Cloning and characterization of a cAMP-specific cyclic nucleotide phosphodiesterase

Abstract

A BSTR ACTCyclic nucleotide phosphodiesterases (PDEs) regulate intracellular levels of cAMP and cGMP by hydrolyzing them to their corresponding 5 monophosphates. We report here the cloning and characterization of a novel cAMP-specific PDE from mouse testis. This unique phosphodiesterase contains a catalytic domain that overall shares <40% sequence identity to the catalytic domain of all other known PDEs. Based on this limited homology, this new PDE clearly represents a previously unknown PDE gene family designated as PDE8. The cDNA for PDE8 is 3,678 nucleotides in length and is predicted to encode an 823 amino acid enzyme. The cDNA includes a full ORF as it contains an in-frame stop codon before the start methionine. PDE8 is specific for the hydrolysis of cAMP and has a K m of 0.15 M. Most common PDE inhibitors are ineffective antagonists of PDE8, including the nonspecific PDE inhibitor 3-isobutyl-1-methylxanthine. Dipyridamole, however, an inhibitor that is generally considered to be relatively specific for the cGMP selective PDEs, does inhibit PDE8 with an IC 50 of 4.5 M. Tissue distribution studies of 22 different mouse tissues indicates that PDE8 has highest expression in testis, followed by eye, liver, skeletal muscle, heart, 7-day embryo, kidney, ovary, and brain in decreasing order. In situ hybridizations in testis, the tissue of highest expression, shows that PDE8 is expressed in the seminiferous epithelium in a stage-specific manner. Highest levels of expression are seen in stages 7-12, with little or no expression in stages 1-6.

T cell activation up-regulates cyclic nucleotide phosphodiesterases 8A1 and 7A3

“…1 A). This sequence is highly homologous to the full-length N terminus of the published mouse PDE8A sequence (10) (Fig. 1 A) but does contain a stretch of about 50 residues at which the sequence diverges from the mouse homolog.…”

“…Currently, 11 major families of PDEs (PDEs 1-11), subdivided on the basis of substrate specificity, kinetic properties, inhibitor profiles, and sequence homology (9)(10)(11)(12)(13), have been identified. PDEs 3B, 4A, 4B, 4D, and 7A1 (14)(15)(16) are reported in human T cells.…”

Development of a Fission Yeast-Based High-Throughput Screen to Identify Chemical Regulators of cAMP Phosphodiesterases

“…[20][21][22] Strains expressing these enzymes regain mating competence, which is deficient in strains lacking PDE activity, 23 indicating that these PDEs are functional when expressed in S. pombe (data not shown).…”

“…No PDE8A inhibition was observed using dipyridamole (data not shown), a known PDE8A inhibitor. 21 As a DNA sequence analysis of the integrated PDE8A gene showed that it encodes the wild-type PDE8A protein, we presume that the failure to observe a response to dipyridamole is due to a permeability problem in fission yeast or a solubility problem with dipyridamole in the growth medium.…”

Phosphodiesterase genes are associated with susceptibility to major depression and antidepressant treatment response

“…families have already been identified based on their substrate specificities, kinetic properties, allosteric regulators, inhibitor sensitivities, and amino acid sequences (1)(2)(3)(4)(5)(6)(7)(8)(9)(10). Within each family, several genes and splice variants have been recognized (2,11).…”