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. 1998 Aug 1;333(Pt 3):693–703. doi: 10.1042/bj3330693

Identification and characterization of the human homologue of the short PDE4A cAMP-specific phosphodiesterase RD1 (PDE4A1) by analysis of the human HSPDE4A gene locus located at chromosome 19p13.2.

M Sullivan 1, G Rena 1, F Begg 1, L Gordon 1, A S Olsen 1, M D Houslay 1
PMCID: PMC1219634  PMID: 9677330

Abstract

The HSPDE4A gene spans 50 kb, consists of at least 17 exons and is orientated 5'-3', telomere to centromere. It is located at chromosome 19p13.2, being 350 kb proximal to the gene encoding TYK2 and 850 kb distal to the gene encoding the low-density lipoprotein receptor. Its structure is consistent with the production of active 'long' and 'short' isoenzymes as the result of alternative mRNA splicing at two splice junctions. Identified is the single alternatively spliced 5' exon encoding the unique N-terminal region of the long isoenzyme HSPDE4A4B (pde46). The upstream conserved regions, UCR1 and UCR2, which form characteristic domains of PDE4 long forms are each encoded by three exons. The PDE4A-subfamily-specific linker region LR1, which joins UCR1 and UCR2, is encoded by two exons, whereas LR2, which joins UCR2 to the catalytic unit, is encoded by a single exon. Identification of exons encoding an enzymically inactive product of this gene, HSPDE4A8A (2el), indicates that this is an authentic gene product. The 5' exon encoding the unique N-terminal region of the human homologue of the rodent isoform RNPDE4A1A (RD1) was located, and the splice junction used to produce this short PDE4A isoform shown to occur at a different position from that seen in both the rat PDE4B and PDE4D genes. Reverse transcriptase PCR analysis indicates that RD1 homologues are conserved across species, having a conserved membrane-targeting region and a hypervariable LR2 region. Human RD1 was expressed transiently in COS-7 cells and detected as an 83 kDa species primarily associated with the high-speed membrane fraction. Human RD1 exhibited a Km for cAMP of about 3 microM, an IC50 value for inhibition by the PDE4-selective inhibitor rolipram of about 0.3 microM and was considerably more thermostable than rat RD1. Human RD1 was generated as a mature 80 kDa species in an in vitro transcription-translation system and shown to be capable of binding to membranes. Knowledge of the gene structure and the associated sequence information should facilitate analysis of the involvement of PDE4A in hereditary disorders that may result from alterations in enzyme expression, activity, regulation and intracellular targeting and serve as a resource for determining authenticity of cloned PDE4A species.

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Selected References

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  1. Aikawa Y., Hara H., Watanabe T. Molecular cloning and characterization of mammalian homologues of the Drosophila retinal degeneration B gene. Biochem Biophys Res Commun. 1997 Jul 30;236(3):559–564. doi: 10.1006/bbrc.1997.7009. [DOI] [PubMed] [Google Scholar]
  2. Ashworth L. K., Batzer M. A., Brandriff B., Branscomb E., de Jong P., Garcia E., Garnes J. A., Gordon L. A., Lamerdin J. E., Lennon G. An integrated metric physical map of human chromosome 19. Nat Genet. 1995 Dec;11(4):422–427. doi: 10.1038/ng1295-422. [DOI] [PubMed] [Google Scholar]
  3. Beavo J. A., Conti M., Heaslip R. J. Multiple cyclic nucleotide phosphodiesterases. Mol Pharmacol. 1994 Sep;46(3):399–405. [PubMed] [Google Scholar]
  4. Bolger G. B., Erdogan S., Jones R. E., Loughney K., Scotland G., Hoffmann R., Wilkinson I., Farrell C., Houslay M. D. Characterization of five different proteins produced by alternatively spliced mRNAs from the human cAMP-specific phosphodiesterase PDE4D gene. Biochem J. 1997 Dec 1;328(Pt 2):539–548. doi: 10.1042/bj3280539. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Bolger G. B., McPhee I., Houslay M. D. 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. doi: 10.1074/jbc.271.2.1065. [DOI] [PubMed] [Google Scholar]
  6. Bolger G. B. Molecular biology of the cyclic AMP-specific cyclic nucleotide phosphodiesterases: a diverse family of regulatory enzymes. Cell Signal. 1994 Nov;6(8):851–859. doi: 10.1016/0898-6568(94)90018-3. [DOI] [PubMed] [Google Scholar]
  7. Bolger G. B., 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. doi: 10.1016/0378-1119(94)90155-4. [DOI] [PubMed] [Google Scholar]
  8. 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. doi: 10.1128/mcb.13.10.6558. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Bradford M. M. 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. doi: 10.1016/0003-2697(76)90527-3. [DOI] [PubMed] [Google Scholar]
  10. Brandriff B. F., Gordon L. A., Fertitta A., Olsen A. S., Christensen M., Ashworth L. K., Nelson D. O., Carrano A. V., Mohrenweiser H. W. Human chromosome 19p: a fluorescence in situ hybridization map with genomic distance estimates for 79 intervals spanning 20 Mb. Genomics. 1994 Oct;23(3):582–591. doi: 10.1006/geno.1994.1546. [DOI] [PubMed] [Google Scholar]
  11. 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. doi: 10.1210/edrv-16-3-370. [DOI] [PubMed] [Google Scholar]
  12. Davis R. L., Takayasu H., Eberwine M., Myres J. Cloning and characterization of mammalian homologs of the Drosophila dunce+ gene. Proc Natl Acad Sci U S A. 1989 May;86(10):3604–3608. doi: 10.1073/pnas.86.10.3604. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Gordon L. A., Bergmann A., Christensen M., Danganan L., Lee D. A., Ashworth L. K., Nelson D. O., Olsen A. S., Mohrenweiser H. W., Carrano A. V. A 30-Mb metric fluorescence in situ hybridization map of human chromosome 19q. Genomics. 1995 Nov 20;30(2):187–194. doi: 10.1006/geno.1995.9886. [DOI] [PubMed] [Google Scholar]
  14. Horton Y. M., Sullivan M., Houslay M. D. Molecular cloning of a novel splice variant of human type IVA (PDE-IVA) cyclic AMP phosphodiesterase and localization of the gene to the p13.2-q12 region of human chromosome 19 [corrected]. Biochem J. 1995 Jun 1;308(Pt 2):683–691. doi: 10.1042/bj3080683. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Houslay M. D., Milligan G. Tailoring cAMP-signalling responses through isoform multiplicity. Trends Biochem Sci. 1997 Jun;22(6):217–224. doi: 10.1016/s0968-0004(97)01050-5. [DOI] [PubMed] [Google Scholar]
  16. Houslay M. D., Sullivan M., Bolger G. B. The multienzyme PDE4 cyclic adenosine monophosphate-specific phosphodiesterase family: intracellular targeting, regulation, and selective inhibition by compounds exerting anti-inflammatory and antidepressant actions. Adv Pharmacol. 1998;44:225–342. doi: 10.1016/s1054-3589(08)60128-3. [DOI] [PubMed] [Google Scholar]
  17. Huston E., Lumb S., Russell A., Catterall C., Ross A. H., Steele M. R., Bolger G. B., Perry M. J., Owens R. J., Houslay M. D. Molecular cloning and transient expression in COS7 cells of a novel human PDE4B cAMP-specific phosphodiesterase, HSPDE4B3. Biochem J. 1997 Dec 1;328(Pt 2):549–558. doi: 10.1042/bj3280549. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Huston E., Pooley L., Julien P., Scotland G., McPhee I., Sullivan M., Bolger G., Houslay M. D. 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. doi: 10.1074/jbc.271.49.31334. [DOI] [PubMed] [Google Scholar]
  19. Jacobitz S., McLaughlin M. M., Livi G. P., Burman M., Torphy T. J. Mapping the functional domains of human recombinant phosphodiesterase 4A: structural requirements for catalytic activity and rolipram binding. Mol Pharmacol. 1996 Oct;50(4):891–899. [PubMed] [Google Scholar]
  20. Laemmli U. K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970 Aug 15;227(5259):680–685. doi: 10.1038/227680a0. [DOI] [PubMed] [Google Scholar]
  21. Livi G. P., Kmetz P., McHale M. M., Cieslinski L. B., Sathe G. M., Taylor D. P., Davis R. L., Torphy T. J., Balcarek J. M. Cloning and expression of cDNA for a human low-Km, rolipram-sensitive cyclic AMP phosphodiesterase. Mol Cell Biol. 1990 Jun;10(6):2678–2686. doi: 10.1128/mcb.10.6.2678. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Loughney K., Martins T. J., Harris E. A., Sadhu K., Hicks J. B., Sonnenburg W. K., Beavo J. A., 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. doi: 10.1074/jbc.271.2.796. [DOI] [PubMed] [Google Scholar]
  23. MacKenzie S. J., Yarwood S. J., Peden A. H., Bolger G. B., Vernon R. G., Houslay M. D. Stimulation of p70S6 kinase via a growth hormone-controlled phosphatidylinositol 3-kinase pathway leads to the activation of a PDE4A cyclic AMP-specific phosphodiesterase in 3T3-F442A preadipocytes. Proc Natl Acad Sci U S A. 1998 Mar 31;95(7):3549–3554. doi: 10.1073/pnas.95.7.3549. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Manganiello V. C., Murata T., Taira M., Belfrage P., Degerman E. Diversity in cyclic nucleotide phosphodiesterase isoenzyme families. Arch Biochem Biophys. 1995 Sep 10;322(1):1–13. doi: 10.1006/abbi.1995.1429. [DOI] [PubMed] [Google Scholar]
  25. Marchmont R. J., Houslay M. D. 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. doi: 10.1042/bj1870381. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Mayer B. J., Baltimore D. Signalling through SH2 and SH3 domains. Trends Cell Biol. 1993 Jan;3(1):8–13. doi: 10.1016/0962-8924(93)90194-6. [DOI] [PubMed] [Google Scholar]
  27. McPhee I., Pooley L., Lobban M., Bolger G., Houslay M. D. 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. doi: 10.1042/bj3100965. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Milatovich A., Bolger G., Michaeli T., Francke U. Chromosome localizations of genes for five cAMP-specific phosphodiesterases in man and mouse. Somat Cell Mol Genet. 1994 Mar;20(2):75–86. doi: 10.1007/BF02290677. [DOI] [PubMed] [Google Scholar]
  29. 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] [Google Scholar]
  30. O'Connell J. C., McCallum J. F., McPhee I., Wakefield J., Houslay E. S., Wishart W., Bolger G., Frame M., Houslay M. D. The SH3 domain of Src tyrosyl protein kinase interacts with the N-terminal splice region of the PDE4A cAMP-specific phosphodiesterase RPDE-6 (RNPDE4A5). Biochem J. 1996 Aug 15;318(Pt 1):255–261. doi: 10.1042/bj3180255. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Olsen A. S., Georgescu A., Johnson S., Carrano A. V. Assembly of a 1-Mb restriction-mapped cosmid contig spanning the candidate region for Finnish congenital nephrosis (NPHS1) in 19q13.1. Genomics. 1996 Jun 1;34(2):223–225. doi: 10.1006/geno.1996.0270. [DOI] [PubMed] [Google Scholar]
  32. Olsen A., Teglund S., Nelson D., Gordon L., Copeland A., Georgescu A., Carrano A., Hammarström S. Gene organization of the pregnancy-specific glycoprotein region on human chromosome 19: assembly and analysis of a 700-kb cosmid contig spanning the region. Genomics. 1994 Oct;23(3):659–668. doi: 10.1006/geno.1994.1555. [DOI] [PubMed] [Google Scholar]
  33. Scotland G., Houslay M. D. 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. doi: 10.1042/bj3080673. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. 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] [Google Scholar]
  35. Shakur Y., Pryde J. G., Houslay M. D. Engineered deletion of the unique N-terminal domain of the cyclic AMP-specific phosphodiesterase RD1 prevents plasma membrane association and the attainment of enhanced thermostability without altering its sensitivity to inhibition by rolipram. Biochem J. 1993 Jun 15;292(Pt 3):677–686. doi: 10.1042/bj2920677. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Shakur Y., Wilson M., Pooley L., Lobban M., Griffiths S. L., Campbell A. M., Beattie J., Daly C., Houslay M. D. 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. doi: 10.1042/bj3060801. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Sharma R. K. Signal transduction: regulation of cAMP concentration in cardiac muscle by calmodulin-dependent cyclic nucleotide phosphodiesterase. Mol Cell Biochem. 1995 Aug-Sep;149-150:241–247. doi: 10.1007/BF01076583. [DOI] [PubMed] [Google Scholar]
  38. Smith K. J., Scotland G., Beattie J., Trayer I. P., Houslay M. D. Determination of the structure of the N-terminal splice region of the cyclic AMP-specific phosphodiesterase RD1 (RNPDE4A1) by 1H NMR and identification of the membrane association domain using chimeric constructs. J Biol Chem. 1996 Jul 12;271(28):16703–16711. doi: 10.1074/jbc.271.28.16703. [DOI] [PubMed] [Google Scholar]
  39. Souness J. E., Rao S. Proposal for pharmacologically distinct conformers of PDE4 cyclic AMP phosphodiesterases. Cell Signal. 1997 May-Jun;9(3-4):227–236. doi: 10.1016/s0898-6568(96)00173-8. [DOI] [PubMed] [Google Scholar]
  40. Sullivan M., Egerton M., Shakur Y., Marquardsen A., Houslay M. D. Molecular cloning and expression, in both COS-1 cells and S. cerevisiae, of a human cytosolic type-IVA, cyclic AMP specific phosphodiesterase (hPDE-IVA-h6.1). Cell Signal. 1994 Sep;6(7):793–812. doi: 10.1016/0898-6568(94)00039-5. [DOI] [PubMed] [Google Scholar]
  41. Szpirer C., Szpirer J., Rivière M., Swinnen J., Vicini E., Conti M. Chromosomal localization of the human and rat genes (PDE4D and PDE4B) encoding the cAMP-specific phosphodiesterases 3 and 4. Cytogenet Cell Genet. 1995;69(1-2):11–14. doi: 10.1159/000133927. [DOI] [PubMed] [Google Scholar]
  42. Templeton N. S., Rodgers L. A., Levy A. T., Ting K. L., Krutzsch H. C., Liotta L. A., Stetler-Stevenson W. G. Cloning and characterization of a novel human cDNA that has DNA similarity to the conserved region of the collagenase gene family. Genomics. 1992 Jan;12(1):175–176. doi: 10.1016/0888-7543(92)90425-r. [DOI] [PubMed] [Google Scholar]
  43. Thompson W. J., Appleman M. M. Multiple cyclic nucleotide phosphodiesterase activities from rat brain. Biochemistry. 1971 Jan 19;10(2):311–316. [PubMed] [Google Scholar]
  44. Torphy T. J., Barnette M. S., Hay D. W., Underwood D. C. Phosphodiesterase IV inhibitors as therapy for eosinophil-induced lung injury in asthma. Environ Health Perspect. 1994 Dec;102 (Suppl 10):79–84. doi: 10.1289/ehp.94102s1079. [DOI] [PMC free article] [PubMed] [Google Scholar]
  45. Vicini E., Conti M. Characterization of an intronic promoter of a cyclic adenosine 3',5'-monophosphate (cAMP)-specific phosphodiesterase gene that confers hormone and cAMP inducibility. Mol Endocrinol. 1997 Jun;11(7):839–850. doi: 10.1210/mend.11.7.9941. [DOI] [PubMed] [Google Scholar]
  46. Weissenbach J., Gyapay G., Dib C., Vignal A., Morissette J., Millasseau P., Vaysseix G., Lathrop M. A second-generation linkage map of the human genome. Nature. 1992 Oct 29;359(6398):794–801. doi: 10.1038/359794a0. [DOI] [PubMed] [Google Scholar]
  47. Wickens M. How the messenger got its tail: addition of poly(A) in the nucleus. Trends Biochem Sci. 1990 Jul;15(7):277–281. doi: 10.1016/0968-0004(90)90054-f. [DOI] [PubMed] [Google Scholar]

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