Cloning and Expression of cDNA for a Human Low-K_m, 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.

Human Recombinant Phosphodiesterase 4B2B Binds (R)-Rolipram at a Single Site with Two Affinities

“…The effective therapeutic concentrations of rolipram in vivo are, however, very similar to the apparent K d of the observed high-affinity (R)-rolipram binding and not to the apparent K i of enzyme inhibition (Bolger et al, 1993). Previous studies using (R)rolipram revealed that some Type 4 PDE enzyme preparations exhibit a competitive inhibition pattern suggesting inhibition at the catalytic site (Nemoz et al, 1989;Livi et al, 1990), whereas other enzyme preparations have complex inhibitory kinetics (Torphy et al, 1992;Bolger et al, 1993;McLaughlin et al, 1993;Amegadzie et al, 1995), suggesting the presence of multiple binding forms of PDE. To determine whether PDE 4 has an intrinsic high-affinity for rolipram, Torphy et al (1992) expressed a human recombinant monocytic Type 4 PDE in yeast cells and determined that high-affinity rolipram binding and cAMP turnover were coexpressed.…”

“…Two Type 4 phosphodiesterases, HSPDE4A and HSPDE4B2B, have been cloned from human brain and expressed in recombinant systems (McLaughlin et al, 1993;Livi et al, 1990). Data from these studies suggest that these proteins contain a high-affinity (R)-rolipram binding site that may represent an allosteric site or one of two distinct catalytic forms of the protein molecule (Torphy et al, 1992).…”

Recombinant Expression of a Type IV, cAMP-Specific Phosphodiesterase: Characterization and Structure−Function Studies of Deletion Mutants

“…This is the first report of a recombinant PDE to be purified from bacteria and obtained in relatively large quantities [although PDE4D1 has been expressed before as a nonfusion protein in E. coli (Swinnen et al, 1989b;Jin et al, 1992) and many PDE4s have been expressed in eukaryotic cells, e.g. Livi et al (1990), Torphy et al (1992), Bates et al (1993), Bolger et al (1993), and Monaco et al (1994)].…”

The Complexity and Multiplicity of the Specific cAMP Phosphodiesterase Family: PDE4, Open New Adapted Therapeutic Approaches

“…Since PDE4 inhibitors act on inflammatory processes, mainly on oxygen-derived free radicals [16], and that moreover some failures (unsolved mortality) were obtained during the PDE3 inhibitor development, PDE4 research was preferentially developed. In another way, the cloning and expression of cDNA for a human low-K m , rolipram-sensitive cAMP phosphodiesterase was firstly reported in 1990 [17] as well as the structure of two rat genes coding for rolipram-sensitive cAMP-PDE [18]. Thus, pharmaceutical companies have conducted many studies to design and develop increasingly potent and specific PDE4 inhibitors (for review see: Houslay et al, 2005 [19].…”