©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Multiple Transcripts for the Human Cardiac Form of the cGMP-inhibited cAMP Phosphodiesterase (*)

Junko Kasuya , Hideki Goko , Yoko Fujita-Yamaguchi (§)

From the (1)Department of Molecular Genetics, Beckman Research Institute of the City of Hope, Duarte, California 91010

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

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 (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 Kvalues for cGMP.


INTRODUCTION

Cyclic nucleotide phosphodiesterases (PDEs)()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) .

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 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.

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.


EXPERIMENTAL PROCEDURES

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.

For expression of the 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% CE, 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).


RESULTS

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) .

When kinetic properties of 80 and 125 kDa recombinant cGI-PDEs for cGMP were compared, significant differences were observed in the K values of the two forms. Kfor 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% CE, 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.




DISCUSSION

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.

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). 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.

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, 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.

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 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) .

Alternative transcription and splicing mechanisms have been reported to generate different protein products from other PDE mRNAs. The Drosophiladunce 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.

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 Kfor cGMP.


FOOTNOTES

*
This work was supported by National Institutes of Health Grants DK27790 and CA33572 and the Juvenile Diabetes Foundation International. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed: Dept. of Molecular Genetics, Beckman Research Institute of the City of Hope, 1450 E. Duarte Rd., Duarte, CA 91010. Tel.: 818-301-8376; Fax: 818-301-8271.

The abbreviations used are: PDE, cyclic nucleotide phosphodiesterase; cGI, cGMP-inhibited; kb, kilobase(s); PCR, polymerase chain reaction; RACE, rapid amplification of cDNA ends; I, inosine; PAGE, polyacrylamide gel electrophoresis; CAT, chloramphenicol acetyltransferase; bp, base pair(s); nt, nucleotide.

cGIP2 and cGIP1 represent the genes that encode cardiac and adipocyte types of cGI-PDE, respectively.

V. C. Manganiello, personal communications.

J. Kasuya, H. Goko, K. Kato, D. Xu, S. Hockman, V. C. Manganiello, and Y. Fujita-Yamaguchi, unpublished data. The numbers of exons indicated in the whole manuscript are tentatively assigned.


ACKNOWLEDGEMENTS

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 (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.

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 and K for cAMP and inhibitor sensitivity to cGMP between ``large'' (full-length) and ``short'' (truncated; 631 amino acids) cGI-PDEs. V and Kfor 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.


REFERENCES
  1. Manganiello, V. C., Smith, C. J., Degerman, E., and Belfrage, P. (1990) in Cyclic Nucleotide Phosphodiesterases: Structure, Regulation and Drug Design (Beavo, J., and Houslay, M. D., eds) pp. 87-116, John Wiley and Sons, Ltd., Chichester
  2. Kono, T., Robinson, F. W., and Sarver, J. A.(1975) J. Biol. Chem.250, 7826-7835 [Abstract]
  3. Loten, E. G., and Sneyd, J. G. T.(1970) Biochem. J.120, 193-197
  4. Manganiello, V., and Vaughan, M.(1973) J. Biol. Chem.248, 7164-7170 [Abstract/Free Full Text]
  5. Zinman, B., and Hollenberg, C. H.(1974) J. Biol. Chem.249, 2182-2187 [Abstract/Free Full Text]
  6. Gettys, T. W., Vine, A. J., Simonds, M. F., and Corbin, J. D.(1988) J. Biol. Chem.263, 10359-10363 [Abstract/Free Full Text]
  7. Shibata, H., and Kono, T.(1990) Biochem. Biophys. Res. Commun.170, 533-539 [Medline] [Order article via Infotrieve]
  8. Smith, C. J., Vasta, V., Degerman, E., Belfrage, P., and Manganiello, V. C.(1991) J. Biol. Chem.266, 13385-13390 [Abstract/Free Full Text]
  9. Butcher, R. W., Sneyd, J. G. T., Park, C. R., and Sutherland, E. W. (1966) J. Biol. Chem.241, 1651-1653 [Abstract/Free Full Text]
  10. Khoo, J. C., Steinberg, D., Thompson, B., and Mayer, S. E.(1973) J. Biol. Chem.248, 3823-3830 [Abstract/Free Full Text]
  11. Kono, T., and Barham, F. W.(1973) J. Biol. Chem.248, 7417-7426 [Abstract/Free Full Text]
  12. Degerman, E., Moos, M., Jr., Rascon, A., Vasta, V., Meacci, E., Smigh, C. J., Lindgren, S., Andersson, K.-E., Belfrage, P., and Manganiello, V.(1994) Biochim. Biophys. Acta1205, 189-198 [Medline] [Order article via Infotrieve]
  13. Grant, P. G., and Colman, R. W.(1984) Biochemistry23, 1801-1807 [Medline] [Order article via Infotrieve]
  14. Degerman, E., Belfrage, P., Newman, A. H., Rice, K. C., and Manganiello, V. C.(1987) J. Biol. Chem.262, 5797-5807 [Abstract/Free Full Text]
  15. Weber, H. W., and Appleman, M. M.(1982) J. Biol. Chem.257, 5339-5341 [Abstract/Free Full Text]
  16. Degerman, E., Manganiello, V. C., Newman, A. H., Rice, K. C., and Belfrage, P.(1989) Adv. Second Messengers Phosphoprotein Res.12, 171-182
  17. Harrison, S. A., Reifsnyder, D. H., Gallis, B., Cado, G. G., and Beavo, J. A.(1986) Mol. Pharmacol.29, 506-514 [Abstract]
  18. Rascon, A., Belfrage, P., Lindgren, S., Andersson, K. E., Stavenow, L., Rice, K., Newman, A., Manganiello, V. C., and Degerman, E.(1992) Biochem. Biophys. Acta1134, 149-152 [Medline] [Order article via Infotrieve]
  19. Degerman, E., Smith, C. J., Tornqvist, H., Vasta, V., Belfrage, P., and Manganiello, V. C.(1990) Proc. Natl. Acad. Sci. U. S. A.87, 533-537 [Abstract]
  20. Macphee, C. H., Reifsnyder, D. H., Moore, T. A., Lerea, K. M., and Beavo, J. A.(1988) J. Biol. Chem.263, 10353-10358 [Abstract/Free Full Text]
  21. Lopez-Aparicio, P., Rascon, A., Manganiello, V. C., Andersson, K.-E., Belfrage, P., and Degerman, E.(1992) Biochem. Biophys. Res. Commun.186, 517-523 [Medline] [Order article via Infotrieve]
  22. Lopez-Aparicio, P., Belfrage, P., Manganiello, V. C., Kono, T., and Degerman, E.(1993) Biochem. Biophys. Res. Commun.193, 1137-1144 [CrossRef][Medline] [Order article via Infotrieve]
  23. Charbonneau, H., Prusti, R. K., LeTrong, H., Sonnenburg, W. K., Mullaney, P. J., Walsh, K. A., and Beavo, J. A.(1990) Proc. Natl. Acad. Sci. U. S. A.87, 288-292 [Abstract]
  24. Chen, C.-N., Denome, S., and Davis, R. L.(1986) Proc. Natl. Acad. Sci. U. S. A.83, 9313-9317 [Abstract]
  25. Li, T., Volpp, K., and Applebury, M. L.(1990) Proc. Natl. Acad. Sci. U. S. A.87, 293-297 [Abstract]
  26. Livi, G. P., Kmetz, P., McHale, M. M., Cieslinski, L. B., Sathe, G. M., Taylor, D. P., Davis, R. L., Torphy, T. T., and Balcarek, J. M.(1990) Mol. Cell. Biol.10, 2678-2686 [Medline] [Order article via Infotrieve]
  27. LeTrong, H., Beier, N., Sonnenburg, W. K., Stroop, S. D., Walsh, K. A., Beavo, J. A., and Charbonneau, H.(1990) Biochemistry29, 10280-10288 [Medline] [Order article via Infotrieve]
  28. Ovchinnkov, Y. A., Gubanov, V. V., Khramtsov, N. V., Ischenko, K. A., Zagranichny, V. E., Muradov, K. G., Shuvaeva, T. M., and Lipkin, V. M. (1987) FEBS Lett.223, 169-173 [CrossRef][Medline] [Order article via Infotrieve]
  29. Higging, D. G., and Applebury, M. L.(1988) Gene (Amst.) 73, 237-244 [CrossRef][Medline] [Order article via Infotrieve]
  30. Meacci, E., Taira, M., Moos, M., Jr., Smith, C. J., Moversusesian, M. A., Degerman, E., Belfrage, P., and Manganiello, V.(1992) Proc. Natl. Acad. Sci. U. S. A.89, 3721-3725 [Abstract]
  31. Taira, M., Hockman, S. C., Calvo, J. C., Taira, M., Belfrage, P., and Manganiello, V. C.(1993) J. Biol. Chem.268, 18573-18579 [Abstract/Free Full Text]
  32. Smith, C. J., Krall, J., Manganiello, V. C., and Moversusesian, M. A. (1993) Biochem. Biophys. Res. Comm.190, 516-521 [CrossRef][Medline] [Order article via Infotrieve]
  33. Xiong, L., LeBon, T. R., and Fujita-Yamaguchi, Y.(1990) Endocrinology126, 2102-2109 [Abstract]
  34. LeBon, T. R., Kasuya, J., Paxton, R. J., Belfrage, P., Hockman, S., Manganiello, V. C., and Fujita-Yamaguchi, Y.(1992) Endocrinology130, 3265-3274 [Abstract]
  35. Sanger, F., Nicklen, S., and Clousen, A. R.(1977) Proc. Natl. Acad. Sci. U. S. A74, 5463-5467 [Abstract]
  36. Chirgwin, J. M., Przybyla, A. E., MacDonald, R. J., and Rutter, W. J. (1979) Biochemistry18, 5294-5298 [Medline] [Order article via Infotrieve]
  37. Laemmli, U. K.(1970) Nature227, 680-685 [Medline] [Order article via Infotrieve]
  38. Zuker, M.(1989) Science244, 48-52 [Medline] [Order article via Infotrieve]
  39. Yan, P.-F., Li, S.-L., Liang, S.-J., Giannini, S., and Fujita-Yamaguchi, Y.(1993) J. Biol. Chem.268, 22444-22449 [Abstract/Free Full Text]
  40. Cheung, P., Xu, H., and Colman, R. W.(1994) FASEB J. (Part I) Experiment Biology Meeting, Abst. 477, April 24-28, Anaheim, CA
  41. Nakajima, N., Horikoshi, M., and Roeder, R. G.(1988) Mol. Cell. Biol.8, 4028-4040 [Medline] [Order article via Infotrieve]
  42. Kozak, M.(1986) J. Cell Biol.108, 229-241 [Abstract]
  43. Kozak, M.(1987) Cell44, 283-292
  44. Grant, P. G., Mannarino, A. F., and Colman, R. W.(1988) Proc. Natl. Acad. Sci. U. S. A.85, 9071-9075 [Abstract]
  45. Shakur, Y., Pryde, J. G., and Houslay, M. D.(1993) Biochem. J.292, 677-686 [Medline] [Order article via Infotrieve]
  46. Carlson, M., and Botstein, D.(1982) Cell28, 145-154 [Medline] [Order article via Infotrieve]
  47. Perlman, D., and Halvorson, H. O.(1981) Cell25, 525-536 [Medline] [Order article via Infotrieve]
  48. Beltzer, J. P., Chang, L.-F. L., Hinkkanen, A. E., and Kohlhaw, G. B. (1986) J. Biol. Chem.261, 5160-5167 [Abstract/Free Full Text]
  49. Wu, M., and Tzagoloff, A.(1987) J. Biol. Chem.262, 12275-12282 [Abstract/Free Full Text]
  50. Ellis, S. R., Morales, M. J., Li, J.-M., Hopper, A. K., and Martin, N. C.(1986) J. Biol. Chem.261, 9703-9709 [Abstract/Free Full Text]
  51. Chiu, M. I., Mason, T. L., and Fink, G. R.(1992) Genetics132, 987-1001 [Abstract/Free Full Text]
  52. Natsoulis, G., Hilger, F., and Fink, G. R.(1986) Cell46, 235-243 [Medline] [Order article via Infotrieve]
  53. Hann, S. R., King, M. W., Bentley, D. L., Anderson, C. W., and Eisenman, R. N.(1988) Cell52, 185-195 [Medline] [Order article via Infotrieve]
  54. Florkiewicz, R. Z., and Sommer, A.(1989) Proc. Natl. Acad. Sci. U. S. A.86, 3978-3981 [Abstract]
  55. Kozak, M.(1986) Cell46, 481-483
  56. Harduin-Lepers, A., Shaper, J. H., and Shaper, N. L.(1993) J. Biol. Chem.268, 14348-14359 [Abstract/Free Full Text]
  57. Kwiatkowski, D. J., Mehl, R., and Yin, H. L.(1988) J. Cell Biol.106, 375-384 [Abstract]
  58. Chretien, S., Dubart, A., Beaupain, D., Raich, N., Grandchamp, B., Rosa, J., Goossens, M., and Romeo, P.-H.(1988) Proc. Natl. Acad. Sci. U. S. A.85, 6-10 [Abstract]
  59. Kastner, P., Krust, A., Turcotte, B., Stropp, U., Tora, L., Gronemeyer, H., and Chambon, P.(1990) EMBO J.9, 1603-1614 [Abstract]
  60. Qiu, Y., and Davis, R. L.(1993) Genes & Dev.7, 1447-1458
  61. Monaco, L., Vicini, E., and Conti, M.(1994) J. Biol. Chem.269, 347-357 [Abstract/Free Full Text]
  62. Obernolte, R., Bhakta, S., Alvarez, R., Bach, C., Zuppan, P., Mulkins, M., Jarnagin, K., and Shelton, E. R.(1993) Gene129, 239-247 [CrossRef][Medline] [Order article via Infotrieve]
  63. Sonnenburg, W. K., Mullaney, P. J., and Beavo, J. A.(1991) J. Biol. Chem.266, 17655-17661 [Abstract/Free Full Text]
  64. Sonnenburg, W. K., Seger, D., and Beavo, J. A.(1993) J. Biol. Chem.268, 645-652 [Abstract/Free Full Text]
  65. Pillai, R., Fluckiger, S., and Colicelli, J.(1994) J. Biol. Chem.269, 30676-30681 [Abstract/Free Full Text]

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