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• W2085041519 abstract "Structural studies on photoreceptor phosphodiesterases type 6 (PDE6s) have been hampered by an inability to express and purify substantial amounts of enzyme. Here we describe bacterial expression and characterization of the chicken cone PDE6 regulatory GAF-A and GAF-B domains. High affinity cGMP binding was found only for GAF-A as predicted from sequence alignments with the GAF domains of PDE2 and PDE5. A homology model of the GAF-A domain of chicken cone PDE6 based on the crystal structure of mouse PDE2A GAF-B was used to identify residues likely to make contact with cGMP. Alanine mutagenesis of 4 of these residues (F123A, D169A, T172A, and T176A) showed that each was absolutely required for cGMP binding. Three of these residues map to the H4 helical structure of the GAF-A domain indicating this region as a key structural component for cGMP binding. Mutagenesis of another residue, S97A, decreased cGMP binding affinity 5-fold. Finally mutagenesis of Glu-124 indicated that it is responsible for part but not all of the high specificity for cGMP binding to PDE6 GAF-A. Since little data is available on the properties of the chicken cone PDE6 holoenzyme, we also characterized the native PDEs of chicken retina. Two histone-activated PDE6 peaks were separated by ion exchange chromatography and identified by mass spectrometry as cone and rod photoreceptor PDE6s, respectively. Both of these PDEs had cGMP binding and kinetic properties similar to their corresponding bovine photoreceptor PDEs. Moreover the cGMP binding properties of chicken cone PDE6 holoenzyme were very similar to those of the bacterially expressed individual GAF-A or GAF-A/B domains. Structural studies on photoreceptor phosphodiesterases type 6 (PDE6s) have been hampered by an inability to express and purify substantial amounts of enzyme. Here we describe bacterial expression and characterization of the chicken cone PDE6 regulatory GAF-A and GAF-B domains. High affinity cGMP binding was found only for GAF-A as predicted from sequence alignments with the GAF domains of PDE2 and PDE5. A homology model of the GAF-A domain of chicken cone PDE6 based on the crystal structure of mouse PDE2A GAF-B was used to identify residues likely to make contact with cGMP. Alanine mutagenesis of 4 of these residues (F123A, D169A, T172A, and T176A) showed that each was absolutely required for cGMP binding. Three of these residues map to the H4 helical structure of the GAF-A domain indicating this region as a key structural component for cGMP binding. Mutagenesis of another residue, S97A, decreased cGMP binding affinity 5-fold. Finally mutagenesis of Glu-124 indicated that it is responsible for part but not all of the high specificity for cGMP binding to PDE6 GAF-A. Since little data is available on the properties of the chicken cone PDE6 holoenzyme, we also characterized the native PDEs of chicken retina. Two histone-activated PDE6 peaks were separated by ion exchange chromatography and identified by mass spectrometry as cone and rod photoreceptor PDE6s, respectively. Both of these PDEs had cGMP binding and kinetic properties similar to their corresponding bovine photoreceptor PDEs. Moreover the cGMP binding properties of chicken cone PDE6 holoenzyme were very similar to those of the bacterially expressed individual GAF-A or GAF-A/B domains. There are two classes of photoreceptors, cones and rods, that differ substantially in their response to light (1Palczewski K. Saari J.C. Curr. Opin. Neurobiol. 1997; 7: 500-504Crossref PubMed Scopus (69) Google Scholar, 2Helmreich E.J. Hofmann K.P. Biochim. Biophys. Acta. 1996; 1286: 285-322Crossref PubMed Scopus (128) Google Scholar, 3Pugh E.N. Cobbs W.H. Vision Res. 1986; 26: 1613-1643Crossref PubMed Scopus (122) Google Scholar). Rods are very sensitive to low levels of light and can respond to a single photon. Cones are about 100 times less sensitive but respond faster, and the light signal can be terminated more rapidly than in rods (3Pugh E.N. Cobbs W.H. Vision Res. 1986; 26: 1613-1643Crossref PubMed Scopus (122) Google Scholar, 4Baylor D.A. Investig. Ophthalmol. Vis. Sci. 1987; 28: 34-49PubMed Google Scholar). Both rod and cone photoreceptors can sense and respond to changes of light through a G-protein-mediated signaling cascade that activates a family of cGMP-specific phosphodiesterases, PDE6s. 1The abbreviations used are: PDE, 3′,5′-cyclic-nucleotide phosphodiesterase; GAF, cGMP-regulated PDEs, Anabaena adenylyl cyclase, E. coli protein FhlA; IBMX, 3-isobutyl-1-methylxanthine; MS, mass spectrometry; MS/MS, tandem mass spectrometry; HPLC, high pressure liquid chromatography; H4, helix α-4. Activation of these PDEs decreases the level of cGMP and thereby modulates the activity of a cyclic nucleotide-gated cation channel. This in turn controls the release of neurotransmitter from the photoreceptor neuron. Therefore, PDE6s play a crucial role in both rod and cone phototransduction. The rod PDE6 holoenzyme has been characterized as a heterotetramer containing one α-subunit (PDE6A), one β-subunit (PDE6B), and two γ-subunits (5Deterre P. Pfister C. Bigay J. Chabre M. Biochimie (Paris). 1987; 69: 365-370Crossref PubMed Scopus (6) Google Scholar). Cone PDE6 is composed of two identical α′-subunits (PDE6C) and two γ-subunits (6Gillespie P.G. Beavo J.A. J. Biol. Chem. 1988; 263: 8133-8141Abstract Full Text PDF PubMed Google Scholar). The α-, β-, and α′-subunits contain the catalytic sites that hydrolyze cGMP. The γ-subunits bind to the holoenzyme and inhibit phosphodiesterase activity. Cone and some rod PDE6s also contain a δ-subunit that may target the normally membrane-bound PDE6 to the cytosol (7Gillespie P.G. Beavo J.A. Proc. Natl. Acad. Sci. U. S. A. 1989; 86: 4311-4315Crossref PubMed Scopus (81) Google Scholar, 8Florio S.K. Prusti R.K. Beavo J.A. J. Biol. Chem. 1996; 271: 24036-24047Abstract Full Text Full Text PDF PubMed Scopus (81) Google Scholar). However, the δ-subunit is also a component of several other proteins and therefore not unique to photoreceptors (8Florio S.K. Prusti R.K. Beavo J.A. J. Biol. Chem. 1996; 271: 24036-24047Abstract Full Text Full Text PDF PubMed Scopus (81) Google Scholar). Although in most vertebrate retinas, cones are much less abundant than rods, the chicken retina is cone-dominant. In this species, cones outnumber rods six to one in the central retina and three to one in the peripheral retina (9Morris V.B. Shorey C.D. J. Comp. Neurol. 1967; 129: 313-340Crossref PubMed Scopus (114) Google Scholar). Most previous biochemical studies of the cone PDE6 isoenzyme have been carried out using bovine retinas due to their large size and availability. Nevertheless a cDNA predicted from homology arguments to encode the chicken cone PDE6 α′-subunit has been isolated and characterized (10Semple-Rowland S.L. Green D.A. Exp. Eye Res. 1994; 59: 365-372Crossref PubMed Scopus (16) Google Scholar). Similarly a cDNA most homologous to the bovine PDE6 β-subunit cDNA also has been reported from chicken pineal gland (11Morin F. Lugnier C. Kameni J. Voisin P. J. Neurochem. 2001; 78: 88-99Crossref PubMed Scopus (35) Google Scholar). PDE6 is a member of the 11 families of Class 1 phosphodiesterases that hydrolyze cyclic nucleotides. Five of these PDE families, PDEs 2, 5, 6, 10, and 11, contain one or two complete GAF domains in their N-terminal regulatory regions (12Beavo J.A. Physiol. Rev. 1995; 75: 725-748Crossref PubMed Scopus (1643) Google Scholar, 13Soderling S.H. Beavo J.A. Curr. Opin. Cell. Biol. 2000; 12: 174-179Crossref PubMed Scopus (649) Google Scholar). GAF domains are regulatory small molecule-binding domains originally named for their presence in cGMP-regulated PDEs, certain adenylyl cyclases and the transcription factor FhlA of bacteria (14Aravind L. Ponting C.P. Trends Biochem. Sci. 1997; 22: 458-459Abstract Full Text PDF PubMed Scopus (488) Google Scholar). Cyclic GMP binds to one of two GAF domains of PDE2, PDE5, and PDE6 (6Gillespie P.G. Beavo J.A. J. Biol. Chem. 1988; 263: 8133-8141Abstract Full Text PDF PubMed Google Scholar, 15Martins T.J. Mumby M.C. Beavo J.A. J. Biol. Chem. 1982; 257: 1973-1979Abstract Full Text PDF PubMed Google Scholar, 16Francis S.H. Bessay E.P. Kotera J. Grimes K.A. Liu L. Thompson W.J. Corbin J.D. J. Biol. Chem. 2002; 277: 47581-47587Abstract Full Text Full Text PDF PubMed Scopus (75) Google Scholar). The catalytic activity of PDE2A is allosterically stimulated by cGMP binding to its GAF-B domain (15Martins T.J. Mumby M.C. Beavo J.A. J. Biol. Chem. 1982; 257: 1973-1979Abstract Full Text PDF PubMed Google Scholar). In PDE5, cGMP binding to the GAF-A domains increases PDE5 catalytic activity and potentiates phosphorylation at an N-terminal serine (17Rybalkin S.D. Rybalkina I.G. Shimizu-Albergine M. Tang X.B. Beavo J.A. EMBO J. 2003; 22: 469-478Crossref PubMed Scopus (197) Google Scholar, 18Corbin J.D. Blount M.A. Weeks II, 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). The functions, regulation, and roles of GAF domains in PDEs have been comprehensively reviewed recently (19Zoraghi R. Corbin J.D. Francis S.H. Mol. Pharmacol. 2004; 65: 267-278Crossref PubMed Scopus (124) Google Scholar). For the amphibian photoreceptor PDE6, it has been found that cGMP occupancy at a GAF domain enhances P-γ binding to the holoenzyme (20Cote R.H. Bownds M.D. Arshavsky V.Y. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 4845-4849Crossref PubMed Scopus (84) Google Scholar, 21Yamazaki M. Li N. Bondarenko V.A. Yamazaki R.K. Baehr W. Yamazaki A. J. Biol. Chem. 2002; 277: 40675-40686Abstract Full Text Full Text PDF PubMed Scopus (26) Google Scholar). Several roles for cGMP binding have been proposed. For example, it has been postulated that non-catalytic cGMP binding to PDE6 may be involved in the recovery from light stimulation and light adaptation (22D'Amours M.R. Cote R.H. Biochem. J. 1999; 340: 863-869Crossref PubMed Scopus (38) Google Scholar). In this case, the GAF sites serve as a cGMP reservoir to buffer cytoplasmic cGMP levels in the dark and accelerate the return of high cGMP to basal levels upon light activation of the PDE. Another model suggests that cGMP binding to a GAF domain regulates the duration of transducin activation of PDE6 by modulating the affinity of P-γ (23Mou H. Grazio III, 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, 24Mou H. Cote R.H. J. Biol. Chem. 2001; 276: 27527-27534Abstract Full Text Full Text PDF PubMed Scopus (72) Google Scholar). These models are not mutually exclusive. Recently the three-dimensional structure of the mouse PDE2A GAF-A/B domains was determined by x-ray diffraction crystallography at 2.9-Å resolution (25Martinez S. Wu A. Glavas N. Tang X. Turley S. Hol W. Beavo J. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 13260-13265Crossref PubMed Scopus (226) Google Scholar). The regulatory PDE2 GAF-A/B domains form a parallel dimer in which only GAF-B binds cGMP. There are 11 amino acid residues that make contact with cGMP and line the binding pocket. In PDE5, 10 of these 11 residues are identical in GAF-A, which therefore allowed the prediction that this would be the cGMP-binding domain of this PDE (25Martinez S. Wu A. Glavas N. Tang X. Turley S. Hol W. Beavo J. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 13260-13265Crossref PubMed Scopus (226) Google Scholar). It was subsequently confirmed that GAF-A of PDE5 is sufficient for high affinity cGMP binding (26Liu L. Underwood T. Li H. Pamukcu R. Thompson W.J. Cell. Signal. 2002; 14: 45-51Crossref PubMed Scopus (40) Google Scholar). A consensus cGMP-binding motif, based on the similarity between PDE2A GAF-B and PDE5 GAF-A domains, has been proposed (Sequence 1, Ref. 25Martinez S. Wu A. Glavas N. Tang X. Turley S. Hol W. Beavo J. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 13260-13265Crossref PubMed Scopus (226) Google Scholar). Mutagenesis in this motif in PDE5 suggested that the Phe in the FD dyad of PDE5 GAF-A domain is essential for cGMP binding (27Sopory S. Balaji S. Srinivasan N. Visweswariah S.S. FEBS Lett. 2003; 539: 161-166Crossref PubMed Scopus (26) Google Scholar). A similar finding has also been shown for the PDE2A GAF-B domain (28Wu A.Y. Tang X.B. Martinez S.E. Ikeda K. Beavo J.A. J. Biol. Chem. 2004; 279: 37928-37938Abstract Full Text Full Text PDF PubMed Scopus (69) Google Scholar). Eight of the 11 residues in mouse PDE2A, (i.e. Ser-424, Phe-438, Asp-439, Val-484, Asp-485, Thr-488, Thr-492, and Glu-512) contact cGMP via side chains. The crystal structure of PDE2 GAF-B and recent mutagenesis studies suggest that its ability to discriminate cGMP versus cAMP is largely determined by Asp-439, which provides positive specificity for cGMP binding via hydrogen bonds between its main chain NH, side chain carboxyl, and the O-6 and N-1 positions of the guanine base of cGMP (25Martinez S. Wu A. Glavas N. Tang X. Turley S. Hol W. Beavo J. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 13260-13265Crossref PubMed Scopus (226) Google Scholar). This residue also provides a negative determinant for cAMP (28Wu A.Y. Tang X.B. Martinez S.E. Ikeda K. Beavo J.A. J. Biol. Chem. 2004; 279: 37928-37938Abstract Full Text Full Text PDF PubMed Scopus (69) Google Scholar). Compared with PDE2 and PDE5, most PDE6s have both higher binding affinity and higher specificity for cGMP (6Gillespie P.G. Beavo J.A. J. Biol. Chem. 1988; 263: 8133-8141Abstract Full Text PDF PubMed Google Scholar, 29Hebert 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). Bovine rod PDE6 binds cGMP with Kd values reported from 25 to 500 nm at a low affinity binding site (23Mou H. Grazio III, 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) and <500 pm at a high affinity site (30.Gillespie, P. G. (1988) Identification, Purification, and Characterization of Bovine Rod and Cone Photoreceptor Phosphodiesterase Isozymes. Ph.D. thesis, University of WashingtonGoogle Scholar). Bovine cone PDE6C appears to have one type of cGMP-binding site with a Kd of about 10 nm (7Gillespie P.G. Beavo J.A. Proc. Natl. Acad. Sci. U. S. A. 1989; 86: 4311-4315Crossref PubMed Scopus (81) Google Scholar). A chimeric bovine PDE6C/PDE5 enzyme also has been reported to contain a single class of non-catalytic cGMP-binding sites with a Kd of 450 nm (31Granovsky A.E. Natochin M. McEntaffer R.L. Haik T.L. Francis S.H. Corbin J.D. Artemyev N.O. J. Biol. Chem. 1998; 273: 24485-24490Abstract Full Text Full Text PDF PubMed Scopus (54) Google Scholar). In this study we report on the bacterial expression of the tandem GAF domains of chicken cone PDE6 and show that the basic features of the cGMP-binding pocket is highly conserved between the cone PDE6C and mouse PDE2A GAF domains. We demonstrate that GAF-A instead of GAF-B contains the single high affinity cGMP-binding domain of PDE6C and that the conserved residues of PDE6C (Phe-123, Asp-169, Thr-172, and Thr-176) each appear to play significant roles in forming a functional cGMP-binding pocket. Finally we report on the isolation and characterization of the chicken PDE6 holoenzymes and show that their cGMP binding characteristics are similar to those of the isolated chicken GAF domains and also to their corresponding mammalian PDE6 counterparts. Chicken eyes were obtained from the Tyson Co. (Little Rock, AK). Bovine eyes were purchased from Schenk Packing (Stanwood, WA). [3H]cGMP was obtained from Amersham Biosciences; cGMP, 3-isobutyl-1-methylxanthine (IBMX), isopropyl β-thioglucopyranoside were from Sigma. Pfu DNA polymerase and the QuikChange® site-directed mutagenesis kits were obtained from Stratagene (La Jolla, CA). Restriction enzymes were purchased from New England Biolabs (Beverly, MA). Isolation and Initial Purification of Chicken Rod and Cone PDE6 Holoenzyme—In a typical experiment 50–100 chicken retinas were dissected in the light, separated from much of the vitreous humor, and homogenized in hypotonic buffer (10 mm Tris, pH 7.5, 1 mm MgCl2, 1 mm dithiothreitol, and 0.2 mm phenylmethylsulfonyl fluoride) using a dozen strokes of a motor-driven Teflon pestle in a glass homogenizer (Potter-Elvehjem tissue grinder). The homogenate was centrifuged at 100,000 × g for 1 h. The supernatant was applied to a 75-ml DE52 anion-exchange column and eluted with a linear NaCl gradient (20–300 mm NaCl, 10 mm Tris, pH 7.5, 1 mm MgCl2) run at a flow rate of 1 ml/min at 4 °C. Sixty fractions of 5 ml each were collected at 4 °C and assayed for histone-activated (2.5 mg/ml, type VIII-S) phosphodiesterase activity by measuring the release of phosphate (7Gillespie P.G. Beavo J.A. Proc. Natl. Acad. Sci. U. S. A. 1989; 86: 4311-4315Crossref PubMed Scopus (81) Google Scholar). cGMP Affinity Column Purification of Chicken PDE6 Holoenzyme— Two histone-activated PDE activity peaks of approximately equal activity were separated on the DE52 anion-exchange column. Approximately 35 ml of the first histone-activated PDE6 peak was pooled and loaded onto an epoxy-Sepharose cGMP affinity column (7Gillespie P.G. Beavo J.A. Proc. Natl. Acad. Sci. U. S. A. 1989; 86: 4311-4315Crossref PubMed Scopus (81) Google Scholar, 15Martins T.J. Mumby M.C. Beavo J.A. J. Biol. Chem. 1982; 257: 1973-1979Abstract Full Text PDF PubMed Google Scholar). The column was washed two times with low salt buffer (20 mm Tris, pH 7.5, 200 mm NaCl, 10 mm dithiothreitol, 0.1 mm EDTA, and 1 mm phenylmethylsulfonyl fluoride), and the cGMP-binding proteins, including chicken photoreceptor PDE, were eluted with 1 mm cGMP in the presence of 2 mm EDTA and 1 mm IBMX in low salt buffer at room temperature. The presence and purity of chicken photoreceptor PDEs were analyzed by SDS-PAGE and silver staining. Immunoprecipitation of Chicken PDE6s—It has been shown previously that the ROS-3 monoclonal antibody recognizes both rod and cone photoreceptor PDE from bovine retina (32Hurwitz R.L. Bunt-Milam A.H. Beavo J.A. J. Biol. Chem. 1984; 259: 8612-8618Abstract Full Text PDF PubMed Google Scholar). The putative chicken cone and rod PDE6 holoenzymes, corresponding to the first or the second histone-activated PDE6 peaks, were immunoprecipitated using the ROS-3 monoclonal antibody bound to Protein G. Typically 15 ml of the DE52 fraction of either chicken rod or cone PDE was mixed with 200 μl of antibody resin (500 μg of antibody bound to 200 μl of Protein G PLUS-agarose beads (Santa Cruz Biotechnology, Inc.) for 1 h at 4 °C) in the presence of 150 mm NaCl. After mixing overnight, the resin was pelleted and washed three times with 1 ml of 10 mm Tris, pH 7.5, 1 mm MgCl2, 300 mm NaCl at 4 °C. Identification of Chicken Rod and Cone PDE6 Using Mass Spectrometry—On-line nano-liquid chromatography/electrospray ionization-MS/MS experiments were performed on an API-US quadrupole time-of-flight mass spectrometer (Micromass) equipped with the CapLC system (Waters, Milford, MA). The stream select module was configured with an OPTI-PAK Symmetry300 C18 trap column (Waters) connected in series with a nanoscale analytical column (75-μm inner diameter × 15 cm, packed with 3.5 μm, XTerra MS C18 particles (Waters)). The eluate from the cGMP affinity column was concentrated to 100 μg/ml using a Centriprep centrifugal filter (Millipore) and then digested with 5 μg/ml trypsin. In other experiments immunoprecipitated proteins were separated by SDS-PAGE and stained with Coomassie Blue. The protein bands corresponding to the molecular weights of rod and cone PDE6 were sliced out from the gel and digested with trypsin for analysis by mass spectrometry (33Shevchenko A. Wilm M. Vorm O. Mann M. Anal. Chem. 1996; 68: 850-858Crossref PubMed Scopus (7818) Google Scholar). Protein digests (5 μl) were injected onto the trap column at 10 μl/min, desalted, and back-flushed to the analytical column at 0.5 μl/min using a gradient elution. The gradient consisted of 5–50% solvent B for 30 min followed by 50% B for 15 min and 50–90% B for 5 min (A = 5% acetonitrile, 0.1% formic acid; B = 95% acetonitrile, 0.1% formic acid). Quadrupole time-of-flight parameters were set as follows: the electrospray potential was set to 3.5 kV, the cone voltage was set to 60 V, the extraction cone was set to 2 V, and the source temperature was set to 80 °C. The MS survey scan was m/z 400–1600 with a scan time of 1 s, and the collision energy was set to 10 eV. When the intensity of a peptide peak rose above a threshold of 20 counts, tandem mass spectra were acquired using the data-dependent algorithm implemented in the MassLynx acquisition software. For operation in the MS/MS mode, the scan time was increased to 2 s, the isolation width was set to include the full isotopic distribution of each peak (3 Da), and the collision energy was set to 15–25 eV. MS/MS spectra were recorded for the doubly, triply, and quadruply charged molecular ions of peptides. All MS/MS spectra were searched against the non-redundant National Center for Biotechnology Information protein data base by using MASCOT (34Perkins D.N. Pappin D.J. Creasy D.M. Cottrell J.S. Electrophoresis. 1999; 20: 3551-3567Crossref PubMed Scopus (6773) Google Scholar) assuming a mass tolerance of 0.3 Da for both the precursor and the fragment ions. cGMP Binding to the Chicken Rod and Cone PDE6 Holoenzymes— Chicken cone PDE6 holoenzyme was immunoprecipitated as described previously except less sample and antibody were used. Usually 1–2 ml of DE52 fraction of chicken rod or cone PDE was mixed with 100–200 μl of antibody resin (100–200 μg of antibody). After incubation overnight, the resin was collected by centrifugation and washed three times with 1 ml of 10 mm Tris, pH 7.5, 1 mm MgCl2, 300 mm NaCl. The PDE immunoprecipitates were then incubated with 5 mm Tris, pH 7.5, 25 mm NaCl, 2 mm EDTA, 1 mm IBMX, 0.1 mg/ml bovine serum albumin, 2–600 nm (7.5 Ci/mmol) [3H]cGMP (200-μl total volume) for 2 h at room temperature. [3H]cGMP bound to the immunoprecipitated PDE was separated from free ligand by filtration on Millipore filters (0.45-μm HA). The Millipore filters were dissolved in Filter-Count® complete liquid scintillation counting mixture (PerkinElmer Life Sciences) overnight and counted in a Packard 1600 TR liquid scintillation analyzer. cGMP Binding and cAMP Binding by Individual Chicken Cone PDE6 GAF Domain Proteins—The binding of cGMP or cAMP to GAF domain proteins was analyzed by the Millipore filter binding assay as described previously (37Yamazaki A. Sen I. Bitensky M.W. J. Biol. Chem. 1980; 255: 11619-11624Abstract Full Text PDF PubMed Google Scholar) usually as a competition assay (28Wu A.Y. Tang X.B. Martinez S.E. Ikeda K. Beavo J.A. J. Biol. Chem. 2004; 279: 37928-37938Abstract Full Text Full Text PDF PubMed Scopus (69) Google Scholar). For the cGMP binding competition assays, 1 nm GAF domain protein was incubated with a fixed amount of [3H]cGMP (usually 1 nm) and various amounts of unlabeled cGMP from 2 to 600 nm for 2 h at room temperature. For the cAMP binding competition assays, 1 nm GAF domain protein was incubated with 1 nm [3H]cGMP and various amounts of unlabeled cAMP from 1 μm to 100 mm for 2 h at room temperature. Most Kd values reported in this study were determined by the homologous or heterologous displacement methods. Care was taken to utilize concentrations of protein that were lower than the measured Kd so that the data represented true binding curves and not a titration analysis. Similarly for all IC50 determinations the concentrations of labeled radioligand was adjusted to be lower than the measured IC50 value so that the IC50 approached the Ki for the cold ligand (35Cheng Y. Prusoff W.H. Biochem. Pharmacol. 1973; 22: 3099-3108Crossref PubMed Scopus (12288) Google Scholar, 36Rovati G.E. Trends Pharmacol. Sci. 1998; 19: 365-369Abstract Full Text Full Text PDF PubMed Scopus (28) Google Scholar). Therefore, the affinity of the ligand can be calculated using the equation of Cheng and Prusoff (35Cheng Y. Prusoff W.H. Biochem. Pharmacol. 1973; 22: 3099-3108Crossref PubMed Scopus (12288) Google Scholar) that states that the equilibrium dissociation constant of the ligand, Ki = IC50/(1 + [Radioligand]/Kd). In the case of homologous displacement, e.g. [3H]cGMP being displaced by cGMP, the equation further simplifies to Ki = IC50 - [Radioligand] since Ki = Kd. Curve fitting was done using GraphPad Prism4 with a one-site competition model constraining the 100 and 0% binding points. Better fits were not obtained with a multiple site model. Enzyme Kinetics of Chicken Rod and Cone PDE6 Holoenzymes—The kinetics for both cGMP and cAMP hydrolysis of the chicken rod and cone PDE6 were determined using fractions from the ion-exchange column. PDE6 activity was assayed in the presence of histone (2.5 mg/ml, type VIII-S, Sigma) by PDE activity assay using either [3H]cGMP or [3H]cAMP as substrate (15Martins T.J. Mumby M.C. Beavo J.A. J. Biol. Chem. 1982; 257: 1973-1979Abstract Full Text PDF PubMed Google Scholar, 32Hurwitz R.L. Bunt-Milam A.H. Beavo J.A. J. Biol. Chem. 1984; 259: 8612-8618Abstract Full Text PDF PubMed Google Scholar). The Km values for cGMP and cAMP hydrolysis were derived by nonlinear regression analysis using GraphPad Prism® from data points obtained using 1–250 μm [3H]cGMP or 50–2500 μm [3H]cAMP as substrate. Three-dimensional Structure Model of Chicken Cone PDE6 GAF-A Domain—A three-dimensional model for the chicken PDE6 GAF-A domain was constructed based upon the crystal structure of the mouse PDE2A GAF-B domain using the Swiss-Model program (38Guex N. Peitsch M.C. Electrophoresis. 1997; 18: 2714-2723Crossref PubMed Scopus (9584) Google Scholar). Sequence alignments were made with the ClustalW program (39Thompson J.D. Higgins D.G. Gibson T.J. Nucleic Acids Res. 1994; 22: 4673-4680Crossref PubMed Scopus (55744) Google Scholar). After adding side chains from a rotamer data base, the working model was energy-minimized using GROMOS 96 (38Guex N. Peitsch M.C. Electrophoresis. 1997; 18: 2714-2723Crossref PubMed Scopus (9584) Google Scholar). Cloning, Expression, and Purification of the GAF Domains of Chicken Cone PDE6 —The pRunH plasmid (a derivative of the pMW172 vector (40Way M. Pope B. Gooch J. Hawkins M. Weeds A.G. EMBO J. 1990; 9: 4103-4109Crossref PubMed Scopus (194) Google Scholar)) containing a C-terminal His6 tag was used as an expression vector for the GAF-A, GAF-B, and GAF-A/B domains of chicken PDE6C. Full-length chicken PDE6C cDNA was a generous gift from Dr. Semple-Rowland (University of Florida). The boundaries of GAF-A/B, GAF-A, and GAF-B domain constructs were amino acids 42–458, 42–238, and 248–458, respectively, based on homology alignment with other PDE GAF domains. DNA coding for these domains was PCR-amplified using the full-length chicken PDE6C as template and primers containing BamHI and XhoI sites. The PCR products were ligated into the pRunH vector downstream of the T7 promoter. Chicken cone PDE6 GAF domain constructs were transformed into C41 competent Escherichia coli. Protein expression was induced by isopropyl β-thioglucopyranoside at 16 °C, and cells were grown overnight. The cell pellets were resuspended in lysis buffer (100 mm NaCl, 20 mm Tris, pH 7.5, 1 mm MgCl2, 10 μg/ml DNase I, 1 μg/ml leupeptin, 1 μg/ml pepstatin) and disrupted through either a French Press or a Microfluidizer® cell disruption apparatus. The lysates were centrifuged at 10,000 × g for 1 h at 4 °C, and the supernatant was incubated with Talon® resin (Clontech) for 2 h at 4 °C. The bound His6-tagged proteins were eluted with buffer containing 150 mm imidazole, 50 mm sodium phosphate (pH 7.0), and 300 mm NaCl. The eluted proteins were concentrated using a Centriprep centrifugal filter (Millipore) and subjected to gel filtration using a Superose-12 column (Amersham Biosciences) to remove aggregated protein and determine the apparent molecular weight of the GAF proteins. SDS gel electrophoresis was carried out to determine the purity of the isolated GAF domain proteins. Site-directed Mutagenesis Studies on Chicken Cone PDE6C GAF-A and GAF-A/B Domains—The QuikChange site-directed mutagenesis kit (Stratagene) was used to make point mutations in chicken cone PDE6 GAF-A and GAF-A/B cDNA constructs. E. coli XL-blue competent cells were used for transformations, and the mutant cDNAs were purified by QIAprep® spin miniprep kit (Qiagen, Valencia, CA). All mutant cDNAs were sequenced to ensure the proper in-frame subcloning and the desired mutation. Modeling of the Chicken Cone PDE GAF-A Domain—Sequence alignments between the GAF-A and GAF-B domains of chicken PDE6C to GAF-B of mouse PDE2A showed that the GAF-A domain of PDE6 had very high homology to PDE2A GAF-B and therefore that the basic architecture of the cGMP-binding pocket might be conserved (Fig. 1A). Of eight side chains known to contact cGMP in PDE2 GAF-B, five are identical in PDE6C GAF-A, and three of these are in the helix α-4 (H4) of PDE2A GAF-B. In addition, Asp-439 of mouse PDE2A GAF-B is conserved as Glu-124 in chicken PDE6C GAF-A. A three-dimensional homology model of chicken cone PDE6 GAF-A based upon the crystal structure of PDE2A GAF-B domain is shown in Fig. 1B. Because of gaps generated by the homology alignment of the two GAF sequences, two extra insertions are introduced as solvent-exposed loops in the modeled structure of chicken cone PDE6 GAF-A. Since both sequences are relatively hydrophobic this does not seem unreasonable. The first insertion resides between the first α-helix and the first β-sheet strand. The second insertion is located between the first and second β-strand. Neither of the two insertions interrupts the secondary structure of the model. For the rest of the sequence, the ove" @default.
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• W2085041519 title "Molecular Determinants of cGMP Binding to Chicken Cone Photoreceptor Phosphodiesterase" @default.
• W2085041519 cites W124547280 @default.
• W2085041519 cites W1503740541 @default.
• W2085041519 cites W1559386011 @default.
• W2085041519 cites W1595517259 @default.
• W2085041519 cites W1653226101 @default.
• W2085041519 cites W1869250499 @default.
• W2085041519 cites W1967882515 @default.
• W2085041519 cites W1974974112 @default.
• W2085041519 cites W1976368031 @default.
• W2085041519 cites W1981593008 @default.
• W2085041519 cites W1987787946 @default.
• W2085041519 cites W1995404969 @default.
• W2085041519 cites W1997353263 @default.
• W2085041519 cites W2002131367 @default.
• W2085041519 cites W2006203702 @default.
• W2085041519 cites W2012304711 @default.
• W2085041519 cites W2014621993 @default.
• W2085041519 cites W2015642465 @default.
• W2085041519 cites W2019102477 @default.
• W2085041519 cites W2025789164 @default.
• W2085041519 cites W2028949252 @default.
• W2085041519 cites W2029733810 @default.
• W2085041519 cites W2031062005 @default.
• W2085041519 cites W2040580243 @default.
• W2085041519 cites W2056233726 @default.
• W2085041519 cites W2067382519 @default.
• W2085041519 cites W2073663150 @default.
• W2085041519 cites W2074631079 @default.
• W2085041519 cites W2078594804 @default.
• W2085041519 cites W2080981029 @default.
• W2085041519 cites W2106882534 @default.
• W2085041519 cites W2118663270 @default.
• W2085041519 cites W2119986687 @default.
• W2085041519 cites W2125865604 @default.
• W2085041519 cites W2137248027 @default.
• W2085041519 cites W2149871191 @default.
• W2085041519 cites W2150744925 @default.
• W2085041519 cites W2165758443 @default.
• W2085041519 cites W2166389885 @default.
• W2085041519 cites W2170589977 @default.
• W2085041519 cites W80090061 @default.
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