FRINK Linked Data Fragments server

Matches in SemOpenAlex for { <https://semopenalex.org/work/W2018916450> ?p ?o ?g. }

• W2018916450 endingPage "29705" @default.
• W2018916450 startingPage "29699" @default.
• W2018916450 abstract "Photoreceptor cGMP phosphodiesterase (PDE6) is the central enzyme in the visual transduction cascade. The PDE6 catalytic subunit contains a catalytic domain and regulatory GAF domains. Unlike most GAF domain-containing cyclic nucleotide phosphodiesterases, little is known about direct allosteric communication of PDE6. In this study, we demonstrate for the first time direct, inter-domain allosteric communication between the GAF and catalytic domains in PDE6. The binding affinity of PDE6 for pharmacological inhibitors or for the C-terminal region of the inhibitory γ subunit (Pγ), known to directly inhibit PDE6 catalysis, was increased ∼2-fold by ligands binding to the GAF domain. Binding of the N-terminal half of Pγ to the GAF domains suffices to induce this allosteric effect. Allosteric communication between GAF and catalytic domains is reciprocal, in that drug binding to the catalytic domain slowed cGMP dissociation from the GAF domain. Although cGMP hydrolysis was not affected by binding of Pγ1–60, Pγ lacking its last seven amino acids decreased the Michaelis constant of PDE6 by 2.5-fold. Pγ1–60 binding to the GAF domain increased vardenafil but not cGMP affinity, indicating that substrate- and inhibitor-binding sites do not totally overlap. In addition, prolonged incubation of PDE6 with vardenafil or sildenafil (but not 3-isobutyl-1-methylxanthine and zaprinast) induced a distinct conformational change in the catalytic domain without affecting the binding properties of the GAF domains. We conclude that although Pγ-mediated regulation plays the dominant role in visual excitation, the direct, inter-domain allosteric regulation described in this study may play a feedback role in light adaptational processes during phototransduction. Photoreceptor cGMP phosphodiesterase (PDE6) is the central enzyme in the visual transduction cascade. The PDE6 catalytic subunit contains a catalytic domain and regulatory GAF domains. Unlike most GAF domain-containing cyclic nucleotide phosphodiesterases, little is known about direct allosteric communication of PDE6. In this study, we demonstrate for the first time direct, inter-domain allosteric communication between the GAF and catalytic domains in PDE6. The binding affinity of PDE6 for pharmacological inhibitors or for the C-terminal region of the inhibitory γ subunit (Pγ), known to directly inhibit PDE6 catalysis, was increased ∼2-fold by ligands binding to the GAF domain. Binding of the N-terminal half of Pγ to the GAF domains suffices to induce this allosteric effect. Allosteric communication between GAF and catalytic domains is reciprocal, in that drug binding to the catalytic domain slowed cGMP dissociation from the GAF domain. Although cGMP hydrolysis was not affected by binding of Pγ1–60, Pγ lacking its last seven amino acids decreased the Michaelis constant of PDE6 by 2.5-fold. Pγ1–60 binding to the GAF domain increased vardenafil but not cGMP affinity, indicating that substrate- and inhibitor-binding sites do not totally overlap. In addition, prolonged incubation of PDE6 with vardenafil or sildenafil (but not 3-isobutyl-1-methylxanthine and zaprinast) induced a distinct conformational change in the catalytic domain without affecting the binding properties of the GAF domains. We conclude that although Pγ-mediated regulation plays the dominant role in visual excitation, the direct, inter-domain allosteric regulation described in this study may play a feedback role in light adaptational processes during phototransduction. The photoreceptor cyclic nucleotide phosphodiesterase (PDE6) 3The abbreviations used are: PDE6, photoreceptor PDE; PDE, cyclic nucleotide phosphodiesterase; Pαβ, catalytic dimer of PDE6 α and β subunits; Pγ, inhibitory γ subunit of PDE6; IBMX, 3-isobutyl-1-methylxanthine. 3The abbreviations used are: PDE6, photoreceptor PDE; PDE, cyclic nucleotide phosphodiesterase; Pαβ, catalytic dimer of PDE6 α and β subunits; Pγ, inhibitory γ subunit of PDE6; IBMX, 3-isobutyl-1-methylxanthine. is the central enzyme in the vertebrate visual signaling pathway in rods and cones. Phototransduction is initiated when light induces the isomerization of the 11-cis-retinal chromophore of rhodopsin, which leads to activation of the photoreceptor-specific G-protein, transducin. Activated transducin then causes activation of PDE6, which results in rapid lowering of cGMP levels, closure of cGMP-gated ion channels, and hyperpolarization of the cell membrane (1Fain G.L. Sensory Transduction. Sinauer Associates, Inc., Sunderland, MA2003Google Scholar, 2Zhang X. Cote R.H. Front. Biosci. 2005; 10: 1191-1204Crossref PubMed Scopus (65) Google Scholar, 3Lamb T.D. Pugh Jr., E.N. Investig. Ophthalmol. Vis. Sci. 2006; 47: 5138-5152Crossref Scopus (224) Google Scholar). Hydrolysis of cGMP by PDE6 must be precisely regulated to control the amplitude and kinetics of the photoresponse. Furthermore, each of these parameters undergoes additional modulation in response to ever-changing conditions of ambient illumination. The PDE6 holoenzyme consists of a catalytic dimer of α and β subunits (Pαβ) and two inhibitory γ subunits (Pγ) that are tightly bound to Pαβ. Transducin activation of PDE6 results from displacement of the inhibitory constraint of Pγ upon activated transducin binding to PDE6. The affinity of Pγ for the Pαβ catalytic dimer is also modulated in a reciprocal manner by noncatalytic cGMP binding to PDE6 at sites distinct from the catalytic site (Ref. 4Arshavsky V.Y. Dumke C.L. Bownds M.D. J. Biol. Chem. 1992; 267: 24501-24507Abstract Full Text PDF PubMed Google Scholar and reviewed in Ref. 5Cote R.H. Beavo J.A. Francis S.H. Houslay M.D. Cyclic Nucleotide Phosphodiesterases in Health and Disease. CRC Press, Inc., Boca Raton, FL2006: 165-193Google Scholar).Photoreceptor PDE6 is one of five members of the class I phosphodiesterase superfamily that contain tandem regulatory GAF domains (i.e. GAFa and GAFb; (6Zoraghi R. Corbin J.D. Francis S.H. Mol. Pharmacol. 2004; 65: 267-278Crossref PubMed Scopus (124) Google Scholar)). The GAF domains were originally named for their presence in cGMP-regulated PDEs, certain adenylyl cyclases, and the transcription factor Fh1A of bacteria (7Aravind L. Ponting C.P. Trends Biochem. Sci. 1997; 22: 458-459Abstract Full Text PDF PubMed Scopus (486) Google Scholar). The GAF domains of the vertebrate PDE members contain a functional cyclic nucleotide binding pocket. cGMP is the ligand for PDE2, PDE5, PDE6, and PDE11 (8Francis S.H. Lincoln T.M. Corbin J.D. J. Biol. Chem. 1980; 255: 620-626Abstract Full Text PDF PubMed Google Scholar, 13Gross-Langenhoff M. Hofbauer K. Weber J. Schultz A. Schultz J.E. J. Biol. Chem. 2006; 281: 2841-2846Abstract Full Text Full Text PDF PubMed Scopus (102) Google Scholar), whereas cAMP is the ligand for PDE10 (13Gross-Langenhoff M. Hofbauer K. Weber J. Schultz A. Schultz J.E. J. Biol. Chem. 2006; 281: 2841-2846Abstract Full Text Full Text PDF PubMed Scopus (102) Google Scholar). For PDE6, the noncatalytic cGMP-binding site has been localized to the N-terminal GAFa domain (14Muradov H. Boyd K.K. Artemyev N.O. Vision Res. 2004; 44: 2437-2444Crossref PubMed Scopus (24) Google Scholar, 15Huang D. Hinds T.R. Martinez S.E. Doneanu C. Beavo J.A. J. Biol. Chem. 2004; 279: 48143-48151Abstract Full Text Full Text PDF PubMed Scopus (47) Google Scholar) (Fig. 1A).Direct allosteric regulation of catalytic activity induced by binding of cyclic nucleotides to the GAF domains has been well documented for PDE2 and PDE5. For both PDE families, cGMP binding to the GAF domains induces a conformational change that relieves inhibition of catalysis in the active sites, causing stimulation of the enzyme (10Martins T.J. Mumby M.C. Beavo J.A. J. Biol. Chem. 1982; 257: 1973-1979Abstract Full Text PDF PubMed Google Scholar, 16Yamamoto T. Manganiello V.C. Vaughan M. J. Biol. Chem. 1983; 258: 12526-12533Abstract Full Text PDF PubMed Google Scholar, 17Rybalkin S.D. Rybalkina I.G. Shimizu-Albergine M. Tang X.B. Beavo J.A. EMBO J. 2003; 22: 469-478Crossref PubMed Scopus (196) Google Scholar). Furthermore, the binding affinity of inhibitors to the catalytic domains of PDE5 is increased by cGMP addition (18Corbin J.D. Blount M.A. Weeks J.L. Beasley A. Kuhn K.P. Ho Y.S. Saidi L.F. Hurley J.H. Kotera J. Francis S.H. Mol. Pharmacol. 2003; 63: 1364-1372Crossref PubMed Scopus (64) Google Scholar, 19Blount M.A. Beasley A. Zoraghi R. Sekhar K.R. Bessay E.P. Francis S.H. Corbin J.D. Mol. Pharmacol. 2004; 66: 144-152Crossref PubMed Scopus (148) Google Scholar). As predicted, this allosteric regulation between the GAF and catalytic domains is reciprocal. For example, in PDE5, some inhibitors enhanced cGMP binding to the GAF domains (11Thomas M.K. Francis S.H. Corbin J.D. J. Biol. Chem. 1990; 265: 14964-14970Abstract Full Text PDF PubMed Google Scholar, 20Turko I.V. Ballard S.A. Francis S.H. Corbin J.D. Mol. Pharmacol. 1999; 56: 124-130Crossref PubMed Scopus (161) Google Scholar). In addition to this direct allosteric communication between GAF and catalytic domains, it has been reported that PDE5 inhibitors can induce a conformational change in the catalytic domain that enhances inhibitor binding affinity in a time-dependent manner (21Blount M.A. Zoraghi R. Bessay E.P. Beasley A. Francis S.H. Corbin J.D. J. Pharmacol. Exp. Ther. 2007; 323: 730-737Crossref PubMed Scopus (20) Google Scholar).Based on the many similarities between PDE5 and PDE6 (22Cote R.H. Int. J. Impot. Res. 2004; 16: S28-S33Crossref PubMed Scopus (131) Google Scholar), direct, inter-domain allosteric communication between the GAF and catalytic domains is predicted for PDE6. However, previous work evaluating whether cGMP binding could influence the catalytic properties of PDE6 has not revealed a direct parallel to the allosteric control exerted on PDE5. For example, cGMP binding to the GAF domains fails to alter either the Km or kcat of the enzyme (4Arshavsky V.Y. Dumke C.L. Bownds M.D. J. Biol. Chem. 1992; 267: 24501-24507Abstract Full Text PDF PubMed Google Scholar, 23D'Amours M.R. Cote R.H. Biochem. J. 1999; 340: 863-869Crossref PubMed Scopus (38) Google Scholar, 24Mou H. Cote R.H. J. Biol. Chem. 2001; 276: 27527-27534Abstract Full Text Full Text PDF PubMed Scopus (72) Google Scholar). Instead, attention has focused on the allosteric control mediated by Pγ on both GAF and catalytic domains. The N-terminal region of Pγ (Fig. 1B) is known to interact with the GAF domains of the Pαβ catalytic dimer with a 50-fold higher affinity than the affinity of the C-terminal region of Pγ for the catalytic domain of Pαβ (24Mou H. Cote R.H. J. Biol. Chem. 2001; 276: 27527-27534Abstract Full Text Full Text PDF PubMed Scopus (72) Google Scholar, 25Artemyev N.O. Hamm H.E. Biochem. J. 1992; 283: 273-279Crossref PubMed Scopus (72) Google Scholar, 26Takemoto D.J. Hurt D. Oppert B. Cunnick J. Biochem. J. 1992; 281: 637-643Crossref PubMed Scopus (42) Google Scholar, 27Guo L.W. Grant J.E. Hajipour A.R. Muradov H. Arbabian M. Artemyev N.O. Ruoho A.E. J. Biol. Chem. 2005; 280: 12585-12592Abstract Full Text Full Text PDF PubMed Scopus (34) Google Scholar, 28Guo L.W. Muradov H. Hajipour A.R. Sievert M.K. Artemyev N.O. Ruoho A.E. J. Biol. Chem. 2006; 281: 15412-15422Abstract Full Text Full Text PDF PubMed Scopus (42) Google Scholar). The C-terminal region of Pγ (Fig. 1B) is responsible for blocking the catalytic activity by binding to the catalytic domains of Pαβ (27Guo L.W. Grant J.E. Hajipour A.R. Muradov H. Arbabian M. Artemyev N.O. Ruoho A.E. J. Biol. Chem. 2005; 280: 12585-12592Abstract Full Text Full Text PDF PubMed Scopus (34) Google Scholar, 31Artemyev N.O. Natochin M. Busman M. Schey K.L. Hamm H.E. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 5407-5412Crossref PubMed Scopus (53) Google Scholar). The ability of Pγ to interact with both GAF and catalytic domains of Pαβ serves to allosterically link the regulatory and catalytic domains of PDE6 in two ways as follows: 1) Pγ binding to the catalytic dimer enhances the binding affinity of cGMP to the GAF domain (32Yamazaki A. Bartucci F. Ting A. Bitensky M.W. Proc. Natl. Acad. Sci. U. S. A. 1982; 79: 3702-3706Crossref PubMed Scopus (81) Google Scholar, 33Cote R.H. Bownds M.D. Arshavsky V.Y. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 4845-4849Crossref PubMed Scopus (84) Google Scholar); 2) cGMP occupancy of the GAF domain enhances Pγ affinity to Pαβ (4Arshavsky V.Y. Dumke C.L. Bownds M.D. J. Biol. Chem. 1992; 267: 24501-24507Abstract Full Text PDF PubMed Google Scholar, 23D'Amours M.R. Cote R.H. Biochem. J. 1999; 340: 863-869Crossref PubMed Scopus (38) Google Scholar, 24Mou H. Cote R.H. J. Biol. Chem. 2001; 276: 27527-27534Abstract Full Text Full Text PDF PubMed Scopus (72) Google Scholar).In this study, we first document that direct allosteric communication between the GAF domains and catalytic domains of PDE6 does indeed occur. Ligand binding to the GAF domains enhances the affinity of inhibitors and Pγ63–87 (i.e. amino acids 63–87 of the Pγ sequence) binding to the catalytic dimer. This inter-domain allosteric mechanism is reciprocal, in that inhibitor binding to catalytic domains increases the binding affinity of cGMP to the GAF domains. The magnitude of this direct allosteric regulation in PDE6 is comparable with that seen in PDE5, and it may play a role in modulating PDE6 activity during persistent activation of rod photoreceptors that occurs during normal daytime illumination conditions.EXPERIMENTAL PROCEDURESMaterials—Bovine retinas were purchased from W. L. Lawson, Inc. Synthetic peptide Pγ63–87 was purchased from New England Peptide. Vardenafil and sildenafil were provided by Bayer Healthcare AG. Ultima Gold scintillation fluid was from PerkinElmer Life Sciences. Filtration membranes were from Millipore, bicinchoninic acid protein assay reagents were from Pierce, and all other chemicals were from Sigma. Stock solutions of PDE inhibitors were prepared in DMSO and diluted to less than 1% final concentration before use in assays.PDE6 and Pαβ Purification and Functional Assays—Bovine rod PDE6 was purified from bovine retinas as described (34Pentia D.C. Hosier S. Collupy R.A. Valeriani B.A. Cote R.H. Methods Mol. Biol. 2005; 307: 125-140PubMed Google Scholar). Pαβ catalytic dimers lacking Pγ were prepared by limited trypsin proteolysis and re-purified by gel filtration chromatography prior to use (34Pentia D.C. Hosier S. Collupy R.A. Valeriani B.A. Cote R.H. Methods Mol. Biol. 2005; 307: 125-140PubMed Google Scholar). PDE6 catalytic activity was measured in 20 mm Tris, 10 mm MgCl2, 0.5 mg/ml bovine serum albumin either with a phosphate release microplate assay or with a radiotracer assay (35Cote R.H. Methods Enzymol. 2000; 315: 646-672Crossref PubMed Google Scholar). The PDE6 concentration was estimated based on the rate of cGMP hydrolysis of trypsin-activated PDE6 and a knowledge of the kcat of the enzyme (5600 mol of cGMP hydrolyzed per Pαβ per s (36Mou H. Grazio H.J. Cook T.A. Beavo J.A. Cote R.H. J. Biol. Chem. 1999; 274: 18813-18820Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar)); this estimate was validated by determining stoichiometric binding of [3H]cGMP to PDE6 with a filter binding assay (37Cote R.H. Methods Mol. Biol. 2005; 307: 141-154PubMed Google Scholar). The inhibition potency (IC50) of PDE5/6 inhibitors or Pγ63–87 was determined in the presence or absence of Pγ mutants using either 2 mm cGMP or 0.1 mm cAMP as substrates.Purification of Pγ and Pγ Mutants—Several Pγ truncation mutants (Fig. 1C) were generated from the full-length coding sequence using standard methods, introduced into the pET11a (Novagen) expression vector, and nucleotide sequences were verified. Recombinant Pγ and mutants (Pγ1–45, Pγ1–60, and Pγ1–80) were expressed in Escherichia coli BL21(DE3). The bacterial extract was partially purified by cation exchange chromatography using SP-Sepharose, followed by C4 reverse-phase high pressure liquid chromatography (38Artemyev N.O. Arshavsky V.Y. Cote R.H. Methods (San Diego). 1998; 14: 93-104Crossref PubMed Scopus (30) Google Scholar). The purity (>95%) and size of these proteins were evaluated by SDS-PAGE. The inhibitory activity of Pγ was assessed by its ability to stoichiometrically inhibit Pαβ catalytic dimers (2 Pγ per Pαβ) (24Mou H. Cote R.H. J. Biol. Chem. 2001; 276: 27527-27534Abstract Full Text Full Text PDF PubMed Scopus (72) Google Scholar). Protein concentrations were determined by the bicinchoninic acid protein assay (39Smith P.K. Krohn R.I. Hermanson G.T. Mallia A.K. Gartner F.H. Provenzano M.D. Fujimoto E.K. Goeke N.M. Olson B.J. Klenk D.C. Anal. Biochem. 1985; 150: 76-85Crossref PubMed Scopus (18445) Google Scholar) using bovine γ-globulin as a standard.Loading cGMP on the GAF Domains of Activated PDE6—Purified Pαβ was preincubated with 10 mm EDTA in binding buffer (100 mm Tris, 2 mm MgCl2, and 0.5 mg/ml bovine serum albumin) for 2 h at 22 °C to inhibit cGMP breakdown before addition of 1 μm [3H]cGMP and N-terminal region Pγ peptides (24Mou H. Cote R.H. J. Biol. Chem. 2001; 276: 27527-27534Abstract Full Text Full Text PDF PubMed Scopus (72) Google Scholar). The amount of cGMP bound to PDE6 under these conditions was verified to be 1.7–2.0 cGMP per Pαβ. The hydrolytic activity of PDE6 was restored by adding 10 mm MgCl2 immediately prior to assaying cyclic nucleotide hydrolysis. For experiments in which [3H]cGMP dissociation kinetics from Pαβ were measured in the absence of Pγ, purified Pαβ was preincubated for 2 h with 10 mm EDTA plus 20 mm dipicolinic acid to abolish residual catalytic activity (40He F. Seryshev A.B. Cowan C.W. Wensel T.G. J. Biol. Chem. 2000; 275: 20572-20577Abstract Full Text Full Text PDF PubMed Scopus (45) Google Scholar). (EDTA alone was unable to protect [3H]cGMP from breakdown by Pαβ (lacking Pγ peptides) during the binding assay.) Release of [3H]cGMP from Pαβ was monitored following addition of 1 mm unlabeled cGMP (containing 10 mm MgCl2 and 5 mm ZnSO4), and the time course of [3H]cGMP dissociation was monitored in the presence or absence of 100 μm vardenafil.Data Analysis—Dose-response experiments were analyzed using nonlinear regression analysis (Sigmaplot) to fit experimental data to a three-parameter logistic dose-response function: y = a/(1 + (x/x0)b), where a is the amplitude, b is the slope factor, and x0 is the IC50 (41Jungbauer A. Graumann K. J. Clin. Ligand Assay. 2001; 24: 270-274Google Scholar). For other experiments, curve-fitting models are described in the figure legends. Except where noted, all experiments were repeated at least three times. Tests of statistical significance for the curve fitting results used the Student's t test to calculate probability values, as indicated in the figure legends.RESULTSBinding of Ligands to the Regulatory GAF Domain Enhances the Ability of Vardenafil to Bind to the Catalytic Domain—Motivated by the analogy of PDE6 with other GAF-containing PDEs, we first explored whether ligand binding to the GAF domains could allosterically alter the properties of the active site of PDE6.Because both cGMP and the N-terminal region of Pγ are known to bind to the GAF domains of the PDE6 catalytic dimer (Pαβ; see Introduction and Fig. 1), we first examined whether the catalytic properties of PDE6 were altered upon binding of these ligands to the GAF domains. The first 60 amino acids of Pγ (Pγ1–60) lack the ability to inhibit catalysis (data not shown), and the primary sites of interaction are confined to the GAF domains (28Guo L.W. Muradov H. Hajipour A.R. Sievert M.K. Artemyev N.O. Ruoho A.E. J. Biol. Chem. 2006; 281: 15412-15422Abstract Full Text Full Text PDF PubMed Scopus (42) Google Scholar, 42Song J. Guo L.W. Muradov H. Artemyev N.O. Ruoho A.E. Markley J.L. Proc. Natl. Acad. Sci. U. S. A. 2008; 105: 1505-1510Crossref PubMed Scopus (79) Google Scholar). We therefore used the truncated mutant Pγ1–60 to determine whether the binding affinity of vardenafil to the catalytic domain of Pαβ was altered when Pγ1–60 or Pγ1–60 plus cGMP were bound. To evaluate catalytic activity while simultaneously testing the effect of occupancy of the GAF domains, cAMP was used as a substrate for this experiment because it binds very poorly to the GAF domain even at high concentrations (12Hebert M.C. Schwede F. Jastorff B. Cote R.H. J. Biol. Chem. 1998; 273: 5557-5565Abstract Full Text Full Text PDF PubMed Scopus (23) Google Scholar). Fig. 2 demonstrates that the inhibitory potency of vardenafil was increased ∼2-fold (IC50 shifted from 4.3 ± 0.3 to 2.2 ± 0.2 nm) by Pγ1–60 binding to the GAF domain. Experiments using a shorter N-terminal fragment of Pγ (Pγ1–45) showed the same 2-fold enhancement of vardenafil binding as were seen with Pγ1–60 (data not shown), confirming that the GAF-interacting region of Pγ (localized to amino acid residues 18–45 (24Mou H. Cote R.H. J. Biol. Chem. 2001; 276: 27527-27534Abstract Full Text Full Text PDF PubMed Scopus (72) Google Scholar)) was responsible for this inter-domain allosterism observed in Fig. 2.FIGURE 2Pγ1–60 binding to the PDE6 GAF domains enhanced vardenafil binding affinity to the catalytic sites. Purified Pαβ (2 nm) was preincubated with 10 mm EDTA for 2 h to inhibit PDE activity, followed by incubation with 1 μm cGMP and 2 μm Pγ1–60 (•), 2 μm Pγ1–60 only (□), or no addition (▴). 10 mm Mg2+ was then added to restore catalytic activity, and the inhibitory potency of vardenafil was measured using 0.1 mm cAMP as substrate with the radiotracer assay (see “Experimental Procedures”). cGMP binding assays confirmed retention of bound cGMP during the experiment. The data are the mean (±S.E.) of five experiments. The solid lines represent the fit to a three-parameter logistic dose-response equation with IC50 values of 4.3 ± 0.3 nm (no addition), 2.2 ± 0.2 nm (Pγ1–60), and 2.3 ± 0.1 nm (Pγ1–60 plus cGMP). The asterisks indicate that the IC50 value was statistically significant (p < 0.05) from the control value.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Inclusion of cGMP with Pγ1–60 so as to occupy the GAFa cGMP binding pocket failed to further enhance the allosteric effect on vardenafil binding to the Pαβ active site (Fig. 2). (Unfortunately, we were unable to directly measure the allosteric effects of cGMP binding in the absence of the Pγ N-terminal region, because the high catalytic rate of Pαβ led to hydrolysis of cGMP unless GAF-interacting Pγ peptides were present to stabilize cGMP binding to the GAFa domain.) It is possible that either Pγ binding to the GAF domain or cGMP occupancy of the GAFa binding pocket induce the same conformational change in the GAF domains that is transmitted to the catalytic domain.Binding of the N-terminal Region of Pγ to the GAF Domains Enhances the Binding Affinity of Pγ63–87 to the Catalytic Domain—The C-terminal region of Pγ inhibits PDE6 catalysis by interacting with amino acid residues lining the entrance to the catalytic pocket (43Granovsky A.E. Artemyev N.O. J. Biol. Chem. 2000; 275: 41258-41262Abstract Full Text Full Text PDF PubMed Scopus (26) Google Scholar); these residues are predicted to be distant from those stabilizing vardenafil binding to the active site (44Card G.L. England B.P. Suzuki Y. Fong D. Powell B. Lee B. Luu C. Tabrizizad M. Gillette S. Ibrahim P.N. Artis D.R. Bollag G. Milburn M.V. Kim S.H. Schlessinger J. Zhang K.Y. Structure (Lond.). 2004; 12: 2233-2247Abstract Full Text Full Text PDF PubMed Scopus (339) Google Scholar). Therefore, we questioned whether binding of the N-terminal region of Pγ to the GAF domains could alter the binding affinity of the C-terminal region of Pγ to the catalytic domains of Pαβ. Fig. 3 shows that the dose-response curve for Pγ63–87 inhibition of cGMP hydrolysis was shifted about 2-fold when Pγ1–45 or Pγ1–60 was bound to the GAF domains. The finding that both vardenafil and Pγ63–87 affinity were increased ∼2-fold when the N-terminal half of Pγ bound to the PDE6 GAF domains indicates that this inter-domain allosteric change is likely to affect the global conformation of the PDE6 catalytic domain.FIGURE 3Binding of Pγ1–45 or Pγ1–60 to the GAF domains equally enhanced the binding affinity of Pγ63–87 to the catalytic domain. Purified Pαβ (0.2 nm) was preincubated with 2 μm Pγ1–45 (□), 2 μm Pγ1–60 (•), or no peptide (▴) for 10 min before adding increasing amounts of Pγ63–87. The PDE activity was measured using 2 mm cGMP as substrate. The data represent the average of four experiments, and curve fitting (solid line) yielded the following IC50 values: 2.2 ± 0.1 μm (no added peptide); 1.2 ± 0.1 μm (Pγ1–45); and 1.5 ± 0.2 μm (Pγ1–60). The asterisks indicate that the IC50 value was statistically significant (p ≤ 0.05) from the control value.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Conformational Changes in Substrate Affinity to the Active Site Require Pγ Interactions with the Catalytic Domain, Not the GAF Domains—These novel allosteric effects of Pγ on vardenafil and Pγ63–87 binding to PDE6 differ from earlier work in which cGMP and/or Pγ1–45 binding to the GAF domains failed to allosterically alter the kinetic parameters for substrate hydrolysis of the Pαβ active site (4Arshavsky V.Y. Dumke C.L. Bownds M.D. J. Biol. Chem. 1992; 267: 24501-24507Abstract Full Text PDF PubMed Google Scholar, 24Mou H. Cote R.H. J. Biol. Chem. 2001; 276: 27527-27534Abstract Full Text Full Text PDF PubMed Scopus (72) Google Scholar). This is unexpected, because cGMP and vardenafil are likely to share some interaction sites within the catalytic pocket, as judged by comparison of crystal structures of PDE5 complexed with 5′-GMP and vardenafil (44Card G.L. England B.P. Suzuki Y. Fong D. Powell B. Lee B. Luu C. Tabrizizad M. Gillette S. Ibrahim P.N. Artis D.R. Bollag G. Milburn M.V. Kim S.H. Schlessinger J. Zhang K.Y. Structure (Lond.). 2004; 12: 2233-2247Abstract Full Text Full Text PDF PubMed Scopus (339) Google Scholar, 45Zhang K.Y. Card G.L. Suzuki Y. Artis D.R. Fong D. Gillette S. Hsieh D. Neiman J. West B.L. Zhang C. Milburn M.V. Kim S.H. Schlessinger J. Bollag G. Mol. Cell. 2004; 15: 279-286Abstract Full Text Full Text PDF PubMed Scopus (247) Google Scholar). To examine this more closely, we measured the Michaelis constant (Km) for cGMP in the presence of two Pγ truncation mutants, Pγ1–60 and Pγ1–80. Pγ1–60 was chosen because it cannot inhibit catalysis, and its primary sites of interaction with Pαβ are within the GAF domain, whereas Pγ1–80 partially inhibits catalysis (∼60% reduction in Vmax; see Ref. 30Skiba N.P. Artemyev N.O. Hamm H.E. J. Biol. Chem. 1995; 270: 13210-13215Abstract Full Text Full Text PDF PubMed Scopus (63) Google Scholar) and has been shown to interact with sites within the catalytic domain (28Guo L.W. Muradov H. Hajipour A.R. Sievert M.K. Artemyev N.O. Ruoho A.E. J. Biol. Chem. 2006; 281: 15412-15422Abstract Full Text Full Text PDF PubMed Scopus (42) Google Scholar, 29Lipkin V.M. Dumler I.L. Muradov K.G. Artemyev N.O. Etingof R.N. FEBS Lett. 1988; 234: 287-290Crossref PubMed Scopus (57) Google Scholar, 42Song J. Guo L.W. Muradov H. Artemyev N.O. Ruoho A.E. Markley J.L. Proc. Natl. Acad. Sci. U. S. A. 2008; 105: 1505-1510Crossref PubMed Scopus (79) Google Scholar) (see Fig. 1). Whereas Pγ1–60 failed to affect the Km (or Vmax) value for cGMP hydrolysis (Fig. 4) (consistent with previous work with Pγ1–45 (24Mou H. Cote R.H. J. Biol. Chem. 2001; 276: 27527-27534Abstract Full Text Full Text PDF PubMed Scopus (72) Google Scholar)), binding of Pγ1–80 to Pαβ increased the Km value for cGMP from 9 ± 0.9 to 23 ± 1.5 μm (Fig. 4). The differences in how cGMP (Fig. 4) and vardenafil (Fig. 2) are affected by Pγ binding to Pαβ may reflect a local conformational change within the catalytic domain upon binding amino acids 61–80 of Pγ that is distinct from the inter-domain communication between the GAF and catalytic domains that is induced by the GAF-interacting region of Pγ.FIGURE 4Pγ1–80 increased the Michaelis-Menten constant of PDE6. Purified Pαβ (20 pm) was preincubated with 200 nm Pγ1–60 (⋄), 200 nm Pγ1–80 (▴) or no peptide (•) for 20 min before addition of increasing amounts of cGMP. The data represent the average of three experiments (except Pγ1–60 where n = 2) and were fit to a two-parameter hyperbolic function with the following parameters: no peptides, Km = 9.1 ± 0.6 μm and Vmax = 6.2 ± 0.1 pmol cGMP/s; Pγ1–60, Km = 9.5 ± 0.9 μm, Vmax = 6.1 ± 0.1 pmol cGMP/s; and Pγ1–80, Km = 22.9 ± 1.5 μm, Vmax = 2.4 ± 0.05 pmol cGMP/s. The asterisk indicates that the Km value for Pγ1–80 was statistically significant (p < 0.05) from the control and Pγ1–60 values.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Binding of Vardenafil to the Catalytic Domains Enhanced cGMP Binding to the GAF Domains—Having established in the previous sections the direct allosteric regulation by the GAF domains on the catalytic domain of PDE6, the principle of allosteric linkage (46Wyman J. Gill S.J. Binding and Linkage: Functional Chemistry of Biological Macromolecules. University Science Books, Mill Valley, CA1990Google Scholar) requires that this allosteric communication be reciprocal; occupancy of the active site should induce conformational changes in the GAF domains. In the earliest study of cGMP binding to the GAF domains of PDE6, the nonspecific PDE inhibitor 3-isobutyl-1-methylxanthine (IBMX) was reported to allosterically enhance cG" @default.
• W2018916450 created "2016-06-24" @default.
• W2018916450 creator A5020344994 @default.
• W2018916450 creator A5035272280 @default.
• W2018916450 creator A5056475217 @default.
• W2018916450 creator A5059322383 @default.
• W2018916450 creator A5090068039 @default.
• W2018916450 date "2008-10-01" @default.
• W2018916450 modified "2023-09-26" @default.
• W2018916450 title "Direct Allosteric Regulation between the GAF Domain and Catalytic Domain of Photoreceptor Phosphodiesterase PDE6" @default.
• W2018916450 cites W1485225530 @default.
• W2018916450 cites W1503740541 @default.
• W2018916450 cites W1506764244 @default.
• W2018916450 cites W1540498597 @default.
• W2018916450 cites W154833763 @default.
• W2018916450 cites W1550626564 @default.
• W2018916450 cites W1551150403 @default.
• W2018916450 cites W1559386011 @default.
• W2018916450 cites W1563092825 @default.
• W2018916450 cites W1607006163 @default.
• W2018916450 cites W1928328877 @default.
• W2018916450 cites W1967786141 @default.
• W2018916450 cites W1968469250 @default.
• W2018916450 cites W1971420793 @default.
• W2018916450 cites W1974974112 @default.
• W2018916450 cites W1995086953 @default.
• W2018916450 cites W2002131367 @default.
• W2018916450 cites W2009620135 @default.
• W2018916450 cites W2013479983 @default.
• W2018916450 cites W2014621993 @default.
• W2018916450 cites W2019102477 @default.
• W2018916450 cites W2024979469 @default.
• W2018916450 cites W2025789164 @default.
• W2018916450 cites W2035974356 @default.
• W2018916450 cites W2040580243 @default.
• W2018916450 cites W2041816586 @default.
• W2018916450 cites W2043392738 @default.
• W2018916450 cites W2047737149 @default.
• W2018916450 cites W2050662518 @default.
• W2018916450 cites W2050780592 @default.
• W2018916450 cites W2052712306 @default.
• W2018916450 cites W2059521359 @default.
• W2018916450 cites W2064110954 @default.
• W2018916450 cites W2073484830 @default.
• W2018916450 cites W2077116876 @default.
• W2018916450 cites W2083106618 @default.
• W2018916450 cites W2085041519 @default.
• W2018916450 cites W2087659427 @default.
• W2018916450 cites W2089677633 @default.
• W2018916450 cites W2093425156 @default.
• W2018916450 cites W2093795363 @default.
• W2018916450 cites W2107307927 @default.
• W2018916450 cites W2111993777 @default.
• W2018916450 cites W2117903697 @default.
• W2018916450 cites W2119774957 @default.
• W2018916450 cites W2132124951 @default.
• W2018916450 cites W2132183423 @default.
• W2018916450 cites W2137248027 @default.
• W2018916450 cites W2142793025 @default.
• W2018916450 cites W2150744925 @default.
• W2018916450 cites W2154558722 @default.
• W2018916450 cites W2165758443 @default.
• W2018916450 cites W2170589977 @default.
• W2018916450 cites W2400385201 @default.
• W2018916450 cites W4232534952 @default.
• W2018916450 doi "https://doi.org/10.1074/jbc.m803948200" @default.
• W2018916450 hasPubMedCentralId "https://www.ncbi.nlm.nih.gov/pmc/articles/2573074" @default.
• W2018916450 hasPubMedId "https://pubmed.ncbi.nlm.nih.gov/18779324" @default.
• W2018916450 hasPublicationYear "2008" @default.
• W2018916450 type Work @default.
• W2018916450 sameAs 2018916450 @default.
• W2018916450 citedByCount "22" @default.
• W2018916450 countsByYear W20189164502012 @default.
• W2018916450 countsByYear W20189164502014 @default.
• W2018916450 countsByYear W20189164502017 @default.
• W2018916450 countsByYear W20189164502019 @default.
• W2018916450 countsByYear W20189164502020 @default.
• W2018916450 countsByYear W20189164502021 @default.
• W2018916450 countsByYear W20189164502023 @default.
• W2018916450 crossrefType "journal-article" @default.
• W2018916450 hasAuthorship W2018916450A5020344994 @default.
• W2018916450 hasAuthorship W2018916450A5035272280 @default.
• W2018916450 hasAuthorship W2018916450A5056475217 @default.
• W2018916450 hasAuthorship W2018916450A5059322383 @default.
• W2018916450 hasAuthorship W2018916450A5090068039 @default.
• W2018916450 hasBestOaLocation W20189164501 @default.
• W2018916450 hasConcept C134306372 @default.
• W2018916450 hasConcept C161790260 @default.
• W2018916450 hasConcept C166342909 @default.
• W2018916450 hasConcept C181199279 @default.
• W2018916450 hasConcept C185592680 @default.
• W2018916450 hasConcept C33923547 @default.
• W2018916450 hasConcept C36503486 @default.
• W2018916450 hasConcept C55493867 @default.
• W2018916450 hasConcept C62826618 @default.
• W2018916450 hasConcept C86803240 @default.
• W2018916450 hasConcept C95444343 @default.
• W2018916450 hasConceptScore W2018916450C134306372 @default.

• next