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Biochem J. 2005 May 15;388(Pt 1):333-42.

Cyclic nucleotide phosphodiesterases in Drosophila melanogaster.

Author information

1
Division of Molecular Genetics, Institute of Biomedical and Life Sciences, University of Glasgow, Glasgow G11 6NU, UK.

Abstract

Cyclic nucleotide PDEs (phosphodiesterases) are important enzymes that regulate intracellular levels of cAMP and cGMP. In the present study, we identify and characterize novel PDEs in the genetic model, Drosophila melanogaster. The Drosophila genome encodes five novel PDE genes in addition to dunce. Predicted PDE sequences of Drosophila show highly conserved critical domains when compared with human PDEs. Thus PDE-encoding genes of D. melanogaster are CG14940-PDE1C, CG8279-PDE6beta, CG5411-PDE8A, CG32648-PDE9 and CG10231-PDE11. Reverse transcriptase-PCRs of adult tissues reveal widespread expression of PDE genes. Drosophila Malpighian (renal) tubules express all the six PDEs: Drosophila PDE1, dunce (PDE4), PDE6, PDE8, PDE9 and PDE11. Antipeptide antibodies were raised against PDE1, PDE6, PDE9 and PDE11. Verification of antibody specificity by Western blotting of cloned and expressed PDE constructs allowed the immunoprecipitation studies of adult Drosophila lysates. Biochemical characterization of immunoprecipitated endogenous PDEs showed that PDE1 is a dual-specificity PDE (Michaelis constant Km for cGMP: 15.3+/-1 microM; Km cAMP: 20.5+/-1.5 microM), PDE6 is a cGMP-specific PDE (Km cGMP: 37+/-13 microM) and PDE11 is a dual-specificity PDE (Km cGMP: 6+/-2 microM; Km cAMP: 18.5+/-5.5 microM). Drosophila PDE1, PDE6 and PDE11 display sensitivity to vertebrate PDE inhibitors, zaprinast (IC50 was 71+/-39 microM for PDE1, 0.65+/-0.015 microM for PDE6 and 1.6+/-0.5 microM for PDE11) and sildenafil (IC50 was 1.3+/-0.9 microM for PDE1, 0.025+/-0.005 microM for PDE6 and 0.12+/-0.06 microM for PDE11). We provide the first characterization of a cGMP-specific PDE and two dual-specificity PDEs in Drosophila, and show a high degree of similarity in structure and function between human and Drosophila PDEs.

PMID:
15673286
PMCID:
PMC1186723
DOI:
10.1042/BJ20050057
[Indexed for MEDLINE]
Free PMC Article

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