Molecular cloning, genomic positioning, promoter identification, and characterization of the novel cyclic amp-specific phosphodiesterase PDE4A10.
We describe the cloning and expression of HSPDE4A10, a novel long form splice variant of the human cAMP phosphodiesterase PDE4A gene. The 825 amino acid HSPDE4A10 contains a unique N terminus of 46 amino acids encoded by a unique 5' exon. Exon-1(4A10) lies approximately 11 kilobase pairs (kb) downstream of exon-1(4A4) and approximately 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-1(4A10) exhibited 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 microM) and IC(50) values for inhibition by rolipram (50 nM) but the V(max) value of the soluble form was approximately 3-fold greater than that of the particulate form. At 55 degrees C, soluble HSPDE4A10 was more thermostable (T(0.5) = 11 min) than the particulate enzyme (T(0.5) = 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.
Rena
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G
Molecular Pharmacology Group, Division of Biochemistry and Molecular Biology, Institute of Biomedical and Life Sciences, University of Glasgow, Glasgow, Scotland, United Kingdom.
Begg
F
F
Ross
A
A
MacKenzie
C
C
McPhee
I
I
Campbell
L
L
Huston
E
E
Sullivan
M
M
Houslay
M D
MD
D015105:3',5'-Cyclic-AMP Phosphodiesterases / Q000235:genetics* / Q000378:metabolism; D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D019556:COS Cells; D003001:Cloning, Molecular; D054703:Cyclic Nucleotide Phosphodiesterases, Type 4; D018076:DNA, Complementary / Q000032:analysis; D016208:Databases, Factual; D004795:Enzyme Stability; D005091:Exons; D015894:Genome, Human; D006801:Humans; D007438:Introns; D007527:Isoenzymes / Q000235:genetics*; D051379:Mice; D008969:Molecular Sequence Data; D011401:Promoter Regions, Genetic / Q000235:genetics*; D051381:Rats; D017386:Sequence Homology, Amino Acid; D014162:Transfection
D018076:DNA, Complementary; D007527:Isoenzymes; D015105:3',5'-Cyclic-AMP Phosphodiesterases; D054703:Cyclic Nucleotide Phosphodiesterases, Type 4; C517355:PDE4A protein, human