Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:3.1.4.1 (phosphodiesterase)
18,767 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Cyclic nucleotide phosphodiesterases (PDEs) regulate all pathways that use cGMP or cAMP as a second messenger. Five of the 11 PDE families have regulatory segments containing GAF domains, 3 of which are known to bind cGMP. In PDE2 binding of cGMP to the GAF domain causes an activation of the catalytic activity by a mechanism that apparently is shared even in the adenylyl cyclase of Anabaena, an organism separated from mouse by 2 billion years of evolution. The 2.9-A crystal structure of the mouse PDE2A regulatory segment reported in this paper reveals that the GAF A domain functions as a dimerization locus. The GAF B domain shows a deeply buried cGMP displaying a new cGMP-binding motif and is the first atomic structure of a physiological cGMP receptor with bound cGMP. Moreover, this cGMP site is located well away from the region predicted by previous mutagenesis and structural genomic approaches.
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PMID:The two GAF domains in phosphodiesterase 2A have distinct roles in dimerization and in cGMP binding. 1227 Nov 24

Cyclic GMP phosphodiesterase (PDE6) in rod photoreceptors, a key enzyme in vertebrate phototransduction, consists of two homologous catalytic subunits (Palpha and Pbeta) and two identical regulatory subunits (Pgammas). Pgamma regulates the PDE activity through its direct interaction with transducin. Here, using electron microscopy and image analysis of single particles, we show the three-dimensional organization of the basic form of bovine PDE, Palphabetagammagamma, and compare its average image with those of Pgamma-released PDE. The structure of Palphabetagammagamma appears to be a flattened bell-shape, with dimensions of 150 x 108 x 60A, and with a handle-like protrusion attached to the top of the structure. Except for the protrusion, the organization consists of two homologous structures arranged side by side, with each structure having three distinct regions, showing pseudo twofold symmetry. These characteristics are consistent with a model in which the overall structure of Palphabetagammagamma is determined by hetero-dimerization of Palpha and Pbeta, with each subunit consisting of one catalytic and two GAF regions. A comparison of the average image of Palphabetagammagamma with those of Pgamma-released PDE suggests that Pgamma release does not affect the overall structure of Palphabeta, and that the Palphabeta C-terminus, but not Pgamma, is a determinant for the Palphabeta orientation on carbon-coated grids. These observations suggest that the basic structure of PDE does not change during its regulation, which implies that Palphabeta is regulated by its regional interaction with Pgamma.
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PMID:Three-dimensional structure of non-activated cGMP phosphodiesterase 6 and comparison of its image with those of activated forms. 1237 17

Recently, we recognized two genes, gbpA and gbpB, encoding putative cGMP-binding proteins with a Zn(2+)-hydrolase domain and two cyclic nucleotide binding domains. The Zn(2+)-hydrolase domains belong to the superfamily of beta-lactamases, also harboring a small family of class II phosphodiesterases from bacteria and lower eukaryotes. Gene inactivation and overexpression studies demonstrate that gbpA encodes the cGMP-stimulated cGMP-phosphodiesterase that was characterized biochemically previously and was shown to be involved in chemotaxis. cAMP neither activates nor is a substrate of GbpA. The gbpB gene is expressed mainly in the multicellular stage and seems to encode a dual specificity phosphodiesterase with preference for cAMP. The enzyme hydrolyses cAMP approximately 9-fold faster than cGMP and is activated by cAMP and cGMP with a K(A) value of approximately 0.7 and 2.3 microM, respectively. Cells with a deletion of the gbpB gene have increased basal and receptor stimulated cAMP levels and are sporogeneous. We propose that GbpA and GbpB hydrolyze the substrate in the Zn(2+)-hydrolase domain, whereas the cyclic nucleotide binding domains mediate activation. The human cGMP-stimulated cAMP/cGMP phosphodiesterase has similar biochemical properties, but a completely different topology: hydrolysis takes place by a class I catalytic domain and GAF domains mediate cGMP activation.
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PMID:Identification and characterization of two unusual cGMP-stimulated phoshodiesterases in dictyostelium. 1242 32

cGMP-specific, cGMP-binding phosphodiesterase (PDE5) regulates such physiological processes as smooth muscle relaxation and neuronal survival. PDE5 contains two N-terminal domains (GAF A and GAF B), but the functional roles of these domains have not been determined. Here we show that recombinant PDE5 is activated directly upon cGMP binding to the GAF A domain, and this effect does not require PDE5 phosphorylation. PDE5 exhibited time- and concentration-dependent reversible activation in response to cGMP, with the highest activation (9- to 11-fold) observed at low substrate concentrations (0.1 micro M cGMP). A monoclonal antibody directed against GAF A blocked cGMP binding, prevented PDE5 activation and decreased basal activity, revealing that PDE5 in its non-activated state has low intrinsic catalytic activity. Activated PDE5 showed higher sensitivity towards sildenafil than non-activated PDE5. The stimulatory effect of cGMP binding on the catalytic activity of PDE5 suggests that this mechanism of enzyme activation may be common among other GAF domain-containing proteins. The data also suggest that development of agonists and antagonists of PDE5 activity based on binding to this site might be possible.
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PMID:PDE5 is converted to an activated state upon cGMP binding to the GAF A domain. 1255 48

In platelets, the nitric oxide (NO)-induced cGMP response is indicative of a highly regulated interplay of cGMP formation and cGMP degradation. Recently, we showed that within the NO-induced cGMP response in human platelets, activation and phosphorylation of phosphodiesterase type 5 (PDE5) occurred. Here, we identify cyclic GMP-dependent protein kinase I as the kinase responsible for the NO-induced PDE5 phosphorylation. However, we demonstrate that cGMP can directly activate PDE5 without phosphorylation in platelet cytosol, most likely via binding to the regulatory GAF domains. The reversal of activation was slow, and was not completed after 60 min. Phosphorylation enhanced the cGMP-induced activation, allowing it to occur at lower cGMP concentrations. Also, in intact platelets, a sustained NO-induced activation of PDE5 for as long as 60 min was detected. Finally, the long-term desensitization of the cGMP response induced by a low NO concentration reveals the physiological relevance of the PDE5 activation within NO/cGMP signaling. In sum, we suggest NO-induced activation and phosphorylation of PDE5 as the mechanism for a long-lasting negative feedback loop shaping the cGMP response in human platelets in order to adapt to the amount of NO available.
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PMID:Direct activation of PDE5 by cGMP: long-term effects within NO/cGMP signaling. 1260 88

Cyclic GMP (cGMP) made in response to atrial natriuretic peptide (ANP) or nitric oxide (NO) is an important regulator of short-term changes in smooth muscle tone and longer-term responses to chronic drug treatment or proliferative signals. The ability of smooth muscle cells (SMCs) to utilize different combinations of phosphodiesterase (PDE) isozymes allows cGMP to mediate these multiple processes. For example, PDE5 as a major cGMP-hydrolyzing PDE effectively controls the development of smooth muscle relaxation. In order for contraction to occur, PDE5 is activated and cGMP falls. Conversely, blockade of PDE5 activity allows the relaxation cycle to be prolonged and enhanced. A recently shown direct activation of PDE5 by cGMP binding to the GAF A domain suggests that this regulatory site might be a target for new drug development. The calcium surge associated with vasoconstrictor initiated contraction also activates a calcium/calmodulin-dependent PDE (PDE1A). Together, PDE5 and PDE1A lower cGMP sufficiently to allow contraction. Longer term, both PDE5 and PDE1A mRNA are induced by chronic stimulation of guanylyl cyclase. This induction is a major cause of the tolerance that develops to NO-releasing drugs. Finally, high levels of cGMP or cAMP also act as a brake to attenuate the proliferative response of SMCs to many mitogens. After vessel damage, in order for SMC proliferation to occur, the levels of cGMP and cAMP must be decreased. In humans, this decrease is caused in large part by induction of another Ca2+/calmodulin-dependent PDE (PDE1C) that allows the brake to be released and proliferation to start.
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PMID:Cyclic GMP phosphodiesterases and regulation of smooth muscle function. 1293 99

Binding of cGMP to the GAF-B domain of phosphodiesterase 2A allosterically activates catalytic activity. We report here a series of mutagenesis studies on the GAF-B domain of PDE2A that support a novel mechanism for molecular recognition of cGMP. Alanine mutations of Phe-438, Asp-439, and Thr-488, amino acids that interact with the pyrimidine ring, decrease cGMP affinity slightly but increase cAMP affinity by up to 8-fold. Each interaction is required to provide for cAMP/cGMP specificity. Mutations of any of the residues that interact with the phosphate-ribose moiety or the imidazole ring abolish cGMP binding. Thus, residues that interact with the pyrimidine ring collectively control cAMP/cGMP specificity, whereas residues that bind the phosphate-ribose moiety and imidazole ring are critical for high affinity binding. Similar decreases in binding were found for mutations made in a bacterially expressed GAF-A/B plus catalytic domain construct. Because these constructs had very high catalytic activity, it appears that these mutations did not cause a global denaturation. The affinities of cAMP and cGMP for wild-type GAF-B alone were approximately 4-fold greater than for the holoenzyme, suggesting that the presence of neighboring domains alters the conformation of GAF-B. More importantly, the PDE2A GAF-B, GAF-A/B, GAF-A/B+C domains, and holoenzyme all bind cGMP with much higher affinity than has previously been reported. This high affinity suggests that cGMP binding to PDE2 GAF-B activates the enzyme rapidly, stoichiometrically, and in an all or none fashion, rather than variably over a large range of cyclic nucleotide concentrations.
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PMID:Molecular determinants for cyclic nucleotide binding to the regulatory domains of phosphodiesterase 2A. 1521 Jun 92

A spinal reflex and the L-arginine-nitric oxide-guanylyl cyclase-cyclic guanosine monophosphate (cGMP) pathway mediate smooth muscle relaxation that results in penile erection. Nerves and endothelial cells directly release nitric oxide in the penis, where it stimulates guanylyl cyclase to produce cGMP and lowers intracellular calcium levels. This triggers relaxation of arterial and trabecular smooth muscle, leading to arterial dilatation, venous constriction, and erection. Phosphodiesterase 5 (PDE5) is the predominant phosphodiesterase in the corpus cavernosum. The catalytic site of PDE5 normally degrades cGMP, and PDE5 inhibitors such as sildenafil potentiate endogenous increases in cGMP by inhibiting its breakdown at the catalytic site. Phosphorylation of PDE5 increases its enzymatic activity as well as the affinity of its allosteric (noncatalytic/GAF domains) sites for cGMP. Binding of cGMP to the allosteric site further stimulates enzymatic activity. Thus phosphorylation of PDE5 and binding of cGMP to the noncatalytic sites mediate negative feedback regulation of the cGMP pathway.
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PMID:Mechanisms of action of PDE5 inhibition in erectile dysfunction. 1522 27

Rod phosphodiesterase (PDE6) is the central effector enzyme in vertebrate visual transduction. Holo-PDE6 consists of two similar catalytic subunits (Palphabeta) and two identical inhibitory subunits (Pgamma). Palphabeta is the only heterodimer in the PDE superfamily, yet its significance for the function of PDE6 is poorly understood. An unequal interaction of Pgamma with Pbeta as compared with Palpha in the PDE6 complex has not been reported. We investigated the interaction interface between full-length Pgamma and Palphabeta, by differentiating Pgamma interaction with each individual Palphabeta subunit through radiolabel transfer from various positions throughout the entire Pgamma molecule. The efficiency of radiolabel transfer indicates that the close vicinity of serine 40 on Pgamma makes a major contribution to the interaction with Palphabeta. In addition, a striking asymmetry of interaction between the Pgamma polycationic region and the Palphabeta subunits was observed when the stoichiometry of Pgamma versus the Palphabeta dimer was below 2. Preferential photolabeling on Pbeta from Pgamma position 40 and on Palpha from position 30 increased while lowering the Pgamma/Palphabeta ratio, but diminished when the Pgamma/Palphabeta ratio was over 2. Our finding leads to the conclusion that two classes of Pgamma binding sites exist on Palphabeta, each composed of GAF domains in both Palpha and Pbeta, differing from the conventional models suggesting that each Pgamma binds only one of the Palphabeta catalytic subunits. This new model leads to insight into how the unique Palphabeta heterodimer contributes to the sophisticated regulation in visual transduction through interaction with Pgamma.
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PMID:Asymmetric interaction between rod cyclic GMP phosphodiesterase gamma subunits and alphabeta subunits. 1566 39

In several species, GAF domains, which are widely expressed small-molecule-binding domains that regulate enzyme activity, are known to bind cyclic nucleotides. However, the molecular mechanism by which cyclic nucleotide binding affects enzyme activity is not known for any GAF domain. In the cyanobacterium, Anabaena, the cyaB1 and cyaB2 genes encode adenylyl cyclases that are stimulated by binding of cAMP to their N-terminal GAF domains. Replacement of the tandem GAF-A/B domains in cyaB1 with the mammalian phosphodiesterase 2A GAF-A/B tandem domains allows regulation of the chimeric protein by cGMP, suggesting a highly conserved mechanism of activation. Here, we describe the 1.9-A crystal structure of the tandem GAF-A/B domains of cyaB2 with bound cAMP and compare it to the previously reported structure of the PDE2A GAF-A/B. Unexpectedly, the cyaB2 GAF-A/B dimer is antiparallel, unlike the parallel dimer of PDE2A. Moreover, there is clear electron density for cAMP in both GAF-A and -B, whereas in PDE2A, cGMP is found only in GAF-B. Phosphate and ribose group contacts are similar to those in PDE2A. However, the purine-binding pockets appear very different from that in PDE2A GAF-B. Differences in the beta2-beta3 loop suggest that this loop confers much of the ligand specificity in this and perhaps in many other GAF domains. Finally, a conserved asparagine appears to be a new addition to the signature NKFDE motif, and a mechanism for this motif to stabilize the cNMP-binding pocket is proposed.
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PMID:Crystal structure of the tandem GAF domains from a cyanobacterial adenylyl cyclase: modes of ligand binding and dimerization. 1570 73


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