• We are sorry, but NCBI web applications do not support your browser and may not function properly. More information
Logo of biochemjBJ Latest papers and much more!
Biochem J. Dec 1, 1997; 328(Pt 2): 539–548.
PMCID: PMC1218953

Characterization of five different proteins produced by alternatively spliced mRNAs from the human cAMP-specific phosphodiesterase PDE4D gene.

Abstract

We have isolated and characterized complete cDNAs for two isoforms (HSPDE4D4 and HSPDE4A5) encoded by the human PDE4D gene, one of four genes that encode cAMP-specific rolipram-inhibited 3',5'-cyclic nucleotide phosphodiesterases (type IVPDEs; PDE4 family). The HSPDE4D4 and HSPDE4D5 cDNAs encode proteins of 810 and 746 amino acids respectively. A comparison of the nucleotide sequences of these two cDNAs with those encoding the three other human PDE4D proteins (HSPDE4D1, HSPDE4D2 and HSPDE4D3) demonstrates that each corresponding mRNA transcript has a unique region of sequence at or near its 5'-end, consistent with alternative mRNA splicing. Transient expression of the five cDNAs in monkey COS-7 cells produced proteins of apparent molecular mass under denaturing conditions of 68, 68, 95, 119 and 105 kDa for isoforms HSPDE4D1-5 respectively. Immunoblotting of human cell lines and rat brain demonstrated the presence of species that co-migrated with the proteins produced in COS-7 cells. COS-cell-expressed and native HSPDE4D1 and HSPDE4D2 were found to exist only in the cytosol, whereas HSPDE4D3, HSPDE4D4 and HSPDE4D5 were found in both cytosolic and particulate fractions. The IC50 values for the selective PDE4 inhibitor rolipram for the cytosolic forms of the five enzymes were similar (0.05-0.14 microM), whereas they were 2-7-fold higher for the particulate forms of HSPDE4D3 and HSPDE4D5 (0.32 and 0.59 microM respectively), than for the corresponding cytosolic forms. Our data indicate that the N-terminal regions of the HSPDE4D3, HSPDE4D4 and HSPDE4D5 proteins, which are derived from alternatively spliced regions of their mRNAs, are important in determining their subcellular localization, activity and differential sensitivity to inhibitors.

Full Text

The Full Text of this article is available as a PDF (558K).

Selected References

These references are in PubMed. This may not be the complete list of references from this article.
  • Bolger GB. Molecular biology of the cyclic AMP-specific cyclic nucleotide phosphodiesterases: a diverse family of regulatory enzymes. Cell Signal. 1994 Nov;6(8):851–859. [PubMed]
  • Beavo JA. Cyclic nucleotide phosphodiesterases: functional implications of multiple isoforms. Physiol Rev. 1995 Oct;75(4):725–748. [PubMed]
  • Conti M, Nemoz G, Sette C, Vicini E. Recent progress in understanding the hormonal regulation of phosphodiesterases. Endocr Rev. 1995 Jun;16(3):370–389. [PubMed]
  • Qiu Y, Davis RL. Genetic dissection of the learning/memory gene dunce of Drosophila melanogaster. Genes Dev. 1993 Jul;7(7B):1447–1458. [PubMed]
  • Qiu YH, Chen CN, Malone T, Richter L, Beckendorf SK, Davis RL. Characterization of the memory gene dunce of Drosophila melanogaster. J Mol Biol. 1991 Dec 5;222(3):553–565. [PubMed]
  • Davis RL. Mushroom bodies and Drosophila learning. Neuron. 1993 Jul;11(1):1–14. [PubMed]
  • Swinnen JV, Joseph DR, Conti M. The mRNA encoding a high-affinity cAMP phosphodiesterase is regulated by hormones and cAMP. Proc Natl Acad Sci U S A. 1989 Nov;86(21):8197–8201. [PMC free article] [PubMed]
  • Jin SL, Swinnen JV, Conti M. Characterization of the structure of a low Km, rolipram-sensitive cAMP phosphodiesterase. Mapping of the catalytic domain. J Biol Chem. 1992 Sep 15;267(26):18929–18939. [PubMed]
  • Sette C, Vicini E, Conti M. The ratPDE3/IVd phosphodiesterase gene codes for multiple proteins differentially activated by cAMP-dependent protein kinase. J Biol Chem. 1994 Jul 15;269(28):18271–18274. [PubMed]
  • Alvarez R, Sette C, Yang D, Eglen RM, Wilhelm R, Shelton ER, Conti M. Activation and selective inhibition of a cyclic AMP-specific phosphodiesterase, PDE-4D3. Mol Pharmacol. 1995 Oct;48(4):616–622. [PubMed]
  • Bolger GB, Rodgers L, Riggs M. Differential CNS expression of alternative mRNA isoforms of the mammalian genes encoding cAMP-specific phosphodiesterases. Gene. 1994 Nov 18;149(2):237–244. [PubMed]
  • Bolger G, Michaeli T, Martins T, St John T, Steiner B, Rodgers L, Riggs M, Wigler M, Ferguson K. A family of human phosphodiesterases homologous to the dunce learning and memory gene product of Drosophila melanogaster are potential targets for antidepressant drugs. Mol Cell Biol. 1993 Oct;13(10):6558–6571. [PMC free article] [PubMed]
  • Némoz G, Zhang R, Sette C, Conti M. Identification of cyclic AMP-phosphodiesterase variants from the PDE4D gene expressed in human peripheral mononuclear cells. FEBS Lett. 1996 Apr 8;384(1):97–102. [PubMed]
  • Verghese MW, McConnell RT, Lenhard JM, Hamacher L, Jin SL. Regulation of distinct cyclic AMP-specific phosphodiesterase (phosphodiesterase type 4) isozymes in human monocytic cells. Mol Pharmacol. 1995 Jun;47(6):1164–1171. [PubMed]
  • Sette C, Iona S, Conti M. The short-term activation of a rolipram-sensitive, cAMP-specific phosphodiesterase by thyroid-stimulating hormone in thyroid FRTL-5 cells is mediated by a cAMP-dependent phosphorylation. J Biol Chem. 1994 Mar 25;269(12):9245–9252. [PubMed]
  • Monaco L, Vicini E, Conti M. Structure of two rat genes coding for closely related rolipram-sensitive cAMP phosphodiesterases. Multiple mRNA variants originate from alternative splicing and multiple start sites. J Biol Chem. 1994 Jan 7;269(1):347–357. [PubMed]
  • Short JM, Fernandez JM, Sorge JA, Huse WD. Lambda ZAP: a bacteriophage lambda expression vector with in vivo excision properties. Nucleic Acids Res. 1988 Aug 11;16(15):7583–7600. [PMC free article] [PubMed]
  • Nishikori M, Hansen H, Jhanwar S, Fried J, Sordillo P, Koziner B, Lloyd K, Clarkson B. Establishment of a near-tetraploid B-cell lymphoma line with duplication of the 8;14 translocation. Cancer Genet Cytogenet. 1984 May;12(1):39–50. [PubMed]
  • Loughney K, Martins TJ, Harris EA, Sadhu K, Hicks JB, Sonnenburg WK, Beavo JA, Ferguson K. Isolation and characterization of cDNAs corresponding to two human calcium, calmodulin-regulated, 3',5'-cyclic nucleotide phosphodiesterases. J Biol Chem. 1996 Jan 12;271(2):796–806. [PubMed]
  • Bolger GB, McPhee I, Houslay MD. Alternative splicing of cAMP-specific phosphodiesterase mRNA transcripts. Characterization of a novel tissue-specific isoform, RNPDE4A8. J Biol Chem. 1996 Jan 12;271(2):1065–1071. [PubMed]
  • Feng DF, Doolittle RF. Progressive sequence alignment as a prerequisite to correct phylogenetic trees. J Mol Evol. 1987;25(4):351–360. [PubMed]
  • MacDonald RJ, Swift GH, Przybyla AE, Chirgwin JM. Isolation of RNA using guanidinium salts. Methods Enzymol. 1987;152:219–227. [PubMed]
  • McPhee I, Pooley L, Lobban M, Bolger G, Houslay MD. Identification, characterization and regional distribution in brain of RPDE-6 (RNPDE4A5), a novel splice variant of the PDE4A cyclic AMP phosphodiesterase family. Biochem J. 1995 Sep 15;310(Pt 3):965–974. [PMC free article] [PubMed]
  • Huston E, Pooley L, Julien P, Scotland G, McPhee I, Sullivan M, Bolger G, Houslay MD. 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. J Biol Chem. 1996 Dec 6;271(49):31334–31344. [PubMed]
  • Kreis TE. Microinjected antibodies against the cytoplasmic domain of vesicular stomatitis virus glycoprotein block its transport to the cell surface. EMBO J. 1986 May;5(5):931–941. [PMC free article] [PubMed]
  • Erdogan S, Houslay MD. Challenge of human Jurkat T-cells with the adenylate cyclase activator forskolin elicits major changes in cAMP phosphodiesterase (PDE) expression by up-regulating PDE3 and inducing PDE4D1 and PDE4D2 splice variants as well as down-regulating a novel PDE4A splice variant. Biochem J. 1997 Jan 1;321(Pt 1):165–175. [PMC free article] [PubMed]
  • Laemmli UK. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970 Aug 15;227(5259):680–685. [PubMed]
  • Lobban M, Shakur Y, Beattie J, Houslay MD. Identification of two splice variant forms of type-IVB cyclic AMP phosphodiesterase, DPD (rPDE-IVB1) and PDE-4 (rPDE-IVB2) in brain: selective localization in membrane and cytosolic compartments and differential expression in various brain regions. Biochem J. 1994 Dec 1;304(Pt 2):399–406. [PMC free article] [PubMed]
  • Thompson WJ, Appleman MM. Multiple cyclic nucleotide phosphodiesterase activities from rat brain. Biochemistry. 1971 Jan 19;10(2):311–316. [PubMed]
  • Marchmont RJ, Houslay MD. A peripheral and an intrinsic enzyme constitute the cyclic AMP phosphodiesterase activity of rat liver plasma membranes. Biochem J. 1980 May 1;187(2):381–392. [PMC free article] [PubMed]
  • Bradford MM. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem. 1976 May 7;72:248–254. [PubMed]
  • Shakur Y, Wilson M, Pooley L, Lobban M, Griffiths SL, Campbell AM, Beattie J, Daly C, Houslay MD. Identification and characterization of the type-IVA cyclic AMP-specific phosphodiesterase RD1 as a membrane-bound protein expressed in cerebellum. Biochem J. 1995 Mar 15;306(Pt 3):801–809. [PMC free article] [PubMed]
  • Baecker PA, Obernolte R, Bach C, Yee C, Shelton ER. Isolation of a cDNA encoding a human rolipram-sensitive cyclic AMP phosphodiesterase (PDE IVD). Gene. 1994 Jan 28;138(1-2):253–256. [PubMed]
  • Kozak M. The scanning model for translation: an update. J Cell Biol. 1989 Feb;108(2):229–241. [PMC free article] [PubMed]
  • Swinnen JV, Tsikalas KE, Conti M. Properties and hormonal regulation of two structurally related cAMP phosphodiesterases from the rat Sertoli cell. J Biol Chem. 1991 Sep 25;266(27):18370–18377. [PubMed]
  • Morena AR, Boitani C, de Grossi S, Stefanini M, Conti M. Stage and cell-specific expression of the adenosine 3',5' monophosphate-phosphodiesterase genes in the rat seminiferous epithelium. Endocrinology. 1995 Feb;136(2):687–695. [PubMed]
  • Nemoz G, Sette C, Hess M, Muca C, Vallar L, Conti M. Activation of cyclic nucleotide phosphodiesterases in FRTL-5 thyroid cells expressing a constitutively active Gs alpha. Mol Endocrinol. 1995 Oct;9(10):1279–1287. [PubMed]
  • Sette C, Conti M. Phosphorylation and activation of a cAMP-specific phosphodiesterase by the cAMP-dependent protein kinase. Involvement of serine 54 in the enzyme activation. J Biol Chem. 1996 Jul 12;271(28):16526–16534. [PubMed]
  • Scotland G, Houslay MD. Chimeric constructs show that the unique N-terminal domain of the cyclic AMP phosphodiesterase RD1 (RNPDE4A1A; rPDE-IVA1) can confer membrane association upon the normally cytosolic protein chloramphenicol acetyltransferase. Biochem J. 1995 Jun 1;308(Pt 2):673–681. [PMC free article] [PubMed]
  • Livi GP, Kmetz P, McHale MM, Cieslinski LB, Sathe GM, Taylor DP, Davis RL, Torphy TJ, Balcarek JM. Cloning and expression of cDNA for a human low-Km, rolipram-sensitive cyclic AMP phosphodiesterase. Mol Cell Biol. 1990 Jun;10(6):2678–2686. [PMC free article] [PubMed]
  • Torphy TJ, Stadel JM, Burman M, Cieslinski LB, McLaughlin MM, White JR, Livi GP. Coexpression of human cAMP-specific phosphodiesterase activity and high affinity rolipram binding in yeast. J Biol Chem. 1992 Jan 25;267(3):1798–1804. [PubMed]
  • McLaughlin MM, Cieslinski LB, Burman M, Torphy TJ, Livi GP. A low-Km, rolipram-sensitive, cAMP-specific phosphodiesterase from human brain. Cloning and expression of cDNA, biochemical characterization of recombinant protein, and tissue distribution of mRNA. J Biol Chem. 1993 Mar 25;268(9):6470–6476. [PubMed]
  • Pillai R, Kytle K, Reyes A, Colicelli J. Use of a yeast expression system for the isolation and analysis of drug-resistant mutants of a mammalian phosphodiesterase. Proc Natl Acad Sci U S A. 1993 Dec 15;90(24):11970–11974. [PMC free article] [PubMed]

Articles from Biochemical Journal are provided here courtesy of The Biochemical Society

Formats:

Related citations in PubMed

See reviews...See all...

Cited by other articles in PMC

See all...

Links

  • Compound
    Compound
    PubChem Compound links
  • Gene
    Gene
    Gene links
  • Gene (nucleotide)
    Gene (nucleotide)
    Records in Gene identified from shared sequence links
  • GEO Profiles
    GEO Profiles
    Related GEO records
  • HomoloGene
    HomoloGene
    HomoloGene links
  • MedGen
    MedGen
    Related information in MedGen
  • Nucleotide
    Nucleotide
    Published Nucleotide sequences
  • OMIM
    OMIM
    OMIM record citing PubMed
  • Pathways + GO
    Pathways + GO
    Pathways, annotations and biological systems (BioSystems) that cite the current article.
  • Protein
    Protein
    Published protein sequences
  • PubMed
    PubMed
    PubMed citations for these articles
  • Substance
    Substance
    PubChem Substance links

Recent Activity

Your browsing activity is empty.

Activity recording is turned off.

Turn recording back on

See more...