Identification and Characterization of PDE4A11, a Novel, Widely Expressed Long Isoform Encoded by the Human PDE4A cAMP Phosphodiesterase Gene

Abstract

PDE4A11 is a novel cAMP-specific phosphodiesterase that is conserved in humans, mouse, rat, pig, and bat. Exon-1 4A11 encodes its unique, 81 amino acid N-terminal region. Reverse-transcriptase polymerase chain reaction performed across the splice junction, plus identification of expressed sequence tags, identifies PDE4A11 as a long isoform possessing UCR1 and UCR2 regulatory domains. Transcript analysis shows that PDE4A11 is widely expressed compared with PDE4A10 and PDE4A4B long isoforms. Truncation analysis identifies a putative promoter in a 250-base pair region located immediately upstream of the start site in Exon-1 4A11. Recombinant PDE4A11, expressed in COS-7 cells, is a 126-kDa protein localized predominantly around the nucleus and in membrane ruffles. PDE4A11 exhibits a K m for cAMP hydrolysis of 4 M, with relative V max similar to that of PDE4A10 and PDE4A4B. PDE4A11 is dose-dependently inhibited by rolipram, 4-[(3-butoxy-4-methoxyphenyl)-methyl]-2-imidazolidinone (Ro 20-1724), cilomilast, roflumilast, and denbufylline, with IC 50 values of 0.7, 0.9, 0.03, 0.004, and 0.3 M, respectively. Soluble and particulate PDE4A11 exhibit distinct rates of thermal inactivation (55°C; T (0.5) ϭ 2.5 and 4.4 min, respectively). Elevating cAMP levels in COS-7 cells activates PDE4A11 concomitant with its phosphorylation at Ser119 by protein kinase A (PKA). PDE4A11 differs from PDE4A4 in sensitivity to cleavage by caspase-3, interaction with LYN SH3 domain, redistribution upon long-term rolipram challenge, and sensitivity to certain PDE4 inhibitors. PDE4A11, PDE4A10, and PDE4A4 all can interact with ␤arrestin.

Overview of PDEs and Their Regulation

“…PDE4A4B transcripts are found in T cells, 52 and PDE4A10 expression is high in the heart and small intestine. 54 PDE4A11 transcripts are widely observed in various tissues 55 with high expression in fetal brain but not in adult brain. 54 In rats, PDE4A transcripts are rich in brain neurons and the olfactory system.…”

cAMP-Specific Phosphodiesterase-4 Enzymes in the Cardiovascular System

“…Specific promoters found immediately 5Ј to the N-terminal coding exon control the expression of individual isoforms. [62][63][64][65] Such minimal PDE4 promoters appear to lack a canonical TATA box but contain CpG-rich islands and a series of perfect stimulating protein 1 (Sp1) consensus binding sites that drive basal promoter activity. Undoubtedly, regions 5Ј to this confer cell type-specific expression and further regulation.…”

PDE4B5, a Novel, Super-Short, Brain-Specific cAMP Phosphodiesterase-4 Variant Whose Isoform-Specifying N-Terminal Region Is Identical to That of cAMP Phosphodiesterase-4D6 (PDE4D6)

“…Confocal imaging for analyzing PDE4 isoforms was performed as described previously (Rena et al, 2001;Shepherd et al, 2003;Wallace et al, 2005). Here, PDE4B5 was transiently overexpressed and visualized in COS cells using PDE4B-specific antisera (Huston et al, 1997).…”

“…Transfection was done using the COS7 simian virus 40-transformed monkey kidney cell line maintained at 37°C in an atmosphere of 5% CO 2 -95% air in complete growth medium containing Dulbecco's modified Eagle's medium supplemented with 0.1% penicillin-streptomycin (10,000 U/ml), glutamine (2 mM), and 10% fetal calf serum. As described previously (Huston et al, 1997;Rena et al, 2001;Wallace et al, 2005), COS7 cells were transfected using DEAE-dextran. The DNA to be transfected (10 g) was mixed and incubated for 15 min with 200 l of 10 mg/ml DEAE-dextran in PBS to give a "DNAdextran" mix.…”