RESUMO
Phosphodiesterase type 3 isoforms, PDE3A and 3B, are expressed primarily in cardiovascular and adipose tissues, respectively. We previously reported a shorter transcript of 4.4-kb PDE3A which is predominantly transcribed in human placenta, whereas a full-length 7. 6-kb transcript corresponding to the cardiac PDE3A cDNA has not been characterized. Due to unfortunate circumstances created by changes in PDE3 nomenclature, PDE3B gene structure previously reported used PDE3A in its title. Here, we describe PDE3A gene structure, which comprises 16 exons spanning over 130 kb on chromosome 12p12. Two PDE3 isoforms share similar gene organization, but localize to different chromosomes. The most distal transcription initiation site of the PDE3A gene is approximately 1071 bases upstream of the ATG site, suggesting that exon 1 consists of 1071 and 960 bp of untranslated and translated sequences, respectively. The proximal 5'-flanking region, which does not contain TATA-like sequences, exhibited weak but significant promoter activity. Results suggest potential involvement of distal promoter/enhancer and translational regulation for expression of the 7.6-kb transcript.
Assuntos
3',5'-AMP Cíclico Fosfodiesterases/genética , Adipócitos/enzimologia , Isoenzimas/genética , Miocárdio/enzimologia , Sequência de Bases , Domínio Catalítico/genética , Mapeamento Cromossômico , Cromossomos Humanos Par 12/genética , Clonagem Molecular , Nucleotídeo Cíclico Fosfodiesterase do Tipo 3 , Primers do DNA/genética , Éxons , Feminino , Humanos , Hibridização in Situ Fluorescente , Íntrons , Dados de Sequência Molecular , Gravidez , Distribuição Tecidual , Transcrição GênicaRESUMO
cDNAs encoding two PDE-3 or cyclic GMP-inhibited (cGI) cyclic nucleotide phosphodiesterase (PDE) isoforms, RPDE-3B (RcGIP1) and HPDE-3A (HcGIP2), were cloned from rat (R) adipose tissue and human (H) heart cDNA libraries. Deletion and N- and C-terminal truncation mutants were expressed in Escherichia coli in order to define their catalytic core. Active mutants of both RPDE-3B and HPDE-3A included the domain conserved among all PDEs plus additional upstream and downstream sequences. An RPDE-3B mutant consisting of the conserved domain alone and one from which the RPDE-3B 44-amino acid insertion was deleted exhibited little or no activity. All active recombinants exhibited a high affinity (< 1 microM) for cyclic AMP (cAMP) and cyclic GMP (cGMP), were inhibited by cAMP, cGMP, and cilostamide, but not by rolipram, and were photolabeled with [32P]-cGMP. The IC50 values for cGMP inhibition of cAMP hydrolysis were lower for HPDE-3A than for RPDE-3B recombinants. The deduced amino acid sequences of HPDE-3A and RPDE-3B catalytic domains are very similar except for the 44-amino acid insertion not found in other PDEs. It is possible that this insertion may not only distinguish PDE-3 catalytic domains from other PDEs and identify catalytic domains of PDE-3 subfamilies or conserved members of the PDE-3 gene family, but may also be involved in the regulation of sensitivity of PDE-3s to cGMP.
Assuntos
3',5'-AMP Cíclico Fosfodiesterases/biossíntese , 3',5'-AMP Cíclico Fosfodiesterases/genética , GMP Cíclico/fisiologia , Isoenzimas/biossíntese , Isoenzimas/genética , Proteínas Recombinantes de Fusão/biossíntese , Proteínas Recombinantes de Fusão/química , 3',5'-AMP Cíclico Fosfodiesterases/antagonistas & inibidores , Animais , Sítios de Ligação/genética , Western Blotting , Catálise , AMP Cíclico/metabolismo , Nucleotídeo Cíclico Fosfodiesterase do Tipo 3 , Ativação Enzimática/efeitos dos fármacos , Ativação Enzimática/genética , Escherichia coli/enzimologia , Escherichia coli/genética , Humanos , Isoenzimas/antagonistas & inibidores , Dados de Sequência Molecular , Mutagênese Insercional , Estrutura Terciária de Proteína , Ratos , Proteínas Recombinantes de Fusão/antagonistas & inibidores , Deleção de SequênciaRESUMO
Two distinct PDE3 [cyclic GMP-inhibited cyclic nucleotide phosphodiesterase (cGI PDE)] isoforms, cGIP1 and cGIP2, have been identified. Here we report cloning of the cDNA and gene encoding human (H)cGIP1 (classified as PDE3B). The cDNA encodes a protein of 1112 amino acids (approximately 123 kDa). Northern blots indicate that its mRNA is expressed in several adipose tissue depots. The human PDE3B gene is composed of 16 exons spanning more than 114 kb and was localized to chromosome 11p15 by in situ hybridization. Exon/intron boundaries were determined, and genetic polymorphism, confirmed by single-strand conformational polymorphism of DNA from 25 healthy subjects, was demonstrated in exon 4 at nucleotide 1389 (A/G). Two polymorphic dinucleotide repeat sequences were identified in introns 5 and 12.
Assuntos
GMP Cíclico/metabolismo , Glicoproteínas/genética , Sequência de Aminoácidos , Animais , Bandeamento Cromossômico , Mapeamento Cromossômico , Cromossomos Humanos Par 11 , DNA Complementar , DNA Satélite , Humanos , Dados de Sequência Molecular , Splicing de RNA , Ratos , Homologia de Sequência de AminoácidosRESUMO
Two distinct but related cGMP-inhibited cyclic nucleotide phosphodiesterase (cGI PDE) cDNAs were cloned from rat adipose tissue cDNA libraries. The open reading frame (3324 base pairs) of RcGIP1 encodes 1108 amino acids, including a hydrophobic membrane-associated domain in the NH2-terminal portion and, in the COOH-terminal portion, a putative catalytic domain conserved among all mammalian PDEs which is preceded by a putative regulatory domain that contains three consensus cAMP-dependent protein kinase phosphorylation sites and followed by a hydrophilic COOH-terminal domain. The carboxyl-terminal portion including the conserved domain was expressed as a glutathione S-transferase fusion protein and exhibited cAMP PDE activity which was inhibited by cilostamide, a specific cGI PDE inhibitor. RcGIP1 cDNA hybridizes strongly with RNA from isolated adipocytes, and its mRNA increases dramatically during differentiation of 3T3-L1 adipocytes. The deduced sequence of the second partial cDNA clone (RcGIP2 clone 53B) is highly homologous to the corresponding region of human cardiac cGI PDE cDNA. RcGIP2 cDNA hybridized strongly with rat cardiac tissue RNA and weakly if at all with RNA from rat adipocytes or 3T3-L1 fibroblasts or adipocytes. We suggest that RcGIP1 represents the hormone-sensitive, membrane-associated rat adipocyte cGI PDE and RcGIP2, a cGI PDE from vascular elements in rat adipose tissue.
Assuntos
3',5'-AMP Cíclico Fosfodiesterases/genética , Tecido Adiposo/enzimologia , Clonagem Molecular , GMP Cíclico/farmacologia , 3',5'-AMP Cíclico Fosfodiesterases/antagonistas & inibidores , 3',5'-AMP Cíclico Fosfodiesterases/química , Sequência de Aminoácidos , Animais , Sequência de Bases , Plaquetas/enzimologia , Sequência Consenso , DNA/química , DNA/genética , Humanos , Dados de Sequência Molecular , Hibridização de Ácido Nucleico , Fosforilação , Quinolonas/farmacologia , RNA Mensageiro/análise , Ratos , Sequências Reguladoras de Ácido Nucleico , Homologia de SequênciaRESUMO
We previously characterized human placental cytosolic cAMP phosphodiesterase (PDE) and found that two low K(m) cAMP PDE isoforms that were very sensitive to inhibition by cGMP and cilostamide were activated by insulin. As a first step toward understanding the mechanisms by which insulin activates this enzyme, we purified the cGMP-inhibited low K(m) cAMP PDE (cGI-PDE) from human placentas. The enzyme was purified 11,700-fold from a pool of 100,000 x g supernatant fractions of 10-15 placentas by ammonium sulfate precipitation, diethylaminoethyl-cellulose chromatography, and affinity chromatography, using an isothiocyanate derivative of cilostamide (CIT-agarose). The specific activity of the affinity-purified enzyme was 432 +/- 17 nmol/min.mg (mean +/- SD; n = 4). Gel permeation chromatography of the CIT-agarose eluates revealed one protein peak that coincided with PDE activity at an elution position of 135,000 daltons. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of this protein peak and CIT-agarose eluates revealed the same patterns, indicating that the purified PDE preparations contained multiple proteins with apparent mol wt of 138K, 83K, 72K, 67K, 63K, and 44K. The 138K form appears to be an intact enzyme; an analogous approximately 135K form has recently been identified in rat adipocyte particulate fractions by specific immunoprecipitation or Western immunoblots. In addition, other smaller forms eluted at 135,000 daltons on gel permeation chromatography, suggesting that, although proteolyzed, they must have been associated by either noncovalent interactions or disulfide bonds. All of the protein bands observed on the sodium dodecyl sulfate-polyacrylamide electrophoresis gel reacted with rabbit antibodies raised against human platelet cGI-PDE. Ten peptides from endoproteinase Lys-C-digests of the affinity-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 a human heart cGI-PDE cDNA, while those of two peptides were not found in the heart enzyme. The sequences of the eight peptides also matched peptide sequences derived from a purified human platelet cGI-PDE. These results provide evidence that the catalytic C-terminal half domain of the placental insulin-sensitive cGI-PDE shares homology with those of human heart and platelet cGI-PDEs. K(m) and maximum velocity values for cAMP and cGMP were 0.57 microM and 862 nmol/min.mg, and 15 microM and 467 nmol/min.mg, respectively. ED50 values for cGMP, cilostamide, and Ro 20-1724 were 0.12, 0.22, and 120 microM, respectively.(ABSTRACT TRUNCATED AT 400 WORDS)
Assuntos
3',5'-AMP Cíclico Fosfodiesterases/isolamento & purificação , GMP Cíclico/farmacologia , Placenta/enzimologia , 3',5'-AMP Cíclico Fosfodiesterases/antagonistas & inibidores , 3',5'-AMP Cíclico Fosfodiesterases/metabolismo , Sequência de Aminoácidos , Western Blotting , Cromatografia de Afinidade , Cromatografia DEAE-Celulose , Cromatografia em Gel , Citosol/enzimologia , Eletroforese em Gel de Poliacrilamida , Feminino , Humanos , Cinética , Dados de Sequência Molecular , Peso Molecular , Fragmentos de Peptídeos/isolamento & purificação , Inibidores da Agregação Plaquetária/farmacologia , Gravidez , Quinolonas/farmacologiaRESUMO
After photolabelling of purified bovine brain particulate cGMP stimulated phosphodiesterase (PDE) with [32P]cGMP, incubation with V8 Endoproteinase Glu-C produced several fragments; most of the [32P] was associated with smaller fragments (approximately 12-14 kDa), and some occasionally with larger fragments (approximately 55-57 kDa). Partial amino acid sequences were determined for the smaller photolabelled fragments and other peptides. On Western immunoblots, affinity-purified antibodies against a synthetic peptide with a sequence matching part of that of the approximately 12-14 kDa photolabelled material reacted with intact PDE and the approximately 12-14 kDa and approximately 55-57 kDa fragments. Several partial cDNA clones encoding the cGMP-stimulated PDE were isolated from a Lambda Zap II bovine brain cDNA library. Deduced amino acid sequence from one cDNA clone, lambda cGS 3-1, as well as the partial sequence of the approximately 12-14 kDa and other fragments, exhibited considerable identity with amino acid residues 311-921 of the cardiac cGMP-stimulated PDE (Trong et al., 1990), including the putative cGMP-binding domain (Charbonneau et al., 1990). These results further define this cGMP-binding domain and suggest that different cGMP-stimulated PDEs will exhibit considerable homology, at least in their cGMP-binding region(s) and catalytic domains.
Assuntos
3',5'-GMP Cíclico Fosfodiesterases/isolamento & purificação , Encéfalo/enzimologia , GMP Cíclico/isolamento & purificação , Miocárdio/enzimologia , 3',5'-GMP Cíclico Fosfodiesterases/genética , 3',5'-GMP Cíclico Fosfodiesterases/imunologia , Marcadores de Afinidade , Sequência de Aminoácidos , Animais , Sequência de Bases , Sítios de Ligação , Western Blotting , Bovinos , DNA/genética , DNA/isolamento & purificação , Eletroforese em Gel de Poliacrilamida , Masculino , Dados de Sequência Molecular , Sondas de Oligonucleotídeos , Oligopeptídeos/síntese química , Oligopeptídeos/genética , Oligopeptídeos/imunologia , Fragmentos de Peptídeos , CoelhosRESUMO
In the absence of detergent, approximately 80-85% of the total cGMP-stimulated phosphodiesterase (PDE) activity in bovine brain was associated with washed particulate fractions; approximately 85-90% of the calmodulin-sensitive PDE was soluble. Particulate cGMP-stimulated PDE was higher in cerebral cortical gray matter than in other regions. Homogenization of the brain particulate fraction in 1% Lubrol increased cGMP-stimulated activity approximately 100% and calmodulin-stimulated approximately 400-500%. Although 1% Lubrol readily solubilized these PDE activities, approximately 75% of the cAMP PDE activity (0.5 microM [3H]cAMP) that was not affected by cGMP was not solubilized. This cAMP PDE activity was very sensitive to inhibition by Rolipram but not cilostamide. Thus, three different PDE types, i.e., cGMP stimulated, calmodulin sensitive, and Rolipram inhibited, are associated in different ways with crude bovine brain particulate fractions. After solubilization and purification by chromatography on cGMP-agarose, heparin-agarose, and Superose 6, the brain particulate cGMP-stimulated PDE cross-reacted with antibody raised against a cGMP-stimulated PDE purified from calf liver supernatant. The brain enzyme exhibited a slightly greater subunit Mr than did soluble forms from calf liver or bovine brain, as evidenced by protein staining or immunoblotting after polyacrylamide gel electrophoresis under denaturing conditions. Incubation of brain particulate and liver soluble cGMP-stimulated PDEs with V8 protease produced several peptides of similar size, as well as at least two distinct fragments of approximately 27 kDa from the brain and approximately 23 kDa from the liver enzyme.(ABSTRACT TRUNCATED AT 250 WORDS)