Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:3.1.4.1 (phosphodiesterase)
18,767 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The products of two phosphodiesterase (PDE) genes (ratPDE3/IVd and ratPDE4/IVb) are present in the rat Sertoli cell in culture, and their expression is under the control of the gonadotropin follicle-stimulating hormone (Swinnen, J.V., Tsikalas, K.E., and Conti, M. (1991) J. Biol. Chem. 266, 18370-18377). To understand the basis of the sequence heterogeneity found in the 5'-region of the different cDNAs thus far characterized, the structure of the coding region of these two cAMP PDE genes was investigated. Analysis of five ratPDE3/IVd and ratPDE4/IVb genomic clones showed that the coding region of these genes expressed in the Sertoli cell is divided into 11 exons distributed over 35-45 kilobases of genomic DNA. The intron/exon boundaries agreed, with some exceptions, with the established consensus sequences and were located in the same position in the coding region of the two genes. Also present were similarities to the exon composition of the Drosophila melanogaster "dunce" gene, the ancestor of these mammalian cAMP PDEs. Multiple AUG codons and short open reading frames were present at the 5'-untranslated end of the ratPDE4/IVb mRNA, but not in the ratPDE3 mRNA. By using polymerase chain reaction amplification or Northern analysis, it was determined that at least two forms of ratPDE3/IVd mRNA are present in rat Sertoli and FRTL-5 thyroid cells, but not in the brain. These mRNA variants are generated by inclusion or removal of an intron sequence that produces a frameshift affecting the position of the initiation AUG codon. Both mRNA species were efficiently translated into cAMP PDE proteins with different molecular masses in a transient transfection assay in COS cells. Polymerase chain reaction amplification demonstrated that heterogeneity of ratPDE4/IVb mRNAs was present in the same location as in the ratPDE3/IVd mRNA. Two ratPDE4/IVb mRNAs with different 5'-ends were expressed in Sertoli and FRTL-5 cells and in the brain. This heterogeneity is caused by the presence of an intron promoter that controls the transcription of this mRNA in Sertoli and FRTL-5 cells, but not in the brain. Upstream exons and additional promoters are probably present and necessary to generate the brain-specific mRNAs. These findings demonstrate that the cAMP-specific PDE genes have complex structure and that cAMP PDE proteins with different amino termini are derived from these genes.
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PMID:Structure of two rat genes coding for closely related rolipram-sensitive cAMP phosphodiesterases. Multiple mRNA variants originate from alternative splicing and multiple start sites. 827 18

The amino acid sequences of all known cGMP-binding phosphodiesterases (PDEs) contain internally homologous repeats (a and b) that are 80-90 residues in length and are arranged in tandem within the putative cGMP-binding domains. In the bovine lung cGMP-binding, cGMP-specific PDE (cGB-PDE or PDE5A), these repeats span residues 228-311 (a) and 410-500 (b). An aspartic acid (residue 289 or 478) that is invariant in repeats a and b of all known cGMP-binding PDEs was changed to alanine by site-directed mutagenesis of cGB-PDE, and wild type (WT) and mutant cGB-PDEs were expressed in COS-7 cells. Purified bovine lung cGB-PDE (native) and WT cGB-PDE displayed identical cGMP-binding kinetics, with approximately 1.8 microM cGMP required for half-maximal saturation. The D289A mutant showed decreased affinity for cGMP (Kd > 10 microM) and the D478A mutant showed increased affinity for cGMP (Kd approximately 0.5 microM) as compared to WT and native cGB-PDE. WT and native cGB-PDE displayed an identical curvilinear profile of cGMP dissociation which was consistent with the presence of distinct slowly dissociating (koff = 0.26 h-1) and rapidly dissociating (koff = 1.00 h-1) sites of cGMP binding. In contrast, the D289A mutant displayed a single koff = 1.24 h-1, which was similar to the calculated koff for the fast site of WT and native cGB-PDE, and the D478A mutant displayed a single koff = 0.29 h-1, which was similar to that calculated for the slow site of WT and native cGB-PDE. These results were consistent with the loss of a slow cGMP-binding site in repeat a of the D289A mutant cGB-PDE, and the loss of a fast site in repeat b of the D478A mutant, suggesting that cGB-PDE possesses two distinct cGMP-binding sites located at repeats a and b, with the invariant aspartic acid being crucial for interaction with cGMP at each site.
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PMID:An essential aspartic acid at each of two allosteric cGMP-binding sites of a cGMP-specific phosphodiesterase. 853 May 5

In order to characterize the structure and regulation of members of the cAMP-specific phosphodiesterase (PDE) family (Type IV PDEs; PDE4 family), we have cloned from the rat a cDNA, pRPDE39, encoding a novel member of this family, which we call RNPDE4A8. Sequencing of the pRPDE39 cDNA shows it to be encoded by the rat PDE4A gene, but to differ from two other PDE4A transcripts, RD1 (pRPDE8; RNPDE4A1) and pRPDE6 (RNPDE4A5), by the presence of a unique region at its 5' end, consistent with alternative mRNA splicing. The pRPDE39 cDNA encodes a predicted protein of 763 amino acids, of which all but 21, located at the extreme amino terminus, are found in the pRPDE6 protein. Expression of pRPDE39 in COS cells produced a protein of 98 +/- 1.4 kDa, as determined by immunoblotting with an antiserum specific to the carboxyl-terminal regions of all PDE4A proteins, compared to a predicted value of 87.5 kDa. RNase protection analysis detected pRPDE39 mRNA only in testis. Immunoblotting of testis extracts demonstrated two bands of 97 +/- 2 and 87 +/- 3 kDa, the larger of which co-migrated with the band seen in COS cells expressing pRPDE39. COS cell expressed pRPDE39 partitioned between a high speed pellet (particulate) fraction (15% of protein; 8% of activity) and a cytosolic fraction. The particulate fraction had a Km for cAMP of 3.3 +/- 0.6 microM, and the cytosolic fraction a Km of 5.4 +/- 2.8 microM. The Vmax values for the pRPDE39 protein, relative to the RD1 protein, were 0.16 +/- 0.06 and 0.29 +/- 0.05 for the particulate and cytosolic forms, respectively. The pRPDE39-encoded PDE activity could not be removed from the particulate fraction by high salt concentrations, or by nonionic detergents. The pRPDE39-encoded enzyme was inhibited by rolipram at an IC50 of 0.5 +/- 0.2 microM for the particulate form and 1.0 +/- 0.2 microM for the cytosolic form, which are values typical of PDE4 family members. The highly tissue-specific distribution of the pRPDE39 mRNA suggest that the pRPDE39 protein functions to modulate a cAMP signaling pathway that is present largely, if not exclusively, in the testis.
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PMID:Alternative splicing of cAMP-specific phosphodiesterase mRNA transcripts. Characterization of a novel tissue-specific isoform, RNPDE4A8. 855 32

Transfection of COS7 cells with a plasmid encoding the human cyclic AMP-specific PDE4A phosphodiesterase PDE-46 (HSPDE4A4B) led to the expression of a rolipram-inhibited PDE4 activity, which contributed approximately 96% of the total COS cell PDE activity. A fusion protein was generated which encompassed residues (788-886) at the extreme C terminus of PDE-46 and was used to generate an antiserum that detected PDE-46 in transfected COS7 cells. Immunoblotting studies identified PDE-46 as a approximately 125-kDa species that was associated with both the soluble and particulate fractions. The relative Vmax of particulate PDE-46 was approximately 56% that of cytosolic PDE-46. Particulate PDE-46 was not solubilized using Triton X-100 or high NaCl concentrations. Immunofluorescence analysis by laser scanning confocal microscopy showed that PDE-46 was located at discrete margins of the cell, indicative of association with membrane cortical regions. The human PDE4A species, h6.1 (HSPDE4A4C), which lacks the N-terminal extension of PDE-46, was found as an entirely soluble species when expressed in COS7 cells. h6.1 was shown to have an approximately 11-fold higher Vmax relative to that of PDE-46. In dose-response studies rolipram inhibited particulate PDE-46 at much lower concentrations (IC50 = 0. 195 microM) than those needed to inhibit the cytosolic enzyme (IC50 = 1.6 microM). The basis of this difference lay in the fact that rolipram served as a simple competitive inhibitor of the cytosol enzyme (Ki = 1.6 microM) but as a partial competitive inhibitor of the particulate enzyme (Ki = 0.037 microM; Ki' = 2.3 microM). Particulate PDE-46 thus showed a approximately 60-fold higher affinity for rolipram than cytosolic PDE-46.
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PMID:The human cyclic AMP-specific phosphodiesterase PDE-46 (HSPDE4A4B) expressed in transfected COS7 cells occurs as both particulate and cytosolic species that exhibit distinct kinetics of inhibition by the antidepressant rolipram. 894 Jan 40

Glucagon elicited a profound increase in the intracellular cAMP concentration of COS-7 cells which had been transiently transfected with a cDNA encoding the rat glucagon receptor and under conditions where cAMP phosphodiesterase activity was fully inhibited. This was achieved in a dose-dependent fashion with an EC50 of 1.8+/-0.4 nM glucagon. In contrast with previous observations made using hepatocytes [Heyworth, Whetton, Kinsella and Houslay (1984) FEBS Lett. 170, 38-42], treatment of transfected COS-7 cells with PMA did not inhibit the ability of glucagon to increase intracellular cAMP levels. PMA-mediated inhibition was not conferred by treatment with okadaic acid, nor by co-transfecting cells with cDNAs encoding various protein kinase C isoforms (PKC-alpha, PKC-betaII and PKC-epsilon) or with the PMA-activated G-protein-receptor kinases GRK2 and GRK3. In contrast, PMA induced the marked inhibition of glucagon-stimulated cAMP production in COS-7 cells that had been co-transfected with a cDNA encoding protein kinase D (PKD). Such inhibition was not due to an action on the catalytic unit of adenylate cyclase, as forskolin-stimulated cAMP production was unchanged by PMA treatment of COS cells that had been co-transfected with both the glucagon receptor and PKD. PKD transcripts were detected in RNA isolated from hepatocytes but not from COS-7 cells. Transcripts for GRK2 were present in hepatocytes but not in COS cells, whereas transcripts for GRK3 were not found in either cell type. It is suggested that PKD may play a role in the regulation of glucagon-stimulated adenylate cyclase.
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PMID:Co-transfection with protein kinase D confers phorbol-ester-mediated inhibition on glucagon-stimulated cAMP accumulation in COS cells transfected to overexpress glucagon receptors. 929 Nov 30

The activity of Na(+)-K(+)-ATPase can be regulated by hormones that activate adenosine 3',5'-cyclic monophosphate-dependent protein kinase (PKA). Here, using a site-directed phosphorylation state-specific antibody, we show that hormonal regulation of Na(+)-K(+)-ATPase can occur via phosphorylation of Ser-943 on its alpha-subunit. cDNAs coding for wild-type rat Na(+)-K(+)-ATPase and Na(+)-K(+)-ATPase in which the PKA phosphorylation site Ser-943 was mutated to Ala were stably and transiently transfected into COS cells. In COS cells expressing wild-type Na(+)-K(+)-ATPase the beta-adrenergic agonist isoproterenol (1 microM) significantly increased the level of phosphorylation of the alpha-subunit. Phosphorylation was accompanied by a significant inhibition of the enzyme activity, as reflected by a decrease in ATP hydrolysis and 86Rb+ transport. The effect of isoproterenol was reproduced by the PKA activator forskolin used in combination with the phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine and was abolished by the specific PKA inhibitor H-89. Okadaic acid, an inhibitor of protein phosphatases 1 and 2A, enhanced phosphorylation and inhibition of Na(+)-K(+)-ATPase induced by isoproterenol. The changes in activity of Na(+)-K(+)-ATPase linearly correlated with the extent of the alpha-subunit of Na(+)-K(+)-ATPase being phosphorylated. When Ser-943 was replaced by alanine, stimulation of the phosphorylation and inhibition of the activity of Na(+)-K(+)-ATPase induced by isoproterenol, alone or in combination with okadaic acid, were not observed. These results indicate that, in intact cells, modulation of the activity of Na(+)-K(+)-ATPase can be achieved by regulation of the state of phosphorylation of Ser-943. Moreover, they provide a biochemical mechanism by which beta-adrenergic agonists can regulate Na(+)-K(+)-ATPase activity.
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PMID:PKA-mediated phosphorylation and inhibition of Na(+)-K(+)-ATPase in response to beta-adrenergic hormone. 931 10

The type 4 phosphodiesterase (PDE) family comprises four enzymes (4A, 4B, 4C and 4D) that are characterized by their specificity for cAMP and selective inhibition by the anti-depressant drug rolipram (4-[3-(cyclopentoxyl)-4-methoxyphenyl]2-pyrrolidone). In common with other PDEs, they consist of a central conserved domain associated with catalytic activity in addition to two N-terminal upstream conserved regions (UCR1 and UCR2) that are unique to the type 4 enzymes. We have isolated a 2 kb cDNA encoding a full-length type 4A PDE{HSPDE4A4B[Bolger, Michaeli, Martins, St.John, Steiner, Rodgers, Riggs, Wigler and Ferguson (1993) Mol. Cell. Biol. 13, 6558-6571]} from a human frontal cortex cDNA library. Northern blot analysis showed that the major PDE4A mRNA of 4.5 kb was widely distributed in different human tissues. The recombinant PDE4A expressed in COS cells had a molecular mass of approx. 117 kDa as revealed by SDS/PAGE/Western blotting with a PDE4A-specific antibody and was specific for cAMP with a Km of 4.8 microM. The enzyme activity was potently inhibited by R-rolipram (IC50 204 nM) and showed a 2.7-fold stereoselectivity over the S enantiomer. Analysis of the kinetics of inhibition indicated that R-rolipram did not behave as a simple competitive inhibitor. Dixon replots suggested that there was more than one mode of interaction consistent with the detection in the enzyme of a high-affinity binding site for R-rolipram with a Kd of 2.3 nM. Truncation of the PDE4A enzyme by deletion mutagenesis showed that neither of the UCRs was required for catalytic activity and identified an approx. 71 kDa core enzyme with a K(m) for cAMP of 3.3 microM. In contrast with the full-length PDE4A, R-rolipram behaved as a simple competitive inhibitor of this form of the enzyme with decreased potency (IC50 1022 nM) and no stereoselectivity. In addition, no high-affinity rolipram-binding site was detected in the truncated enzyme, indicating that this interaction involves sequences upstream of the catalytic domain of the enzyme.
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PMID:Human phosphodiesterase 4A: characterization of full-length and truncated enzymes expressed in COS cells. 933 50

Phosphodiesterase I (EC 3.1.4.1)/nucleotide pyrophosphatase (EC 3.6.1.9) enzymes are a family of type II transmembrane proteins that catalyze the cleavage of phosphodiester and phosphosulfate bonds of a variety of molecules, including deoxynucleotides, NAD, and nucleotide sugars. The human genes for two members of this family have been cloned and designated PC-1 (PDNP1) and PD-Ialpha/autotaxin (PDNP2). We have now cloned the third member of this family from a human prostate cDNA library and designated it human phosphodiesterase-Ibeta (PD-Ibeta). The PD-Ibeta cDNA contains a 2625-bp-long open reading frame which encodes an 875-amino-acid protein. COS-7 cells transfected with an expression vector, pBK-CMV, containing PD-Ibeta cDNA had high phosphodiesterase I activity compared to the mock-transfected cells. By using in situ hybridization to human metaphase chromosomes, we have assigned the locus for the PD-Ibeta (PDNP3) gene to the q22 region of human chromosome 6.
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PMID:Molecular cloning and chromosomal localization of PD-Ibeta (PDNP3), a new member of the human phosphodiesterase I genes. 934 68

5'-Rapid amplification of cDNA ends, done on poly(A)+ RNA from human U87 cells, was used to identify 420 bp of novel 5' sequence of a PDE4B cAMP-specific phosphodiesterase (PDE). This identified an open reading frame encoding a putative 721-residue 'long-form' PDE4B splice variant, which we term HSPDE4B3. HSPDE4B3 differs from the two known PDE4B forms by virtue of its unique 79-residue N-terminal region, compared with the unique N-terminal regions of 94 and 39 residues found in HSPDE4B1 and HSPDE4B2 respectively. In transfected COS7 cells the two long forms, HSPDE4B1 and HSPDE4B3, had molecular masses of approx. 104 and approx. 103 kDa respectively. Expressed in COS-7 cells, the three HSPDE4B isoforms were found in the high-speed supernatant (cytosol) fraction as well as both the high-speed pellet (P2) and low-speed pellet (P1) fractions. All isoforms showed similar Km values for cAMP hydrolysis (1.5-2.6 microM). The maximal activities of the soluble cytosolic activity of the two long forms were very similar, whereas that of the short form, HSPDE4B2, was approx. 4-fold higher. Particulate-associated HSPDE4B1 and HSPDE4B2 were less active (approx. 40%) than their cytosol forms, whereas particulate HSPDE4B3 was similar in activity to its cytosolic form. Particulate and cytosolic forms of HSPDE4B1 and HSPDE4B3 were similarly inhibited by rolipram {4-[3-(cyclopentoxyl)-4-methoxyphenyl]-2-pyrrolidone}, the selective inhibitor of PDE4 (IC50 0.05-0.1 microM), whereas particulate-associated HSPDE4B2 was profoundly (approx. 10-fold) more sensitive (IC50 0.02 microM) to rolipram inhibition than its cytosolic form (IC50 0.2 microM). The various particulate-associated HSPDE4B isoforms showed very different susceptibilities to solubilization with the detergent Triton X-100 and high NaCl concentration. A novel cDNA, called pRPDE74, was obtained by screening a rat olfactory lobe cDNA library. This contained an open reading frame encoding a 721-residue protein that showed approx. 96% amino acid identity with HSPDE4B3 and is proposed to reflect the rat homologue of this human enzyme and is thus called RNPDE4B3. Alternative splicing of mRNA generated from both the human and rat PDE4B genes produces long and short splice variants that have unique N-terminal splice regions. It is suggested that these alternatively spliced regions determine changes in the maximal catalytic activity of the isoforms, their susceptibility to inhibition by rolipram and mode of interaction with particulate fractions.
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PMID:Molecular cloning and transient expression in COS7 cells of a novel human PDE4B cAMP-specific phosphodiesterase, HSPDE4B3. 937 14

We report the cloning, expression, and characterization of a new family of cyclic nucleotide phosphodiesterase (PDE) that has unique kinetic and inhibitor specificities. A clone corresponding to the C terminus of this PDE was initially identified by a bioinformatic approach and used to isolate a cDNA that is likely full-length. This novel PDE, designated as MMPDE9A1, shows highest mRNA expression in kidney with lower levels in liver, lung, and brain. The mRNA size by Northern blot analysis is approximately 2.0 kilobases, and the cDNA encoding PDE9A1 is 1929 base pairs in length. The largest open reading frame predicts a protein of 534 amino acids with a molecular mass of 62,000 Da. When expressed in COS-7 cells, PDE9A1 activity was not inhibited well by either the nonselective inhibitor 3-isobutyl-1-methyl-xanthine or the new selective PDE5 inhibitor, sildenafil, but it is inhibited by the PDE1/5 inhibitor (+)-cis-5,6a, 7,8,9 hyl] phenylmethyl]-5-methyl-cylopent[4,5]imidao[2, 1-b]purin-49(3H)one (SCH51866) with an IC50 of 1.55 microM. This new phosphodiesterase is highly specific for cGMP. Its Km of approximately 0.07 microM for cGMP is the lowest yet reported for a PDE, being at least 40-170 times lower than that of PDE5 and PDE6, respectively.
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PMID:Identification and characterization of a novel family of cyclic nucleotide phosphodiesterases. 962 45


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