We have isolated cDNAs encoding PDE4B4, a new cAMP-specific phosphodiesterase (PDE4) isoform with novel properties. The amino acid sequence of PDE4B4 demonstrates that it is encoded by the PDE4B gene, but that it differs from the previously isolated PDE4B1, PDE4B2 and PDE4B3 isoforms by the presence of a novel N-terminal region of 17 amino acids. PDE4B4 contains both of the upstream conserved region 1 (UCR1) and UCR2 regulatory units that are characteristic of ‘long’ PDE4 isoforms. RNase protection demonstrated that PDE4B4 mRNA is expressed preferentially in liver, skeletal muscle and various regions of the brain, which differs from the pattern of tissue distribution of the other known PDE4B long forms, PDE4B1 and PDE4B3. Expression of PDE4B4 cDNA in COS7 cells produced a protein of 85kDa under denaturing conditions. Subcellular fractionation of recombinant, COS7-cell expressed PDE4B4 showed that the protein was localized within the cytosol, which was confirmed by confocal microscopic analysis of living COS7 cells transfected with a green fluorescent protein—PDE4B4 chimaera. PDE4B4 exhibited a Km for cAMP of 5.4μM and a Vmax, relative to that of the long PDE4B1 isoform, of 2.1. PDE4B4 was inhibited by the prototypical PDE4 inhibitor rolipram {4-[3-(cyclopentoxyl)-4-methoxyphenyl]-2-pyrrolidinone} with an IC50 of 83nM. Treatment of COS7 cells with forskolin, to elevate cAMP levels, produced activation of PDE4B4, which was associated with the phosphorylation of PDE4B4 on Ser-56 within UCR1. The unique tissue distribution and intracellular targeting of PDE4B4 suggests that this isoform may have a distinct functional role in regulating cAMP levels in specific cell types.
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March 2003
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Research Article|
March 01 2003
Molecular cloning and subcellular distribution of the novel PDE4B4 cAMP-specific phosphodiesterase isoform
Malcolm SHEPHERD;
Malcolm SHEPHERD
∗Molecular Pharmacology Group, Division of Biochemistry and Molecular Biology, Institute of Biology and Life Sciences, University of Glasgow, Davidson Building, Glasgow G12 8QQ, Scotland, U.K.
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Theresa McSORLEY;
Theresa McSORLEY
∗Molecular Pharmacology Group, Division of Biochemistry and Molecular Biology, Institute of Biology and Life Sciences, University of Glasgow, Davidson Building, Glasgow G12 8QQ, Scotland, U.K.
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Aileen E. OLSEN;
Aileen E. OLSEN
†Veterans Affairs Medical Center, Huntsman Cancer Institute, Departments of Medicine (Division of Oncology) and Oncological Science, University of Utah Health Science Center, Salt Lake City, UT 84148, U.S.A.,
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Lee Ann JOHNSTON;
Lee Ann JOHNSTON
∗Molecular Pharmacology Group, Division of Biochemistry and Molecular Biology, Institute of Biology and Life Sciences, University of Glasgow, Davidson Building, Glasgow G12 8QQ, Scotland, U.K.
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Neil C. THOMSON;
Neil C. THOMSON
‡Department of Respiratory Medicine, Western Infirmary, Glasgow, Scotland, U.K.
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George S. BAILLIE;
George S. BAILLIE
∗Molecular Pharmacology Group, Division of Biochemistry and Molecular Biology, Institute of Biology and Life Sciences, University of Glasgow, Davidson Building, Glasgow G12 8QQ, Scotland, U.K.
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Miles D. HOUSLAY;
Miles D. HOUSLAY
∗Molecular Pharmacology Group, Division of Biochemistry and Molecular Biology, Institute of Biology and Life Sciences, University of Glasgow, Davidson Building, Glasgow G12 8QQ, Scotland, U.K.
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Graeme B. BOLGER
Graeme B. BOLGER
1
†Veterans Affairs Medical Center, Huntsman Cancer Institute, Departments of Medicine (Division of Oncology) and Oncological Science, University of Utah Health Science Center, Salt Lake City, UT 84148, U.S.A.,
1To whom correspondence should be addressed: University of Alabama at Birmingham, Comprehensive Cancer Center, WTI 520A, 1530 3rd Avenue South, Birmingham, AL 35294-3300, U.S.A. (e-mail Graeme.Bolger@ccc.uab.edu).
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Publisher: Portland Press Ltd
Received:
July 10 2002
Revision Received:
November 19 2002
Accepted:
November 20 2002
Accepted Manuscript online:
November 20 2002
Online ISSN: 1470-8728
Print ISSN: 0264-6021
The Biochemical Society, London ©2003
2003
Biochem J (2003) 370 (2): 429–438.
Article history
Received:
July 10 2002
Revision Received:
November 19 2002
Accepted:
November 20 2002
Accepted Manuscript online:
November 20 2002
Citation
Malcolm SHEPHERD, Theresa McSORLEY, Aileen E. OLSEN, Lee Ann JOHNSTON, Neil C. THOMSON, George S. BAILLIE, Miles D. HOUSLAY, Graeme B. BOLGER; Molecular cloning and subcellular distribution of the novel PDE4B4 cAMP-specific phosphodiesterase isoform. Biochem J 1 March 2003; 370 (2): 429–438. doi: https://doi.org/10.1042/bj20021082
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