Molecular cloning, genomic positioning, promoter identification, and characterization of the novel cyclic AMP-specific phosphodiesterase PDE4A10

被引:58
作者
Rena, G [1 ]
Begg, F [1 ]
Ross, A [1 ]
Mackenzie, C [1 ]
Mcphee, I [1 ]
Campbell, L [1 ]
Huston, E [1 ]
Sullivan, M [1 ]
Houslay, MD [1 ]
机构
[1] Univ Glasgow, Inst Biomed & Life Sci, Div Biochem & Mol Biol, Mol Pharmacol Grp, Glasgow G12 8QQ, Lanark, Scotland
关键词
D O I
10.1124/mol.59.5.996
中图分类号
R9 [药学];
学科分类号
1007 ;
摘要
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 similar to 11 kilobase pairs (kb) downstream of exon-1(4A4) and similar to 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 59 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 muM) and IC50 values for inhibition by rolipram (50 nM) but the V-max value of the soluble form was similar to3- fold greater than that of the particulate form. At 55 degreesC, 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.
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收藏
页码:996 / 1011
页数:16
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