Cloning and expression of cDNA for a human low-Km, rolipram-sensitive cyclic AMP phosphodiesterase.

Abstract

We have isolated cDNA clones representing cyclic AMP (cAMP)-specific phosphodiesterases (PDEases) from a human monocyte cDNA library. One cDNA clone (hPDE-1) defines a large open reading frame of ca. 2.1 kilobases, predicting a 686-amino-acid, ca. 77-kilodalton protein which contains significant homology to both rat brain and Drosophila cAMP PDEases, especially within an internal conserved domain of ca. 270 residues. Amino acid sequence divergence exists at the NH2 terminus and also within a 40- to 100-residue domain near the COOH-terminal end. hPDE-1 hybridizes to a major 4.8-kilobase mRNA transcript from both human monocytes and placenta. The coding region of hPDE-1 was engineered for expression in COS-1 cells, resulting in the overproduction of cAMP PDEase activity. The hPDE-1 recombinant gene product was identified as a low-Km cAMP phosphodiesterase on the basis of several biochemical properties including selective inhibition by the antidepressant drug rolipram. Known inhibitors of other PDEases (cGMP-specific PDEase, cGMP-inhibited PDEase) had little or no effect on the hPDE-1 recombinant gene product. Human genomic Southern blot analysis suggests that this enzyme is likely to be encoded by a single gene. The presence of the enzyme in monocytes may be important for cell function in inflammation. Rolipram sensitivity, coupled with homology to the Drosophila cAMP PDEase, which is required for learning and memory in flies, suggests an additional function for this enzyme in neurobiochemistry.

Expression of three mammalian cDNAs that interfere with RAS function in Saccharomyces cerevisiae.

“…JC44, the cDNA capable of suppressing pdel -pde2-yeast, is a human homolog of the rat dunce protein-like phosphodiesterase, which we isolated in a previous screen. Its sequence appears to be identical to a recently reported human phosphodi-JC265 esterase gene (30) We searched existing data banks for nucleotide or amino acid sequences similar to JC99, JC265, and JC310. No striking similarities were found to other genes in the pairwise searches of sequence data bases.…”

The inhibition of antigen‐induced eosinophilia and bronchoconstriction by CDP840, a novel stereo‐selective inhibitor of phosphodiesterase type 4

“…Briefly, full length cDNAs encoding PDE 4B and D were isolated by the polymerase chain reaction from a human eosinophil cDNA library using published sequence data (McLaughlin et al, 1993;Bolger et al, 1993). A human PDE 4A cDNA was obtained from a human frontal cortex cDNA library (Clontech) by hybridization with a partial cDNA isolated from the human monocytic cell line U937 (Livi et al, 1990). A combination of reverse transcription coupled to PCR and 5' RACE methods (Edwards et al, 1991) was used to isolate a PDE 4C cDNA from the human glioblastoma cell line U87 (Engels et al, 1994).…”

Identification of cyclic AMP‐phosphodiesterase variants from the PDE4D gene expressed in human peripheral mononuclear cells

“…Four PDE4 genes (PDE4A, PDE4B, PDE4C, PDE4D) are expressed in rat [16][17][18][19], mouse [20], and human [21][22][23][24][25], and their characterization has shown a close relationship between genes from two different species [14]. Furthermore, for three out of four rat and human genes, multiple mRNA variants and corresponding protein products have been detected [13,14].…”