Abstract
We describe the cloning and expression of HSPDE4A10, a novel long form splice variant of the human cAMP phosphodiesterase PDE4Agene. The 825 amino acid HSPDE4A10 contains a unique N terminus of 46 amino acids encoded by a unique 5′ exon. Exon-14A10 lies ∼11 kilobase pairs (kb) downstream of exon-14A4 and ∼13.5 kb upstream of the PDE4A common exon 2. We identify a rat PDE4A10 ortholog and reveal a murine ortholog by nucleotide sequence database searching. PDE4A10 transcripts were detected in various human cell lines and tissues. The 5′ sequence flanking exon-14A10exhibited promoter activity with the minimal functional promoter region being highly conserved in the corresponding mouse genomic sequence. Transient expression of the engineered human PDE4A10 open reading frame in COS7 cells allowed detection of a 121-kDa protein in both soluble and particulate fractions. PDE4A10 was localized primarily to the perinuclear region of COS7 cells. Soluble and particulate forms exhibited similar K m values for cAMP hydrolysis (3–4 μM) and IC50 values for inhibition by rolipram (50 nM) but the V max value of the soluble form was ∼3-fold greater than that of the particulate form. At 55°C, soluble HSPDE4A10 was more thermostable (T0.5 = 11 min) than the particulate enzyme (T0.5 = 5 min). HSPDE4A10 and HSPDE4A4B are shown here to be similar in size and exhibit similar maximal activities but differ with respect to sensitivity to inhibition by rolipram, thermostability, interaction with the SRC homology 3 domain of LYN, an SRC family tyrosyl kinase, and subcellular localization. We suggest that the unique N-terminal regions of PDE4A isoforms confer distinct properties upon them.
Footnotes
- Received August 18, 2000.
- Accepted January 12, 2001.
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Send reprint requests to: Prof. Miles Houslay, Molecular Pharmacology Group, Division of Biochemistry and Molecular Biology, Davidson Building, Institute of Biomedical and Life Sciences, University of Glasgow, Glasgow G12 8QQ, Scotland, UK. E-mail:m.houslay{at}bio.gla.ac.uk
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↵1 Current address: MRC Protein Phosphorylation Unit, Department of Biochemistry, University of Dundee, Dundee DD1 5EH, Scotland.
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↵2 Current address: Grant Management, 20-23 Woodside Place, Glasgow G3 7QF, Scotland, UK.
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↵3 Current address: Department of Botany, University of Cambridge, Cambridge, CB12 1QW, England, UK.
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↵4 Current address: Scottish Biomedical, Block H, Ground Floor, Telford Pavilion, Todd Campus West of Scotland Science Park, Glasgow G20 0XA, Scotland, UK.
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↵5 Current address: AstraZeneca R&D Charnwood, Bakewell Road, Loughborough, Leicestershire, LE11 5RH, England, UK.
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This work was supported financially by the Medical Research Council (UK).
- The American Society for Pharmacology and Experimental Therapeutics
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