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 spli ce variant of the human cAMP phosphodiesterase PDE4A gene. The 825 amino ac id HSPDE4A10 contains a unique N terminus of 46 amino acids encoded by a un ique 5' exon. Exon-1(4A10) lies similar to 11 kilobase pairs (kb) downstrea m 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 nucleo tide sequence database searching. PDE4A10 transcripts were detected in vari ous human cell lines and tissues. The 59 sequence flanking exon-1(4A10) exh ibited promoter activity with the minimal functional promoter region being highly conserved in the corresponding mouse genomic sequence. Transient exp ression of the engineered human PDE4A10 open reading frame in COS7 cells al lowed detection of a 121-kDa protein in both soluble and particulate fracti ons. PDE4A10 was localized primarily to the perinuclear region of COS7 cell s. Soluble and particulate forms exhibited similar K-m values for cAMP hydr olysis (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 thermosta ble (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 max imal activities but differ with respect to sensitivity to inhibition by rol ipram, 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.