Interrupted Glucagon Signaling Reveals Hepatic α Cell Axis and Role for L-Glutamine in α Cell Proliferation.

Decreasing glucagon action lowers the blood glucose and may be useful therapeutically for diabetes. However, interrupted glucagon signaling leads to α cell proliferation. To identify postulated hepatic-derived circulating factor(s) responsible for α cell proliferation, we used transcriptomics/proteomics/metabolomics in three models of interrupted glucagon signaling and found that proliferation of mouse, zebrafish, and human α cells was mTOR and FoxP transcription factor dependent. Changes in hepatic amino acid (AA) catabolism gene expression predicted the observed increase in circulating AAs. Mimicking these AA levels stimulated α cell proliferation in a newly developed in vitro assay with L-glutamine being a critical AA. α cell expression of the AA transporter Slc38a5 was markedly increased in mice with interrupted glucagon signaling and played a role in α cell proliferation. These results indicate a hepatic α islet cell axis where glucagon regulates serum AA availability and AAs, especially L-glutamine, regulate α cell proliferation and mass via mTOR-dependent nutrient sensing.

Catalytic site amino acids of PKGI-alpha influence allosteric cGMP binding.

Ser-64, an autophosphorylation site in the autoinhibitory subdomain of cGMP-dependent protein kinase type I-alpha (PKGI-alpha), lowers affinity for cGMP and suppresses catalytic activity (1). Using the structure of homologous cAMP-dependent protein kinase as a model, three conserved residues (Gln-401, His-404, Cys-518) in the PKGI-alpha catalytic site are predicted to be juxtaposed to Ser-64 (2). Individual point mutants (Q401A, H404A and C518A) and a double mutant (S64A/H404A) have been generated. cGMP or cAMP affinities (K(a)) of each mutant protein for phosphotransferase activation and allosteric (3H)cGMP-binding affinity (K(D)) of each mutant protein are significantly improved over those of wild-type (WT) PKGI-alpha. However, affinities (K(m)) of the mutant PKGs for peptide substrates or ATP are unaltered. Kinase activity ratio (-GMP/+cGMP) of H404A is greater than that for WT, Q401A, or C518A, and similar to that for S64A and S64A/H404A. These results reveal a unique mechanism whereby catalytic domain residues predicted to be spatially close to Ser-64 of the regulatory domain weaken the intrinsically high affinity of PKGI-alpha for cGMP and provide for autoinhibition of catalytic activity.

Calcineurin regulates homologous desensitization of natriuretic peptide receptor-A and inhibits ANP-induced testosterone production in MA-10 cells.

Receptor desensitization is a ubiquitous regulatory mechanism that defines the activatable pool of receptors, and thus, the ability of cells to respond to environmental stimuli. In recent years, the molecular mechanisms controlling the desensitization of a variety of receptors have been established. However, little is known about the molecular mechanisms that underlie desensitization of natriuretic peptide receptors, including natriuretic peptide receptor-A (NPR-A). Here we report that calcineurin (protein phosphatase 2B, PP2B, PPP3C) regulates homologous desensitization of NPR-A in murine Leydig tumor (MA-10) cells. We demonstrate that both pharmacological inhibition of calcineurin activity and siRNA-mediated suppression of calcineurin expression potentiate atrial natriuretic peptide (ANP)-induced cGMP synthesis. Treatment of MA-10 cells with inhibitors of other phosphoprotein phosphatases had little or no effect on ANP-induced cGMP accumulation. In addition, overexpression of calcineurin blunts ANP-induced cGMP synthesis. We also present data indicating that the inhibition of calcineurin potentiates ANP-induced testosterone production. To better understand the contribution of calcineurin in the regulation of NPR-A activity, we examined the kinetics of ANP-induced cGMP signals. We observed transient ANP-induced cGMP signals, even in the presence of phosphodiesterase inhibitors. Inhibition of both calcineurin and phosphodiesterase dramatically slowed the decay in the response. These observations are consistent with a model in which calcineurin mediated dephosphorylation and desensitization of NPR-A is associated with significant inhibition of cGMP synthesis. PDE activity hydrolyzes cGMP, thus lowering intracellular cGMP toward the basal level. Taken together, these data suggest that calcineurin plays a previously unrecognized role in the desensitization of NPR-A and, thereby, inhibits ANP-mediated increases in testosterone production.

PDE5 inhibitors: targeting erectile dysfunction in diabetics.

Erectile dysfunction (ED) is strongly linked to cardiovascular disease (CVD), especially in diabetics. ED is associated with deleterious changes in the overall vasculature and is recognized as an indicator of higher risk for adverse cardiovascular events. Endothelial dysfunction, vascular smooth muscle changes and increased fibrosis are indicated as major players in both ED and CVD. ED in diabetics is more refractory to acute treatment with phosphodiesterase-5 (PDE5) inhibitors (Viagra, Cialis, Levitra, Zydena) than in non-diabetics, but recent studies indicate that chronic administration of these drugs improves endothelial function, preserves vascular smooth muscle and decreases fibrotic changes. Use of PDE5 inhibitors in pre-diabetic and diabetic men may protect cardiovascular health, including vascular function in penile tissues.

Conformational conversion of PDE5 by incubation with sildenafil or metal ion is accompanied by stimulation of allosteric cGMP binding.

Phosphodiesterase-5 (PDE5) is a dimer containing a cGMP-specific catalytic domain and an allosteric cGMP-binding subdomain (GAF A) on each subunit. PDE5 exhibits three conformational forms that can be separated by Native PAGE and are denoted as Bands 1, 2, and 3 in decreasing order of mobility. A preparation comprised mainly of Band 2 PDE5 was partially converted to Band 3 PDE5 by 1h incubation with cGMP or the PDE5-specific inhibitors sildenafil, vardenafil, or tadalafil, but not with cAMP, milrinone (PDE3-specific), or rolipram (PDE4-specific). Band 2 PDE5 was converted almost entirely to Band 3 PDE5 by overnight incubation with sildenafil at 30°C. This time-dependent conversion was accompanied by a 7-fold increase in allosteric cGMP-binding activity, suggesting that Band 3 PDE5 is a much more active form than Band 2 PDE5 for allosteric cGMP binding. Conversion of Band 2 PDE5 to Band 3 PDE5 occurred faster by pre-incubation with cGMP, which binds to both the allosteric and catalytic sites of PDE5, than with catalytic site-specific sildenafil. Overnight incubation of a Band 2/Band 3 PDE5 mixture with EDTA caused time-dependent conversion to Band 1 PDE5 (apoenzyme), and this conversion was accompanied by a 50% loss in cGMP-binding activity. After incubation with EDTA, addition of Mn(++) or Mg(++) caused reversion of Band 1 to a Band 2/Band 3 PDE5 mixture in which Band 3 PDE5 predominated. This reversion was accompanied by a 3-fold increase in allosteric cGMP-binding activity. The combination of results implied that physiological conversion of Band 2 to Band 3 PDE5 by cGMP and/or divalent metal ion occupancy of the catalytic domain would increase allosteric cGMP binding to the enzyme. This conversion would produce a greater negative feedback effect on cGMP action by increasing sequestration of cGMP at the allosteric cGMP-binding site of PDE5 and by increasing cGMP degradation at the catalytic site of the enzyme. This conversion would also increase PDE5 inhibitor binding to the enzyme.

Mammalian cyclic nucleotide phosphodiesterases: molecular mechanisms and physiological functions.

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.

Development of a high throughput screen for allosteric modulators of melanocortin-4 receptor signaling using a real time cAMP assay.

The melanocortin MC(4) receptor is a potential target for the development of drugs for both obesity and cachexia. Melanocortin MC(4) receptor ligands known thus far are orthosteric agonists or antagonists, however the agonists, in particular, have generally exhibited unwanted side effects. For some receptors, allosteric modulators are expected to reduce side-effect profiles. To identify allosteric modulators of the melanocortin MC(4) receptor, we created HEK293 cell lines coexpressing the human melanocortin MC(4) receptor and a modified luciferase-based cAMP sensor. Monitoring luminescence as a readout of real-time intracellular cAMP concentration, we demonstrate that this cell line is able to report melanocortin agonist responses, as well as inverse agonist response to the physiological AgRP peptide. Based on the MC4R-GLO cell line, we developed an assay that was shown to meet HTS standards (Z'=0.50). A pilot screen run on the Microsource Spectrum compound library (n=2000) successfully identified 62 positive modulators. This screen identified predicted families of compounds: ß(2)AR agonists - the ß(2)AR being endogenously expressed in HEK293 cells, an adenylyl cyclase activator and finally a distribution of phosphodiesterase (PDE) inhibitors well characterized or recently identified. In this last category, we identified a structural family of coumarin-derived compounds (imperatorin, osthol and prenyletin), along with deracoxib, a drug in veterinary use for its COX2 inhibitory properties. This latter finding unveiled a new off-target mechanism of action for deracoxib as a PDE inhibitor. Overall, these data are the first report of a HTS for allosteric modulators for a Gs protein coupled receptor.

Metal ion stimulators of PDE5 cause similar conformational changes in the enzyme as does cGMP or sildenafil.

Purified PDE5 preparations exhibited variable proportions of two mobility forms (Bands 2 and 3) by native PAGE. Treatment of recombinant or native PDE5 with either cGMP or a substrate analog such as sildenafil, each of which is known to produce stimulatory effects on enzyme functions, caused a similar native PAGE band-shift to the lower mobility form (shift of Band 2 to Band 3). Incubation of PDE5 with Mg(++) or Mn(++), which is known to stimulate activity, caused a similar shift of the enzyme from Band 2 to Band 3 as did cGMP or sildenafil, but incubation with EDTA caused a time- and concentration-dependent shift to higher mobility (shift of Bands 2 and 3 to Band 1). A slow time course of the EDTA-induced band-shift suggested removal of a pre-bound metal ion (Me(++)) with affinity of ~0.1 nM, which was similar to the previously determined affinity of PDE5 for Zn(++). The EDTA-treated enzyme (Band 1) could be shifted to Bands 2 and 3 by addition of cGMP, sildenafil, or Me(++); however, the cGMP- or sildenafil-induced shift was inhibited and the Me(++)-induced shift was facilitated by treatment with EDTA. Results suggested that Me(++) removal from PDE5 produces a unique apoenzyme form (Band 1, more globular, negatively charged, or both) of PDE5 that can be partially converted to forms (Band 2, less globular or negatively charged, or both; and Band 3, more elongated/positively charged, or both) by addition of Me(++), substrate, or substrate analog. It is concluded that Me(++) causes conversion of PDE5 to similar conformational forms as caused by substrate or inhibitor binding to the catalytic site.

Inhibition of cyclic nucleotide phosphodiesterases by methylxanthines and related compounds.

Naturally occurring methylxanthines were the first inhibitors of cyclic nucleotide (cN) phosphodiesterases (PDEs) to be discovered. To improve potency and specificity for inhibition of various PDEs in research and for treatment of diseases, thousands of compounds with related structures have now been synthesized. All known PDE inhibitors contain one or more rings that mimic the purine in the cN substrate and directly compete with cN for access to the catalytic site; this review focuses on inhibitors that contain a nucleus that is closely related to the xanthine ring of theophylline and caffeine and the purine ring of cNs. The specificity and potency of these compounds for blocking PDE action have been improved by appending groups at positions on the rings as well as by modification of the number and distribution of nitrogens and carbons in those rings. Several of these inhibitors are highly selective for particular PDEs; potent and largely selective PDE5 inhibitors are used clinically for treatment of erectile dysfunction [sildenafil (Viagra™), tadalafil (Cialis™) and vardenafil (Levitra™)] and pulmonary hypertension [sildenafil (Revatio™) and tadalafil (Adenocirca)]. Related compounds target other PDEs and show therapeutic promise for a number of maladies.

cGMP-dependent protein kinases and cGMP phosphodiesterases in nitric oxide and cGMP action.

To date, studies suggest that biological signaling by nitric oxide (NO) is primarily mediated by cGMP, which is synthesized by NO-activated guanylyl cyclases and broken down by cyclic nucleotide phosphodiesterases (PDEs). Effects of cGMP occur through three main groups of cellular targets: cGMP-dependent protein kinases (PKGs), cGMP-gated cation channels, and PDEs. cGMP binding activates PKG, which phosphorylates serines and threonines on many cellular proteins, frequently resulting in changes in activity or function, subcellular localization, or regulatory features. The proteins that are so modified by PKG commonly regulate calcium homeostasis, calcium sensitivity of cellular proteins, platelet activation and adhesion, smooth muscle contraction, cardiac function, gene expression, feedback of the NO-signaling pathway, and other processes. Current therapies that have successfully targeted the NO-signaling pathway include nitrovasodilators (nitroglycerin), PDE5 inhibitors [sildenafil (Viagra and Revatio), vardenafil (Levitra), and tadalafil (Cialis and Adcirca)] for treatment of a number of vascular diseases including angina pectoris, erectile dysfunction, and pulmonary hypertension; the PDE3 inhibitors [cilostazol (Pletal) and milrinone (Primacor)] are used for treatment of intermittent claudication and acute heart failure, respectively. Potential for use of these medications in the treatment of other maladies continues to emerge.

Probing the catalytic sites and activation mechanism of photoreceptor phosphodiesterase using radiolabeled phosphodiesterase inhibitors.

Retinal photoreceptor phosphodiesterase (PDE6) is unique among the phosphodiesterase enzyme family not only for its catalytic heterodimer but also for its regulatory gamma-subunits (Pgamma) whose inhibitory action is released upon binding to the G-protein transducin. It is generally assumed that during visual excitation both catalytic sites are relieved of Pgamma inhibition upon binding of two activated transducin molecules. Because PDE6 shares structural and pharmacological similarities with PDE5, we utilized radiolabeled PDE5 inhibitors to probe the catalytic sites of PDE6. The membrane filtration assay we used to quantify [(3)H]vardenafil binding to PDE6 required histone II-AS to stabilize drug binding to the active site. Under these conditions, [(3)H]vardenafil binds stoichiometrically to both the alpha- and beta-subunits of the activated PDE6 heterodimer. [(3)H]vardenafil fails to bind to either the PDE6 holoenzyme or the PDE6 catalytic dimer reconstituted with Pgamma, consistent with Pgamma blocking access to the drug-binding sites. Following transducin activation of membrane-associated PDE6 holoenzyme, [(3)H]vardenafil binding increases in proportion to the extent of PDE6 activation. Both [(3)H]vardenafil binding and hydrolytic activity of transducin-activated PDE6 fail to exceed 50% of the value for the PDE6 catalytic dimer. However, adding a 1000-fold excess of activated transducin can stimulate the hydrolytic activity of PDE6 to its maximum extent. These results demonstrate that both subunits of the PDE6 heterodimer are able to bind ligands to the enzyme active site. Furthermore, transducin relieves Pgamma inhibition of PDE6 in a biphasic manner, with only one-half of the maximum PDE6 activity efficiently attained during visual excitation.

cGMP-dependent protein kinase Ialpha associates with the antidepressant-sensitive serotonin transporter and dictates rapid modulation of serotonin uptake.

BACKGROUND: The Na(+)/Cl(-)-dependent serotonin (5-hydroxytryptamine, 5-HT) transporter (SERT) is a critical element in neuronal 5-HT signaling, being responsible for the efficient elimination of 5-HT after release. SERTs are not only targets for exogenous addictive and therapeutic agents but also can be modulated by endogenous, receptor-linked signaling pathways. We have shown that neuronal A3 adenosine receptor activation leads to enhanced presynaptic 5-HT transport in vitro and an increased rate of SERT-mediated 5-HT clearance in vivo. SERT stimulation by A3 adenosine receptors derives from an elevation of cGMP and subsequent activation of both cGMP-dependent protein kinase (PKG) and p38 mitogen-activated protein kinase. PKG activators such as 8-Br-cGMP are known to lead to transporter phosphorylation, though how this modification supports SERT regulation is unclear. RESULTS: In this report, we explore the kinase isoform specificity underlying the rapid stimulation of SERT activity by PKG activators. Using immortalized, rat serotonergic raphe neurons (RN46A) previously shown to support 8-Br-cGMP stimulation of SERT surface trafficking, we document expression of PKGI, and to a lower extent, PKGII. Quantitative analysis of staining profiles using permeabilized or nonpermeabilized conditions reveals that SERT colocalizes with PKGI in both intracellular and cell surface domains of RN46A cell bodies, and exhibits a more restricted, intracellular pattern of colocalization in neuritic processes. In the same cells, SERT demonstrates a lack of colocalization with PKGII in either intracellular or surface membranes. In keeping with the ability of the membrane permeant kinase inhibitor DT-2 to block 8-Br-cGMP stimulation of SERT, we found that DT-2 treatment eliminated cGMP-dependent kinase activity in PKGI-immunoreactive extracts resolved by liquid chromatography. Similarly, treatment of SERT-transfected HeLa cells with small interfering RNAs targeting endogenous PKGI eliminated 8-Br-cGMP-induced regulation of SERT activity. Co-immunoprecipitation studies show that, in transporter/kinase co-transfected cells, PKGIalpha specifically associates with hSERT. CONCLUSION: Our findings provide evidence of a physical and compartmentalized association between SERT and PKGIalpha that supports rapid, 8-Br-cGMP-induced regulation of SERT. We discuss a model wherein SERT-associated PKGIalpha supports sequentially the mobilization of intracellular transporter-containing vesicles, leading to enhanced surface expression, and the production of catalytic-modulatory SERT phosphorylation, leading to a maximal enhancement of 5-HT clearance capacity.

Allosteric-site and catalytic-site ligand effects on PDE5 functions are associated with distinct changes in physical form of the enzyme.

Native phosphodiesterase-5 (PDE5) homodimer contains distinct non-catalytic cGMP allosteric sites and catalytic sites for cGMP hydrolysis. Purified recombinant PDE5 was activated by pre-incubation with cGMP. Relatively low concentrations of cGMP produced a Native PAGE gel shift of PDE5 from a single band position (lower band) to a band with decreased mobility (upper band); higher concentrations of cGMP produced a band of intermediate mobility (middle band) in addition to the upper band. Two point mutations (G659A and G659P) near the catalytic site that reduced affinity for cGMP substrate retained allosteric cGMP-binding affinity like that of WT PDE5 but displayed cGMP-induced gel shift only to the middle-band position. The upper band could represent a form produced by cGMP binding to the catalytic site, while the middle band could represent a form produced by cGMP binding to the allosteric site. Millimolar cGMP was required for gel shift of PDE5 when added to the pre-incubation before Native PAGE, presumably due to removal of most of the cGMP during electrophoresis, but micromolar cGMP was sufficient for this effect if cGMP was included in the native gel buffer. cGMP-induced gel shift was associated with stimulation of PDE5 catalytic activity, and the rates of onset and reversibility of this effect suggested that it was due to cGMP binding to the allosteric site. Incubation of PDE5 with non-hydrolyzable, catalytic site-specific, substrate analogs such as the inhibitors sildenafil and tadalafil, followed by dilution, did not produce activation of catalytic activity like that obtained with cGMP, although both inhibitors produced a similar gel shift to the upper band as that obtained with cGMP. This implied that occupation of the catalytic site alone can produce a gel shift to the upper band. PDE5 activation or gel shift was reversed by lowering cGMP with dilution followed by at least 1h of incubation. Such slow reversibility could prolong effects of cGMP on PDE5 in cells after decline of this nucleotide. Reversal was also achieved by Mg(++) addition to the pre-incubation mixture to promote cGMP degradation, but Mg(++) addition did not reverse the gel shift caused by sildenafil, which is not hydrolyzed by PDE5. Upon extensive dilution, the effect of tadalafil, a potent PDE5 inhibitor, to enhance catalytic-site affinity for this inhibitor was rapidly reversed. Thus, kinetic effect of binding of a high-affinity PDE5 inhibitor to the catalytic site is more readily reversible than that obtained by cGMP binding to the allosteric site. It is concluded that cGMP or PDE5 inhibitor binding to the catalytic site, or ligand binding to both the catalytic site and allosteric site simultaneously, changes PDE5 to a similar physical form; this form is distinct from that produced by cGMP binding to the allosteric site, which activates the enzyme and reverses more slowly.

Interactions between cyclic nucleotide phosphodiesterase 11 catalytic site and substrates or tadalafil and role of a critical Gln-869 hydrogen bond.

Poor understanding of the topography of cyclic nucleotide (CN) phosphodiesterase (PDE) catalytic sites compromises development of potent, selective inhibitors for therapeutic use. In the X-ray crystal structures of the catalytic domains of some PDEs, an invariant glutamine hydrogen bonds with groups at C6 and N1 or N7 on catalytic products or analogous positions of some inhibitors, inferring similar bonds with CNs (Nature 425:98-102, 2003; J Mol Biol 337:355-365, 2004; Mol Cell 15:279-286, 2004). A site-directed mutant (Q869A) lacking this invariant Gln in cGMP-/cAMP-hydrolyzing PDE11 had unaltered catalytic activity and affinity for sildenafil; but cGMP/cAMP or tadalafil affinity was reduced approximately 50- or 140-fold, respectively, and calculated free energy of binding suggested one hydrogen bond for each. A cGMP analog lacking the C6 oxygen had approximately 80-fold weakened affinity, modifications at N(2), N7, or 2'-OH diminished affinity approximately 16-fold, and analogs with groups appended at N1 had only 2- to 6-fold weakened affinity. Analogs with C8 substitutions were ineffective inhibitors, suggesting that cGMP binds in the anti conformation. Calculated decline in free energy of binding was consistent with that for one hydrogen bond only in the analog lacking binding potential at C6. In conclusion, Gln-869 interacts strongly with cGMP/cAMP and tadalafil, but not with sildenafil; interactions with CN analogs suggest a hydrogen bond only between Gln-869 and the C6 substituent. The results define interactions between the PDE11 catalytic site and substrates/inhibitors and advance potential for inhibitor design.

cGMP-hydrolytic activity and its inhibition by sildenafil in normal and failing human and mouse myocardium.

In mouse models of cardiac disease, the type 5 (PDE5)-selective cyclic nucleotide phosphodiesterase inhibitor sildenafil has antihypertrophic and cardioprotective effects attributable to the inhibition of cGMP hydrolysis. To investigate the relevance of these findings to humans, we quantified cGMP-hydrolytic activity and its inhibition by sildenafil in cytosolic and microsomal preparations from the left ventricular myocardium of normal and failing human hearts. The vast majority of cGMP-hydrolytic activity was attributable to PDE1 and PDE3. Sildenafil had no measurable effect on cGMP hydrolysis at 10 nM, at which it is selective for PDE5, but it had a marked effect on cGMP and cAMP hydrolysis at 1 microM, at which it inhibits PDE1. In contrast, in preparations from the left ventricles of normal mice and mice with heart failure resulting from coronary artery ligation, the effects of sildenafil on cGMP hydrolysis were attributable to inhibition of both PDE5 and PDE1; PDE5 comprised approximately 22 and approximately 43% of the cytosolic cGMP-hydrolytic activity in preparations from normal and failing mouse hearts, respectively. These differences in PDE5 activities in human and mouse hearts call into question the extent to which the effects of sildenafil in mouse models are likely to be applicable in humans and raise the possibility of PDE1 as an alternative therapeutic target.

Cyclic GMP specifically suppresses Type-Ialpha cGMP-dependent protein kinase expression by ubiquitination.

Type I cGMP-dependent protein kinase (PKG-I) mediates nitric oxide (NO) and hormone dependent smooth muscle relaxation and stimulates smooth muscle cell-specific gene expression. Expression of PKG-I in cultured smooth muscle cells depends on culture conditions and is inhibited by inflammatory cytokines such as interleukin-I and tumor necrosis factor-alpha, which are known to stimulate Type II NO synthase (iNOS) expression. We report here that the suppression of PKG-I protein levels in smooth muscle cells is triggered by the ubiquitin/26S proteasome pathway. Incubation of vascular smooth muscle cells with phosphodiesterase-resistant cyclic GMP analogs (e.g., 8-bromo-cGMP) decreases PKG-I protein level in a time- and concentration-dependent manner. To study this process, we tested the effects of 8-Br-cGMP on PKG-I protein level in Cos7 cells, which do not express endogenous type I PKG mRNA. 8-Br-cGMP induced the ubiquitination and down-regulation of PKG-Ialpha, but not PKG-Ibeta. Treatment of cells with the 26S proteasome inhibitor, MG-132, increased ubiquitination of PKG. Blocking PKG-I catalytic activity using the cell-permeant specific PKG-I inhibitor, DT-2, inhibited cGMP-induced PKG-I ubiquitination and down-regulation, suggesting that PKG catalytic activity and autophosphorylation were required for suppression of PKG-I level. Mutation of the known autophosphorylation sites of PKG-Ialpha to alanine uncovered a specific role for autophosphorylation of serine-64 in cGMP-dependent ubiquitination and suppression of PKG-I level. The results suggest that chronic elevation of cGMP, as seen in inflammatory conditions, triggers ubiquitination and degradation of PKG-Ialpha in smooth muscle.

Phosphorylation increases affinity of the phosphodiesterase-5 catalytic site for tadalafil.

Phosphodiesterase-5 (PDE5) is phosphorylated at a single serine residue by cyclic nucleotide-dependent protein kinases. To test for a direct effect of phosphorylation on the PDE5 catalytic site, independent of cGMP binding to the allosteric sites of the enzyme, binding of the catalytic site-specific substrate analog [(3)H]tadalafil to PDE5 was measured. Phosphorylation increased [(3)H]tadalafil binding 3-fold, whereas cGMP caused a 1.6-fold increase. Combination of both treatments caused more than 4-fold increase in [(3)H]tadalafil binding, and effects were additive only at submaximal stimulation. Consistent with the increase in affinity, phosphorylation slowed the [(3)H]tadalafil exchange-dissociation rate from PDE5 more than 6-fold. Finally, phosphorylation increased affinity for hydrolysis of a catalytic site-specific cGMP analog, 2'-O-anthraniloyl-cGMP, by approximately 3-fold. The combined results showed that phosphorylation activates PDE5 catalytic site independently of cGMP binding to the allosteric sites. The results suggested that phosphorylation acts in concert with allosteric cGMP binding to stimulate the PDE5 catalytic site, which should promote negative feedback regulation of the cGMP pathway in intact cells. By increasing the affinity of the catalytic site, phosphorylation should also consequently increase the potency and duration of PDE5 inhibitor action.

Critical amino acids in phosphodiesterase-5 catalytic site that provide for high-affinity interaction with cyclic guanosine monophosphate and inhibitors.

The molecular bases for phosphodiesterase 5 (PDE5) catalytic-site affinity for cyclic guanosine monophosphate (cGMP) and potency of inhibitors are poorly understood. Cocrystal structures of PDE5 catalytic (C) domain with inhibitors reveal a hydrogen bond and hydrophobic interactions with Tyr-612, hydrogen bonds with Gln-817, a hydrophobic clamp formed by Phe-820 and Val-782, and contacts with His-613, Leu-765, and Phe-786 [Sung et al. (2003) Nature 425, 98-102; Huai et al. (2004) J. Biol. Chem. 279, 13095-13101]. Present results of point mutations of full-length PDE5 showed that maximum catalysis was decreased 2650-fold in H613A and 55-fold in F820A. Catalytic-site affinities for cGMP, vardenafil, sildenafil, tadalafil, or 3-isobutyl-1-methylxanthine (IBMX) were respectively weakened 14-, 123-, 30-, 51-, and 43-fold for Y612A; 63-, 511-, 43-, 95- and 61-fold for Q817A; and 59-, 448-, 71-, 137-, and 93-fold for F820A. The data indicate that these three amino acids are major determinants of affinity for cGMP and potency of selective and nonselective inhibitors, and that higher vardenafil potency over sildenafil and tadalafil results from stronger contacts with Tyr-612, Gln-817, and Phe-820. Affinity of V782A for cGMP, vardenafil, sildenafil, tadalafil, or IBMX was reduced 5.5-, 23-, 10-, 3-, and 12-fold, respectively. Change in affinity for cGMP, vardenafil, sildenafil, or IBMX in Y612F, H613A, L765A, or F786A was less, but affinity of H613A or F786A for tadalafil was weakened 37- and 17-fold, respectively. The results quantify the role of PDE5 catalytic-site residues for cGMP and inhibitors, indicate that Tyr-612, Gln-817, and Phe-820 are the most important cGMP or inhibitor contacts studied, and identify residues that contribute to selectivity among different classes of inhibitors.

Conversion of phosphodiesterase-5 (PDE5) catalytic site to higher affinity by PDE5 inhibitors.

Phosphodiesterase-5 (PDE5) specifically hydrolyzes cGMP, thereby contributing to modulation of intracellular levels of this nucleotide. In the present study, preincubation with cGMP increased PDE5 catalytic activity for cGMP degradation, and it converted the PDE5 catalytic site to a form that was more potently inhibited by each of the three PDE5 catalytic site-specific inhibitors: sildenafil, vardenafil, and tadalafil. These results implied that elevated cGMP initiates a physiological negative feedback on the cGMP pathway by increasing the affinity of the PDE5 catalytic site for cGMP. This increase in catalytic site activity or affinity for inhibitors could be caused by binding of cGMP to either the PDE5 allosteric sites, catalytic site, or both. Whether occupation of the catalytic site alone could mediate the effect was examined using radiolabeled PDE5 inhibitors in the absence of cGMP. Exchange-dissociation of [(3)H]sildenafil (Viagra), [(3)H]vardenafil (Levitra), or [(3)H]tadalafil (Cialis) from full-length PDE5 or isolated catalytic domain revealed two kinetic components (slow and fast). Extended preincubation of full-length PDE5, but not isolated catalytic domain, with (3)H inhibitors converted the biphasic pattern to a single slow (high-affinity) component. Studies of amino-terminally truncated PDE5 established that full-length mammalian GAF-B (cGMP-binding phosphodiesterase, Anabaena adenylyl cyclases, Escherichia coli FhlA) subdomain conjoined with the catalytic domain was sufficient for this conversion. In conclusion, binding of substrate or substrate analogs such as PDE5 inhibitors to the catalytic site converts a fast (low-affinity) inhibitor dissociation component of the PDE5 catalytic site to a slow (high-affinity) inhibitor dissociation component. This effect is predicted to improve the substrate affinity or inhibitory potencies of these compounds in intact cells.