existence of tissue-specifically altered 5`-ends in transcripts for
both Type I and II PDEs. Thus, tissue-specific regulation of gene
expression by alternative transcription/splicing mechanisms resulting
in heterogeneity in the N-terminal region but conserving catalytic
regions seems to be a common phenomena in PDE gene families.
for cGMP.
125 kDa)
baculovirus, and Drs. Thomas LeBon, Judy Singer-Sam, and Susan Germaand
for critical reading of this manuscript. Secretarial assistance
provided by Faith Sorensen is greatly appreciated.
and K
for cAMP and inhibitor sensitivity to
cGMP between ``large'' (full-length) and ``short''
(truncated; 631 amino acids) cGI-PDEs. V
and K
for cAMP of truncated cGI-PDEs
increased by 16- and 4.2-fold, respectively. cGMP inhibited the
truncated cGI-PDE less potently (
2-fold) than the full-length
cGI-PDE. This additional information and our conclusion thus indicate
that the N-terminal domain plays significant roles in membrane
association and biochemical properties of cGI-PDE.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.