Cgmp activate pkg

Whether post-transcriptional modifications of the target protein or PKG e. This interaction is purportedly required for PKG phosphorylation of the myosin-binding subunit and activation of myosin light chain phosphatase Surks et al. It is not known whether phosphorylation of SERT is directly mediated by PKG or involves its action on another kinase, such as the pmitogen-activated protein kinase, and whether the effect involves an intermediary protein Zhu et al.

Casteel et al. These differences highlight subtleties in the interaction of PKGs with target proteins and potentially provide a basis for a spectrum of selective physiological effects mediated by PKGI isozymes. This suggests that interactions with other proteins contribute to its subcellular distribution. The requirement or advantage for colocalization of PKGI isozymes with certain substrates could be due to a number of factors Koller et al. Proximity of PKGI to the target residue could increase efficiency of phosphate transfer, as has been suggested for autophosphorylation events described in section II.

Colocalization increases the concentration of the kinase and its substrate in that locale, which would increase the rate of phosphorylation assuming that the substrate concentration in the unbound state is below that required to achieve V max. Alternatively, interaction of the target protein with PKGI could elicit a conformational change that uncovers or approximates the phosphorylatable residue.

In some instances, PKGI isozymes reversibly relocate in response to changes in cGMP and can thus phosphorylate protein substrate s at the new location Wyatt et al. Molecular mechanisms that provide for these transient PKG targeting events are not known. A cGMP-induced exposure of a site s on PKG could either work independently or in combination with contacts provided by the leucine zipper to foster the localization. PKG exit from the nucleus is slow, and it is not known if nuclear GKIPs bind the activated PKG, thereby preserving nuclear localization, or how reversal of this translocation is regulated.

Consequently, it is entirely plausible that a number of events could alter characteristics of proteins e. These possibilities add substantially to the potential complexity of the cellular response to a NO signal. Despite the findings cited above in sections II. Weber et al.

The discordance between reports such as this one and others that suggest strict specificity between a PKGI isozyme and its substrate is difficult to reconcile. At the outset, it suggests that PKG substrate selectivity in the intact animal may be less rigorously controlled than in isolated cultured cells. On the surface, the apparent lack of PKGI isozyme specificity seems to seriously compromise potential benefits of medications that might target one or the other PKGI or its anchoring.

It is clear that functions of this pathway are integrally intertwined with many other signaling pathways, and the influence of these other pathways will almost assuredly differ to some extent among cell types. Full treatment of these topics is beyond the scope of this review. Described sections III. The contractile state of smooth muscle is largely determined by the state of phosphorylation and activity of the myosin light chain; phosphorylation of the myosin light chain at Ser is mediated by the myosin light-chain kinase and leads to increased myosin ATPase activity and muscle contraction Kamm and Stull, ; Somlyo and Somlyo, ; Ding et al.

Myosin light-chain phosphatase reverses this by catalyzing the dephosphorylation of the myosin light chain. A recent report indicates that L-type channels expressed in human embryonic kidney cells or cardiomyocytes are phosphorylated by PKGI, which is also associated with inhibition of channel function Yang et al.

Whether this mechanism significantly affects calcium current in smooth muscle cells has not been determined. Signaling through certain receptors, the actions of which are mediated through calcium, can also be attenuated by direct PKGI action Christensen and Mendelsohn, Role of PKGI in modulating intracellular calcium level to promote smooth muscle relaxation.

PKGI phosphorylation of RhoA at Ser blocks the action of this protein in myriad processes; these include signaling through Rho-associated protein kinases to foster increased contraction in vascular smooth muscle, regulation of cellular adhesion, and relief of inhibition of insulin receptor substrate-1 to enhance insulin signaling through activation of the downstream phosphoinositide 3-kinase-Akt cascade Ellerbroek et al.

In vascular smooth muscle in the absence of cGMP signaling, RhoA translocates from the cytosol to the plasma membrane, where it increases the activity of the Rho-associated protein kinase. The activated Rho-kinase then phosphorylates Thr of the myosin-binding subunit of myosin light-chain phosphatase, thereby inhibiting phosphatase activity toward myosin light chain and fostering contraction Amano et al.

PKGI phosphorylation of RhoA promotes its dissociation from the plasma membrane and its return to the cytosol. Activated PKG also catalyzes phosphorylation of Ser on the myosin light-chain phosphatase regulatory subunit, which in turn blocks Rho kinase inactivation of the phosphatase Nakamura et al.

PKGI can phosphorylate cAMP response element-binding protein in vitro, but whether this occurs physiologically is unclear Colbran et al. NO-mediated increases in cGMP and PKG activation are reported to increase mitochondrial biogenesis and abundance of the uncoupling protein in brown fat Haas et al.

PKGI-mediated regulation of gene expression and protein levels is implicated in some models of neuronal plasticity and learning, as well as in pathological conditions such as Alzheimer's disease and schizophrenia Monfort et al. Phospho-NFAT is cytosolic; when dephosphorylated, it translocates to the nucleus and promotes gene transcription, which in the case of cardiomyocytes seem to be pro-hypertrophic genes Hogan et al. Activation of TRPC channels either by stretch or a G q -coupled agonist increases influx of calcium into the cell and concomitantly activates many proteins that can dephosphorylate NFAT Crabtree and Olson, ; Nilius et al.

Increased nuclear NFAT has been proposed to directly promote expression of proteins that are associated with cardiac hypertrophy as well as to activate a feed-forward mechanism for increased expression of TRPC6, which would further increase intracellular calcium and foster persistent nuclear NFAT relocation Kuwahara et al.

Phosphorylation of VASP is correlated with 1 changes in platelet cytoskeletal organization, 2 inhibition of platelet activation Horstrup et al. PKGI phosphorylates and activates the cGMP-specific PDE5, the first recognized physiological substrate of known enzymatic function, in platelets, smooth muscle cells, and cerebellar Purkinje cells Wyatt et al. Cytoprotective effects have been observed in heart cells Qin et al. Precise mechanisms for these protective effects are undoubtedly complex.

PKC-mediated phosphorylation and activation of this channel produces rapid potassium influx into the mitochondria and increased production of reactive oxygen species e.

Major tissue damage to intact hearts or cardiomyocytes subjected to ischemia and reperfusion conditions is mediated in part by the proapoptotic pathway involving p38 MAPK Ma et al. Phosphorylation at this site by PKA and other kinases has been correlated with decreased apoptosis and increased cell viability. Ser is located in the region that is required for dimerization with and blocking of Bcl-xL action, a cytoprotective protein.

Phosphorylation at Ser blocks Bad binding to Bcl-xL, thereby fostering cytoprotection Datta et al. These effects have prompted efforts to use inhibitors of cGMP-hydrolyzing PDEs for treatment of certain types of cancers. With such a complex system, there are myriad possibilities for physiological or pathophysiological factors to impact the signaling process; these factors may vary among tissues. In some instances, alteration of function at one point in the pathway may elicit changes at another point, thereby emphasizing the interconnectedness of these components and complicating understanding of mechanisms in play.

Signals that stimulate PKGI-mediated phosphorylations are countered by actions of phosphoprotein phosphatases and PDEs that reverse the phosphorylations or terminate the signals, respectively. Moreover, both cGMP-binding sites on PKGI monomers contribute to activation of PKG phosphotransferase activity so that significant occupation of the sites is necessary to achieve optimum phosphotransferase activity Francis and Corbin, The concentration of intracellular receptors for cGMP varies widely.

In porcine vascular smooth muscle, PKGI dimer has been determined to be 0. A number of problems can decrease generation of the NO signal. However, development of drugs particularly by Bayer Schering Pharma AG that directly activate NO-GC, thereby circumventing the NO-generation step, shows promise for treatment of patients who are plagued with problems in this portion of the pathway.

NO-GC is inactivated by NO-mediated nitrosation of its cysteines, and physiologically relevant levels of glutathione protect against this Sayed et al. Studies suggest that tolerance to nitrovasodilators could be due in part to desensitization of NO-GC, decreased metabolic processing of nitrovasodilators to generate NO, or increased cGMP breakdown.

NO-mediated vasodilation that is blunted in a model of chronic hypoxia is associated with decreased NO-GC catalytic activity, although the NO-GC protein level is unchanged Pearce et al. Tight regulation of cGMP level, as with any biological signal, is required to maintain the sensitivity of the response to the incoming signal and rapid adjustment to changes in that signal.

An increase in cGMP sufficient to saturate its intracellular receptors i. Under such conditions, the signaling pathway would be functioning at full throttle and would be temporarily unresponsive to changes in the incoming signal, such as a decline in NO production. It is noteworthy that cGMP concentration in response to NO in platelets and vascular smooth muscle is biphasic and quickly returns to near basal levels primarily by the action of cGMP-hydrolyzing PDEs.

Therefore, interpretation of results involving PDE action should seriously consider these factors before making extrapolations to prospects for development of potential therapies that would intersect with these pathways.

With some exceptions, the absolute concentration of PDEs within a cell is low because the catalytic rate for most PDEs is quite high k cat values range from 0. The biological advantage of such a diverse group of enzymes with broadly similar function is not clear, but selective compartmentation of some PDEs within cells, the apparent actions of these PDEs on particular pools of cGMP, and the capacity for diverse population of PDEs to deal with a broad range of cellular cGMP concentrations are now recognized Conti and Beavo, Overlap in affinity of various PDEs for cGMP and the uncertainties about compartmentation of these enzymes make it difficult to assign exclusive function to one PDE even when relatively selective PDE inhibitors are used.

Little is known about the efficiency with which PDE inhibitors access the interior of the cell and whether they are selectively localized in particular regions of those cells Thompson, ; Wunder et al. This problem continues to confound studies of PDE actions. Values compiled from Bender and Beavo a. The range of values is likely to be due to the different assay conditions and preparations of the respective enzymes in various laboratories.

Handb Exp Pharmacol — As alluded to in section IV. D, PDE6 expression is restricted to photoreceptor and pineal cells. Involvement of PDE5 in many other processes continues to be a major point of interest to the scientific and medical communities Rutten et al.

PDE9 expression has been reported in retinal pigment epithelial cells and in certain neurons Diederen et al. However, the population of PDEs in any given portion of a cell or even in cells of the same type in the same or different species can vary Shimizu-Albergine et al. Some PDEs are primarily located in the cytosol e. Development of compounds that selectively block the catalytic function of particular PDEs continues to be a major interest of the pharmaceutical industry, academicians, and clinicians.

The potential impact of such compounds has already been demonstrated with the therapeutic and marketing success of three PDE5-selective inhibitors, sildenafil, vardenafil, and tadalafil, which bind to the PDE5 catalytic site, thereby blocking access and hydrolysis of cGMP Jeremy et al. More of these PDE5 inhibitors are making their way into the commercial market Kouvelas et al.

The availability of X-ray crystal structures of the catalytic domains of several cGMP-hydrolyzing PDEs, some in complex with an inhibitor or catalytic product, has provided meaningful direction for synthesis of potent and selective new drugs Sung et al. However, to date, all potent drugs that inhibit cGMP-hydrolyzing PDEs have been identified using the traditional medicinal chemistry approach of modifying a starting scaffold.

Thus, understanding influences of the regulatory domains on catalytic site functions for most PDEs still relies on biochemical approaches. These allosteric cGMP-binding sites in PDEs 2 and 5 are also considered to be good drug targets, but progress on this front continues to lag behind that made in identifying ligands for catalytic sites.

Several structures of the allosteric cGMP-binding sites have now been reported and provide invaluable information about the topography of these pockets Martinez et al. In addition to the concept of developing compounds that target only one protein, there is potential for development of compounds that act at several points in the cGMP-signaling pathway as noted in the discussion of cN analogs in section II.

Such compounds could potentially work at low levels and elicit synergistic effects. Overall, little is known about regulation of the levels of PDE proteins, although changes in both the activities and levels of particular cGMP-hydrolyzing PDEs have been demonstrated in association with cellular proliferation and differentiation, as well as with tissue dysfunction Maclean et al.

Both short-term and long-term regulation of the action of PDEs allows for fine control of the amplitude and duration of cGMP elevation and provides for refined blunting or enhancement of the signal Gopal et al. As a result, under many conditions, the hydrolytic rate of PDEs would be less than maximal and increased cellular cGMP would be predicted to increase hydrolytic rate of these PDEs simply as a result of mass action.

The cGMP-hydrolyzing activity of PDE1 is largely determined by intracellular calcium level and perhaps by specific pools of calcium that enter the cell in response to different stimuli Miller et al. At least a portion of the cardiomyocyte PDE2 is presumed to be located near the cell membrane because it largely accounts for breakdown of cGMP pools in this compartment. PDE5, whose presence in cardiomyocytes continues to be a matter of debate, is largely cytosolic and apparently more involved in breakdown of cytosolic cGMP pools generated by NO-GC activation Palmer and Maurice, ; Corbin et al.

The catalytic activities of the PDE3 family have long been recognized as having important impact on lipolysis, glycogenolysis, insulin secretion, and cardiac function Degerman et al.

Increases in PDE1 and PDE5 activities in cardiomyocytes have been reported to be associated with cardiac hypertrophy, and suppression of these activities produces an antihypertrophic effect Corbin et al.

Using immunohistochemistry, the Kass group Takimoto et al. Persistence of PDE5 localization at the z-band in the absence of NO signaling is insufficient for the antiadrenergic effect of sildenafil. In another model of cardiomyopathy, overexpression of NO-GC or sildenafil inhibition of PDE5 in dystrophin-deficient hearts improves contractile function, protects cardiomyocytes against workload-induced damage of the sarcolemma, improves the profile of mitochondrial metabolism, and mitigates progression of cardiac disease Khairallah et al.

Evidence is mounting that cGMP activation of PKGI and the resulting reduction in intracellular calcium blunts expression of genes associated with cardiomyocyte hypertrophy, perhaps in part through diminished calcineurin activity Hogan et al. Although it is generally agreed that elevation of cGMP in heart tissues is frequently associated with antihypertrophic and antifibrotic effects, the role of PKGI in mediating these effects is still unclear, and the PDE s that are responsible for controlling cGMP level is also unclear.

Some have suggested that effects of agents e. Lukowski et al. This group found that absence of PKGI in cardiomyocytes does not increase the hypertrophy induced by either process and does not increase basal heart size; this suggests that proteins other than PKGI may mediate the antihypertrophic effects of cGMP. Moreover, no changes in cGMP-hydrolyzing PDE activities or protein levels in these heart tissues could be discerned, and no PDE5 activity or protein could be detected in cardiomyocytes from any of these animals.

These results raise questions about the mode of action reported for the antihypertrophic effects of PDE5 inhibitors and are difficult to reconcile with the well documented reports of others who have provided compelling evidence for the functional importance of PKGI and PDE5 in cardiomyocytes. However, differences in the models and conditions that have been used to study these processes and the overall complexity of signaling in the heart in response to various agonists and functional challenges may account for some of the conflicting results.

The authors suggest that each pool of PDE5 is involved in controlling cGMP in select microdomains, and that localized, rather than global, changes in cGMP in response to NO or PDE5 inhibitors selectively affect calcium mobilization and its proaggregatory effects.

This suggests that cGMP would occupy the allosteric sites and induce the more active state before significant interaction of cGMP with the respective catalytic sites.

Because allosteric cGMP binding to PDEs activates the catalytic sites, this would sustain an activated state of cGMP breakdown for a significant time, thereby contributing to negative feedback action Mullershausen et al. Moreover, the cGMP-binding affinities of some of the allosteric sites e. From these considerations, it is evident that cGMP produced within a given cell or a particular microdomain in that cell is likely to encounter multiple sites that compete for cGMP binding based on affinity and abundance.

The total concentration of the respective binding sites will also affect the cGMP distribution, thereby influencing signaling efficacy and persistence of the signal.

Because the affinities of these proteins for cGMP are in the same range, cGMP would be predicted to immediately distribute into all of these sites. A number of inhibitors of cGMP-hydrolyzing PDEs are currently in clinical use for treatment of maladies associated with cardiovascular health. Cilostazol Pletal , a relatively selective inhibitor of PDE3, exhibits vasodilatory and antiaggregatory effects on vascular smooth muscle and platelets, respectively, and is marketed to treat intermittent claudication Kambayashi et al.

Milrinone Primacor is also a selective inhibitor of PDE3 and is used in treatment of acute heart failure Stehlik and Movsesian, The antiaggregatory effect of dipyridamole in platelets involves its effect on adenosine uptake, but at therapeutic levels achieved in plasma, it could also inhibit PDE5 activity Serebruany et al. Both effects would promote platelet disaggregation.

The PDE5-selective inhibitors vardenafil, sildenafil, and tadalafil have gained wide visibility as treatments for erectile dysfunction. Sildenafil marketed as Revatio and tadalafil marketed as Adcirca are also approved for treatment of pulmonary hypertension Francis et al.

PDE5 inhibitors have also been shown to be effective in treatment of Raynaud's disease, although they are currently not approved for this use Fries et al. The outstanding safety profiles of the PDE5 inhibitors have encouraged consideration of use of these medications for treatment of other maladies.

F, it is likely that there will soon be trials examining potential clinical applications for this problem. PDE5 inhibitors may also be useful in treatment of benign prostate syndrome and lower urinary tract symptoms LUTS , which are common maladies in aging men. PDE5 is expressed in human prostate and bladder, and PDE5 inhibitors elicit relaxation of tone in tissues from both organs. Investigators are testing the efficacy of PDE5-selective inhibitors for reducing symptoms associated with these maladies.

In addition, use of PDE inhibitors is being tested for treatment of premature ejaculation, Peyronie's disease, and ureteral relaxation for eased passage of kidney stones Gratzke et al. There is some evidence that elevation of cGMP by use of PDE5 inhibitors is atheroprotective in the vasculature Kemp-Harper and Schmidt, and may improve endothelial health Rosano et al. Despite the small effect of the approved PDE5 inhibitors on systemic blood pressure, investigators still consider development of pharmacotherapies that increase cGMP in the vasculature good candidates for treatment of hypertension Kemp-Harper and Schmidt, , and there continues to be some interest in usefulness of these therapies for treatment of female sexual dysfunction.

Finally, reports have suggested that PDE5 inhibitors may speed circadian adaptation to changes in light schedules as experienced in transcontinental travel Agostino et al. Negative feedback regulation is a common control for accelerating the demise of second messenger signals, including that of cGMP.

Kono, S. Francis, and J. Corbin, unpublished observations; Kim et al. These events occur within seconds after NO-stimulation and are pivotal in returning cGMP to near-basal levels in the short term.

Moreover, these same events increase the affinity of the PDE5 catalytic site for inhibitors, which then facilitates potency and efficacy of the pharmacological action of these compounds Blount et al. In these instances, prior exposure to a stimulus that elevates cGMP causes rapid activation of PDE5 that persists and desensitizes the cell to subsequent challenges by the activator.

In both vascular smooth muscle cells and platelets, evidence indicates that cGMP binding to the PDE5 allosteric site is sufficient to activate the enzyme and that this cGMP dissociates slowly so that the activation of catalytic function is sustained.

Responsiveness to a second NO signal is slowly recovered as indicated by the progressive increase in both the magnitude of the increase in cGMP and the time course of its elevation Fig. However, in patients taking nitroglycerin, NO exposure would be ongoing and could contribute to NO resistance. Strips of aorta were preincubated in the absence [Control No Pretreatment ] or presence of a NO donor for 10 min; tissue was washed to remove the NO donor.

After either 30 or 60 min, the tissue was rechallenged with high levels of the NO donor. Tissues were harvested at indicated times and analyzed for cGMP content. Mol Pharmacol — Phosphorylation of PDE5 enhances the desensitization but is not necessary for the persistent and diminished changes in cGMP in response to a NO signal Mullershausen et al. However, phospho-PDE5 persists for at least an hour in some instances, indicating that phosphoprotein phosphatases do not rapidly dephosphorylate the enzyme under these conditions Wyatt et al.

Agents that directly activate PKG potently relax the tissues, indicating that the desensitization is not due to changes in PKG function and is consistent with the interpretation that desensitization is due to an effect on PDE5 Mullershausen et al. Corbin, unpublished observations Fig. This is accompanied by a decline in PKGI activity. Cultured rat vascular smooth muscle cells were exposed to 0. PDE5 catalytic activity was measured using 0. Prolonged exposure to 0.

Sellak et al. The molecular basis for these changes in PKGI and PDE5 are still poorly understood and are likely to be mediated by a number of processes, including changes in gene transcription, mRNA stabilization, protein synthesis, protein degradation, or state of activation of the respective proteins. For PKG, prolonged activation fosters autophosphorylation, which has been shown to promote ubiquitination and protein breakdown Dey et al.

PKG expression is also down-regulated by RhoA and up-regulated by Rac1, but whether these processes are in play with prolonged elevation of cGMP is not known Zeng et al. Nevertheless, unlike the tolerance associated with NO therapies, no tachyphylaxis after use of PDE5 inhibitors for treatment of erectile dysfunction has been reported despite prolonged and regular use in many instances. So far, however, most of these inhibitors have been taken sporadically once or twice a week. A daily regimen has been approved for tadalafil so that drug exposure will be more sustained; long-term follow-up on these patients with particular attention to an increase in tachyphylaxis would be prudent.

An unexpected persistence of the pharmacological effects of PDE5 inhibitors has been reported in men being treated for erectile dysfunction and in experimental protocols that model cardiac damage. This suggests that sustained high level of the inhibitors in plasma is not required for continued therapeutic efficacy.

However, the molecular mechanism underlying this phenomenon is not understood Francis et al. One possibility is that the biochemical processes and changes elicited by elevation of cGMP in penile vascular smooth muscle are retained for many hours after PDE5 inhibitors are cleared.

This scenario is supported by results reported by Kukreja and colleagues Salloum et al. A single dose of sildenafil in these mice has cardioprotective effects even at 24 h. Hearts from sildenafil-treated animals have significantly elevated levels of eNOS and inducible NOS proteins, the activities of which are implicated in the protective effects Salloum et al. Alternatively, tissue sensitivity to a subsequent cGMP signal may be enhanced by a feed-forward process.

These inhibitors have very high affinity for PDE5 0. Exposure of purified PDE5 to sildenafil, vardenafil, or tadalafil for 5 to 12 h causes a time-dependent conversion of PDE5 to a form that has higher affinity for these compounds Blount et al.

Thus, the time of PDE5 exposure to these medications is within the range of the time required for PDE5 conversion to a higher-affinity conformation.

Moreover, the smooth muscle in the penile vasculature of a man who has been sexually aroused after taking one of these medications will experience an increase in cGMP synthesis as a result of NO released from penile nerves and endothelial cells and a decrease in cGMP breakdown as a result of these medications to block PDE5 activity.

PDE5 conversion to a higher-affinity form would be enhanced not only by the time of exposure to the inhibitors before and after sexual arousal but also by the NO-induced rise in cGMP in response to sexual arousal. This would foster increased cGMP binding to PDE5 allosteric sites and phosphorylation at Ser human in its regulatory site, both of which increase the affinity of PDE5 interaction with the respective inhibitors Corbin et al.

All components would favor a conformation of PDE5 that has significantly higher affinity for the inhibitors. As these medications are cleared from the plasma, the amount that is free in the cytosol will decline in parallel. However, the component that is bound to PDE5 both higher and lower affinity states will dissociate slowly; once dissociated, it is likely to be bound again either by the same PDE5 molecule or by another PDE5 that has a free catalytic site, thereby retarding clearance of the drug from the cell and providing for persistence of its inhibition of PDE5 beyond the predicted therapeutic time frame based on plasma clearance time Francis et al.

B for platelets and aortic vascular smooth muscle, in this setting, the penile vascular smooth muscle seems to have enhanced responsiveness to a subsequent NO signal from penile nerves and endothelium.

This apparently disparate response of different smooth muscles to NO-induced increased cGMP may indicate that there are distinct mechanisms at play even when tissues are closely related. The fact that vascular smooth muscle cells in the penile corpus cavernosum are continually exposed to a low level of endothelially derived NO, as are other vascular smooth muscle cells and platelets, but experience a brief surge of neuronally derived NO during sexual arousal may be a partial explanation for this different response.

Many of the mechanisms involved in NO signaling through cGMP and PKG are now well established, and roles of this pathway in physiological and pathological processes continue to be discovered. The impact of the action of cGMP-hydrolyzing PDEs on this signaling pathway is also well established, and the potential for therapeutic intervention has been demonstrated by the success of PDE5 inhibitors sildenafil, tadalafil, and vardenafil for treatment of erectile dysfunction and pulmonary hypertension.

A full grasp of its mechanisms will undoubtedly open new avenues for treating medical consequences of its dysfunction. National Center for Biotechnology Information , U. Pharmacological Reviews. Pharmacol Rev. Sharron H. Francis , Jennifer L. Busch , and Jackie D.

Author information Copyright and License information Disclaimer. Corresponding author. Address correspondence to: Sharron H. Francis, Dept. This article has been cited by other articles in PMC. Abstract 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.

Introduction The identification of nitric oxide NO 1 , a small gaseous molecule, as a key biological signal was a landmark event in understanding regulation of many physiological functions. Open in a separate window. Cross-Talk among Cyclic Nucleotide Phosphodiesterases. Interaction with cGMP. Structural Features. Autoinhibition and Activation. Han et al. Michalewski et al. Angelone et al. Alioua et al. Sausbier et al. Soh et al. Nakamura et al. Fotis et al. Holmgreen et al. Chalimoniuk et al.

Xia et al. Kang et al. Takasago et al. Wu et al. Andric et al. Layland et al. Wyatt et al. Requirement for Selective Role of cGMP-dependent Protein Kinase I Isozymes and Subcellular Targeting for Phosphorylation of Substrates The shared specifications for consensus sequence in phosphorylation of synthetic peptides by PKGI isozymes suggests that either could phosphorylate a target protein in intact cells, but this is now a controversial concept Wolfe et al.

Regulation of Phosphodiesterase-5 PKGI phosphorylates and activates the cGMP-specific PDE5, the first recognized physiological substrate of known enzymatic function, in platelets, smooth muscle cells, and cerebellar Purkinje cells Wyatt et al.

Dual-Specificity Phosphodiesterases. Negative Feedback Negative feedback regulation is a common control for accelerating the demise of second messenger signals, including that of cGMP. Conclusions Many of the mechanisms involved in NO signaling through cGMP and PKG are now well established, and roles of this pathway in physiological and pathological processes continue to be discovered.

References Agostino et al. Ahmad et al. Aitken et al. Amino acid sequences at the two phosphorylation sites. Aizawa et al. Alderton et al. Alverdi et al. Amano et al. Ammendola et al. Antl et al. Blood — [ PubMed ] [ Google Scholar ]. Aswad and Greengard, Aswad DW, Greengard P. Purification and characterization. Aversa et al. Barber and Butcher, Barber R, Butcher RW. Barger et al. Barman et al. Bazhin et al. Beals et al. Science — [ PubMed ] [ Google Scholar ].

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A slit smearing correction was used in gnom to deconvolute the effect of the beam slit height. Custom software for carrying out the calculations was written in the c programming language. In addition to two-state modeling, we used SVD analysis see, e. Previous application of SVD analysis to small-angle scattering data includes detection of structural intermediates in lysozyme unfolding A matrix was constructed with rows indexing the Q values and columns indexing the titration points. The singular value spectrum was analyzed to determine what fraction of the signal could be explained by the first two components, and the left singular vectors were inspected to determine whether individual scattering components were dominated by noise.

Assumption a is reasonable because PKG concentrations were in the neighborhood of 75, nM, which is 2—3 orders of magnitude larger than the measured cGMP-binding site dissociation constants 55 and nM.

Assumption b has not been experimentally confirmed but is a hypothesis that is tested by our model. We are interested in three functions: i the fraction of the population that has cGMP bound to site A, including binding to both sites, A R x ; ii the fraction of the population that has cGMP bound to site B, including binding to both sites, B R x ; and iii the fraction of the population that has cGMP bound to both sites AB R x.

A perfectly spherical protein of the same molecular weight as the deletion mutant P R distributions from all experiments. The apparent isosbestic point in the P R distributions suggests a mixture of just two molecular species in solution. Scattering data obtained from the 0. Scattering data from the 9. The sample measured with excess cGMP 9.

The apparent isosbestic point in the P R distributions Fig. A linear least-squares fit was performed to optimize the fit of each titration point by selection of relative weights of the compact and extended state. This simple two-state model accurately reproduces the data at each titration point Fig. Singular value decomposition analysis Fig. Two-state model fit overlayed with 1. States are annotated with respect to size and activity.

Right Extended state occupancy vs. Singular value decomposition analysis of cGMP titration data. Left Normalized singular values are shown for SVD of the titration data. Right The first three SVD components of the scattering signal for the titration data are shown offset, sorted top-to-bottom. The third component and higher are dominated by noise.

Combined, these plots provide evidence that the titration data are explained by two underlying signals, consistent with a two-state model of the elongation. For this purpose, we developed a simple model of cGMP binding without cooperativity Fig. The model has no free parameters. The model shows that the occupancy of the high-affinity site alone is poorly correlated with occupancy of the extended state of the molecule Fig. The occupancy of the low-affinity site is much better correlated with the occupancy of the extended state.

The occupancy of both states combined is also well correlated with the occupancy of the extended state. Although the data do not distinguish elongation on binding to the low-affinity site vs.

The PKG and PKA molecules have high enough sequence identity for homology modeling, and are expected to have the same overall shape. Our data are therefore consistent with the model of the interaction between the R subunit and C subunit of PKA, and can provide insight into mechanisms of cyclic nucleotide activation of both enzymes.

Dissociation of PKA-I is greatly enhanced when exactly two cAMP molecules are bound to each R subunit 25 , suggesting that two cAMP molecules are required to release the autoinhibitory pseudosubstrate sequence of the R subunit from the catalytic cleft of the C subunit and disrupt all other interactions between the PKA subunits.

Indeed, for both the PKAs and the PKGs, cyclic nucleotide binding releases the autoinhibitory sequence from the catalytic cleft see ref. Support for these arguments comes from considering the model of the complex between the PKA R subunit and C subunit Our scattering data suggest that the interactions between the R and C domains in PKG are similar to those between the subunits in PKA, and so we predict that the low-affinity domain of the PKG R domain has the smaller set of interactions with the C domain.

In addition, all biochemical studies of the PKGs and PKAs support involvement of both cyclic nucleotide-binding sites in activation of the enzymes, regardless of their order in the primary structure. Because activation requires release of pseudosubstrate from the catalytic cleft see ref.

Conversely, the effect of elongation or dissociation on activity in cyclic nucleotide-dependent protein kinases is currently unknown. Although binding of cAMP was originally assumed to cause dissociation of the R and C subunits in PKA 27 — 29 , fluorescent resonance energy transfer measurements suggest that dissociation may require interactions with the PKA inhibitor protein 30 — 33 , and question whether dissociation is required for activation 34 , The amount of elongation in the dimer can thus be easily accounted for by the elongation of two monomers.