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The superfamily of cyclic nucleotide (cN) phosphodiesterases (PDEs) is comprised of 11 families of enzymes. PDEs break down cAMP and/or cGMP and are major determinants of cellular cN levels and, consequently, the actions of cN-signaling pathways. PDEs exhibit a range of catalytic efficiencies for breakdown of cAMP and/or cGMP and are regulated by myriad processes including phosphorylation, cN binding to allosteric GAF domains, changes in expression levels, interaction with regulatory or anchoring proteins, and reversible translocation among subcellular compartments. Selective PDE inhibitors are currently in clinical use for treatment of erectile dysfunction, pulmonary hypertension, intermittent claudication, and chronic pulmonary obstructive disease; many new inhibitors are being developed for treatment of these and other maladies. Recently reported x-ray crystallographic structures have defined features that provide for specificity for cAMP or cGMP in PDE catalytic sites or their GAF domains, as well as mechanisms involved in catalysis, oligomerization, autoinhibition, and interactions with inhibitors. In addition, major advances have been made in understanding the physiological impact and the biochemical basis for selective localization and/or recruitment of specific PDE isoenzymes to particular subcellular compartments. The many recent advances in understanding PDE structures, functions, and physiological actions are discussed in this review.

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In addition to its cGMP-selective catalytic site, cGMP-binding cGMP-specific phosphodiesterase (PDE5) contains two allosteric cGMP-binding sites and at least one phosphorylation site (Ser92) on each subunit [Thomas, M.K., Francis, S.H. & Corbin, J.D. (1990) J. Biol. Chem. 265, 14971±14978]. In the present study, prior incubation of recombinant bovine PDE5 with a phosphorylation reaction mixture [cGMP-dependent protein kinase (PKG) or catalytic subunit of cAMP-dependent protein kinase (PKA), MgATP, cGMP, 3-isobutyl-1-methylxanthine], shown earlier to produce Ser92 phosphorylation, caused a 50±70% increase in enzyme activity and also increased the affinity of cGMP binding to the allosteric cGMP-binding sites. Both effects were associated with increases in its phosphate content up to 0.6 mol per PDE5 subunit. Omission of any one of the preincubation components caused loss of stimulation of catalytic activity. Addition of the phosphorylation reaction mixture to a crude bovine lung extract, which contains PDE5, also produced a significant increase in cGMP PDE catalytic activity. The increase in recombinant PDE5 catalytic activity brought about by phosphorylation was time-dependent and was obtained with 0.2±0.5 mm PKG subunit, which is approximately the cellular level of this enzyme in vascular smooth muscle. Significantly greater stimulation was observed using cGMP substrate concentrations below the K m value for PDE5, although stimulation was also seen at high cGMP concentrations. Considerably higher concentration of the catalytic subunit of PKA than of PKG was required for activation. There was no detectable difference between phosphorylated and unphosphorylated PDE5 in median inhibitory concentration for the PDE5 inhibitors, sildenafil, or zaprinast 3-isobutyl-1-methylxanthine. Phosphorylation reduced the cGMP concentration required for half-maximum binding to the allosteric cGMP-binding sites from 0.13 to 0.03 mm. The mechanism by which phosphorylation of PDE5 by PKG could be involved in physiological negative-feedback regulation of cGMP levels is discussed.

Intracellular cAMP and cGMP levels are increased in response to a variety of hormonal and chemical stimuli; these nucleotides play key roles as second messenger signals in modulating myriad physiological processes. The cAMP-dependent protein kinase and cGMP-dependent protein kinase are major intracellular receptors for these nucleotides, and the actions of these enzymes account for much of the cellular responses to increased levels of cAMP or cGMP. This review summarizes many studies that have contributed significantly to an improved understanding of the catalytic, regulatory, and structural properties of these protein kinases. These accumulated findings provide insights into the mechanisms by which these enzymes produce their specific physiological effects and are helpful in considering the actions of other protein kinases as well.

Cyclic nucleotide phosphodiesterases (PDEs) are a superfamily of enzymes controlling cellular concentrations of the second messengers cAMP and cGMP. Crystal structures of the catalytic domains of cGMP-specific PDE5A1 and cAMP-specific PDE4D2 in complex with the nonselective inhibitor 3-isobutyl-1-methylxanthine have been determined at medium resolution. The catalytic domain of PDE5A1 has the same topological folding as that of PDE4D2, but three regions show different tertiary structures, including residues 79 -113, 208 -224 (H-loop), and 341-364 (M-loop) in PDE4D2 or 535-566, 661-676, and 787-812 in PDE5A1, respectively. Because H-and M-loops are involved in binding of the selective inhibitors, the different conformations of the loops, thus the distinct shapes of the active sites, will be a determinant of inhibitor selectivity in PDEs. IBMX binds to a subpocket that comprises key residues Ile-336, Phe-340, Gln-369, and Phe-372 of PDE4D2 or Val-782, Phe-786, Gln-817, and Phe-820 of PDE5A1. This subpocket may be a common site for binding nonselective inhibitors of PDEs.Cyclic nucleotide phosphodiesterases (PDEs) 1 hydrolyze cAMP and cGMP to 5Ј-AMP and 5Ј-GMP. The second messengers, cAMP and cGMP, mediate the response of cells to a wide variety of hormones and neurotransmitters and modulate many metabolic processes such as cardiac and smooth muscle contraction, glycogenolysis, platelet aggregation, secretion, lipolysis, ion channel conductance, apoptosis, and growth control (1-6).The human genome encodes 21 PDE genes and over 60 PDE isoforms categorized into 11 families (7-16). PDE molecules contain three regions: an N-terminal splicing region, a regulatory domain, and a catalytic domain near the C terminus. The 11 PDE families share a conserved catalytic domain of about 300 amino acids but rare homology in other regions across families. The function of the N-terminal splicing region of the PDE families is unknown. The regulatory domains of PDEs contain various structural motifs such as a calmodulin-binding domain in PDE1, upstream conserved region in PDE4, PAS (period clock protein, aryl hydrocarbon receptor nuclear translocator, and single-minded protein) domain in PDE8, GAF (cGMP-specific PDE, adenylyl cyclase, and Fh1A) domain in PDE2, -5, -6, -10, and -11. The regulatory domains have been shown to play roles in the regulation of the catalytic activity of PDEs or to participate in cross-talk with other signaling pathways (16 -18).PDEs share high degree (25-49%) of amino acid conservation in the catalytic domains, implying a similar three-dimensional structure of the catalytic domains. However, PDE families and isoforms within the respective family have varying substrate preferences for cAMP and cGMP. The PDE4, -7, and -8 families prefer to hydrolyze cAMP, whereas PDE5, -6, and -9 are cGMP-specific. PDE1, -2, -3, -10, and -11 show activities toward both substrates but have distinct K m values for cAMP and cGMP (16). In addition, many PDE families possess selective inhibitors that bind to the conserved active sit...