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An Interface of Interaction between Photoreceptor cGMP Phosphodiesterase Catalytic Subunits and Inhibitory γ Subunits

1996; Elsevier BV; Volume: 271; Issue: 33 Linguagem: Inglês

10.1074/jbc.271.33.19964

1083-351X

Michael Natochin, Nikolai O. Artemyev,

Nitric Oxide and Endothelin Effects

Cyclic guanosine 5′-monophosphate (cGMP) phosphodiesterase (PDE) regulates the level of cGMP on transduction of a visual signal in vertebrate photoreceptor cells. Two identical inhibitory PDE γ subunits (Pγs) block catalytic activity of PDE-α and -β subunits (Pαβ) in the dark. The primary regions of Pγ involved in the interaction with Pαβ are a central polycationic region, Pγ-24-45, and a C-terminal region of Pγ. Recently, we have shown that the C-terminal region of Pγ, which is the major Pγ inhibitory domain, blocks PDE activity by binding to the catalytic site of PDE (Artemyev, N. O., Natochin, M., Busman, M., Schey, K. L., and Hamm, H. E. (1996) Proc. Natl. Acad. Sci. U. S. A. 93, 5407-5412). Here, we localize the site on the rod cGMP PDE α subunit that binds to the central polycationic domain of Pγ. This site is located within a region that links a second noncatalytic cGMP binding site with the catalytic domain of PDE. A polypeptide coresponding to this region, Pα-461-553, expressed as a glutathione S-transferase fusion protein in Escherichia coli and isolated after cleavage of the fusion protein with thrombin, blocks inhibition of PDE activity by Pγ. In addition, Pα-461-553 binds to the Pγ-24-45 region (Kd, 7 µM), as measured by a fluorescent increase in a Pγ-24-45Cys peptide labeled with 3-(bromoacetyl)-7-diethylaminocoumarin. The Pα-461-553 region was further characterized by using a set of synthetic peptides. A peptide corresponding to residues 517-541 of Pα (Pα-517-541) effectively suppressed inhibition of PDE activity by Pγ and bound to Pγ-24-45Cys labeled with 3-(bromoacetyl)-7-diethylaminocoumarin (Kd, 22 µM). Pα-517-541 also competes with the activated rod G-protein α-subunit for binding to Pγ labeled with lucifer yellow vinyl sulfone. This suggests that light activation of rod PDE by the G-protein transducin involves competition between transducin α-guanosine 5′-triphosphate and Pα-517-541 for binding to the Pγ-24-45 region. Based on the results, we propose a linear model of interactions between catalytic and inhibitory PDE subunits. Cyclic guanosine 5′-monophosphate (cGMP) phosphodiesterase (PDE) regulates the level of cGMP on transduction of a visual signal in vertebrate photoreceptor cells. Two identical inhibitory PDE γ subunits (Pγs) block catalytic activity of PDE-α and -β subunits (Pαβ) in the dark. The primary regions of Pγ involved in the interaction with Pαβ are a central polycationic region, Pγ-24-45, and a C-terminal region of Pγ. Recently, we have shown that the C-terminal region of Pγ, which is the major Pγ inhibitory domain, blocks PDE activity by binding to the catalytic site of PDE (Artemyev, N. O., Natochin, M., Busman, M., Schey, K. L., and Hamm, H. E. (1996) Proc. Natl. Acad. Sci. U. S. A. 93, 5407-5412). Here, we localize the site on the rod cGMP PDE α subunit that binds to the central polycationic domain of Pγ. This site is located within a region that links a second noncatalytic cGMP binding site with the catalytic domain of PDE. A polypeptide coresponding to this region, Pα-461-553, expressed as a glutathione S-transferase fusion protein in Escherichia coli and isolated after cleavage of the fusion protein with thrombin, blocks inhibition of PDE activity by Pγ. In addition, Pα-461-553 binds to the Pγ-24-45 region (Kd, 7 µM), as measured by a fluorescent increase in a Pγ-24-45Cys peptide labeled with 3-(bromoacetyl)-7-diethylaminocoumarin. The Pα-461-553 region was further characterized by using a set of synthetic peptides. A peptide corresponding to residues 517-541 of Pα (Pα-517-541) effectively suppressed inhibition of PDE activity by Pγ and bound to Pγ-24-45Cys labeled with 3-(bromoacetyl)-7-diethylaminocoumarin (Kd, 22 µM). Pα-517-541 also competes with the activated rod G-protein α-subunit for binding to Pγ labeled with lucifer yellow vinyl sulfone. This suggests that light activation of rod PDE by the G-protein transducin involves competition between transducin α-guanosine 5′-triphosphate and Pα-517-541 for binding to the Pγ-24-45 region. Based on the results, we propose a linear model of interactions between catalytic and inhibitory PDE subunits. INTRODUCTIONIn vertebrate photoreceptor cells, light-activated rhodopsin catalyzes GDP-GTP exchange on the rod G-protein α-subunit (Gtα), 1The abbreviations used are: Gtαα subunit of the photoreceptor G-protein transducinPDErod outer segment cGMP phosphodiesterasePα, Pβ, and Pγα, β, and γ subunits of PDEtaPDEtrypsin-activated PDEBC3-(bromoacetyl)-7-diethylaminocoumarinLYlucifer yellow vinyl sulfoneHPLChigh performance liquid chromatographyGSTglutathione S-transferaseGTPγSguanosine 5′-O-(thiotriphosphate)PBSphosphate-buffered salinecGB-PDEcGMP-binding, cGMP-specific PDE. which in turn activates cGMP phosphodiesterase (PDE). PDE activation leads to the rapid hydrolysis of cytoplasmic cGMP and closure of sodium channels, resulting in hyperpolarization of the rod and cone cells (for review, see Chabre and Deterre (10Chabre M. Deterre P. Eur. J. Biochem. 1989; 179: 255-266Crossref PubMed Scopus (219) Google Scholar) and Stryer (35Stryer L. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 557-559Crossref PubMed Scopus (46) Google Scholar)). Rod photoreceptor PDE is composed of two large homologous catalytic α and β subunits (Pα and Pβ) of nearly identical size (Mr 99,261 and 98,308, respectively) and two copies of an inhibitory γ subunit (Pγ, Mr 9700) (Ovchinnikov et al., 28Ovchinnikov Y.A. Lipkin V.M. Kumarev V.P. Gubanov V.V. Khramtsov N.V. Akhmedov N.B. Zagranichny V.E. Muradov K.G. FEBS Lett. 1986; 204: 288-292Crossref PubMed Scopus (89) Google Scholar, 29Ovchinnikov Y.A. Gubanov V.V. Khramtsov K.A. Ischenko K.A. Zagranichny V.E. Muradov K.G. Shuvaeva T.M. Lipkin V.M. FEBS Lett. 1987; 223: 169-173Crossref PubMed Scopus (78) Google Scholar; Deterre et al., 11Deterre P. Bigay J. Forquet F. Robert M. Chabre M. Proc. Natl. Acad. Sci. U. S. A. 1988; 85: 2424-2428Crossref PubMed Scopus (166) Google Scholar; Lipkin et al., 21Lipkin V.M. Khramtsov N.V. Vasilevskaya I.A. Atabekova N.V. Muradov K.G. Gubanov V.V. Li T. Johnston J.P. Volpp K.J. Applebury M.L. J. Biol. Chem. 1990; 265: 12955-12959Abstract Full Text PDF PubMed Google Scholar). The primary structures of Pα, Pβ, and cone-specific Pα′ subunits (Li et al., 18Li T. Volpp K. Applebury M.L. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 293-297Crossref PubMed Scopus (98) Google Scholar) revealed that these PDEs constitute one family, that of PDE6 (Beavo et al., 6Beavo J.A. Conti M. Heaslip R.J. Mol. Pharmacol. 1994; 46: 399-405PubMed Google Scholar). The photoreceptor PDEs belong to a broader group of cGMP-binding PDEs, which contain two noncatalytic cGMP binding sites located N-terminally to the conserved PDE catalytic domain (Yamazaki et al., 40Yamazaki A. Sen I. Bitensky M.W. Casnellie J. Greengard P. J. Biol. Chem. 1980; 255: 11619-11624Abstract Full Text PDF PubMed Google Scholar; Gillespie and Beavo, 12Gillespie P.G. Beavo J.A. Proc. Natl. Acad. Sci. U. S. A. 1989; 86: 4311-4315Crossref PubMed Scopus (81) Google Scholar; Lipkin et al., 21Lipkin V.M. Khramtsov N.V. Vasilevskaya I.A. Atabekova N.V. Muradov K.G. Gubanov V.V. Li T. Johnston J.P. Volpp K.J. Applebury M.L. J. Biol. Chem. 1990; 265: 12955-12959Abstract Full Text PDF PubMed Google Scholar; Trong et al., 38Trong H.L. Beier N. Sonnenburg W.K. Stroop S.D. Walsh K.A. Beavo J.A. Charbonneau H. Biochemistry. 1990; 29: 10280-10288Crossref PubMed Scopus (51) Google Scholar; McAllister-Lucas et al., 23McAllister-Lucas L.M. Sonnenburg W.K. Kadlecek A. Seger D. Trong H.L. Colbran J.L. Thomas M.K. Walsh K.A. Francis S.H. Corbin J.D. Beavo J.A. J. Biol. Chem. 1993; 268: 22863-22873Abstract Full Text PDF PubMed Google Scholar). Unique features of photoreceptor PDEs are their high kcat/Km parameter and their ability to be inhibited by Pγ and activated by rod and cone G-protein α subunits.Interactions between PDE catalytic and inhibitory subunits and the mechanism of PDE inhibition have been studied extensively. Two regions of Pγ, polycationic region Pγ-24-45 and the C terminus of Pγ, have been shown to participate in the interaction with Pαβ (Lipkin et al., 20Lipkin 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; Artemyev and Hamm, 1Artemyev N.O. Hamm H.E. Biochem. J. 1992; 283: 273-279Crossref PubMed Scopus (72) Google Scholar; Brown, 8Brown R.L. Biochemistry. 1992; 31: 5918-5925Crossref PubMed Scopus (61) Google Scholar; Takemoto et al., 36Takemoto D.J. Hurt D. Oppert B. Cunnick J. Biochem. J. 1992; 281: 637-643Crossref PubMed Scopus (42) Google Scholar). Both of these Pγ domains bind to Pαβ, allowing effective inhibition of PDE activity by the Pγ C terminus. Initial studies indicated that the major sites of Pα and Pβ interaction with Pγ are different and located in the N-terminal regions (Pα, 16-30 and 78-90; Pβ, 91-110 and 211-230) in areas with a high level of dissimilarity between catalytic subunits (Oppert et al., 27Oppert B. Cunnick J.M. Hurt D. Takemoto D.J. J. Biol. Chem. 1991; 266: 16607-16613Abstract Full Text PDF PubMed Google Scholar; Oppert and Takemoto, 26Oppert B. Takemoto D.J. Biochem. Biophys. Res. Commun. 1991; 178: 474-479Crossref PubMed Scopus (9) Google Scholar).More recently, using a cross-linking approach we have demonstrated that the C terminus of Pγ interacts with region Pα-751-763 located within the PDE catalytic domain (Artemyev et al., 4Artemyev 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). In this study, to identify a Pα region for binding to Pγ-24-45, we have expressed several large domains of Pα as GST fusion proteins in Escherichia coli. One of these fusion proteins contained a region unique for photoreceptor PDEs that links a second noncatalytic cGMP binding site with the catalytic domain. This protein was used to obtain a polypeptide, Pα-461-553, which blocks inhibition of PDE activity by Pγ and binds to Pγ-24-45. Characterization of the Pα-461-553 region using a set of synthetic peptides revealed that the Pα-517-541 site is primarily responsible for Pα binding to the polycationic region of Pγ.DISCUSSIONIn rod photoreceptor cells, GTP-bound transducin α subunits interact with a PDE complex of two catalytic αβ subunits and two inhibitory γ subunits, leading to displacement of the inhibitory subunits and subsequent PDE activation. The interfaces of critical interactions between Pγ and Pαβ or Pγ and GtαGTP have been analyzed in a number of studies (Lipkin et al., 20Lipkin 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; Artemyev and Hamm, 1Artemyev N.O. Hamm H.E. Biochem. J. 1992; 283: 273-279Crossref PubMed Scopus (72) Google Scholar; Artemyev et al., 2Artemyev N.O. Rarick H.M. Mills J.S. Skiba N.P. Hamm H.E. J. Biol. Chem. 1992; 267: 25067-25072Abstract Full Text PDF PubMed Google Scholar, 3Artemyev N.O. Mills J.S. Thornburg K.R. Knapp D.R. Schey K.L. Hamm H.E. J. Biol. Chem. 1993; 268: 23611-23615Abstract Full Text PDF PubMed Google Scholar; Oppert et al., 27Oppert B. Cunnick J.M. Hurt D. Takemoto D.J. J. Biol. Chem. 1991; 266: 16607-16613Abstract Full Text PDF PubMed Google Scholar; Brown, 8Brown R.L. Biochemistry. 1992; 31: 5918-5925Crossref PubMed Scopus (61) Google Scholar; Takemoto et al., 36Takemoto D.J. Hurt D. Oppert B. Cunnick J. Biochem. J. 1992; 281: 637-643Crossref PubMed Scopus (42) Google Scholar; Skiba et al., 31Skiba 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, 32Skiba N.P. Bae H. Hamm H.E. J. Biol. Chem. 1996; 271: 413-424Abstract Full Text Full Text PDF PubMed Scopus (144) Google Scholar). Although functional sites of Pγ have been elucidated, the interactive surfaces on GtαGTP and Pαβ are not well defined.The central polycationic region of Pγ, residues 24-45, together with the C terminus, is involved in the interaction between Pγ and the PDE catalytic subunits. The primary role of the Pγ-24-45 region is to enhance the affinity of the Pγ-Pαβ interaction. To map the Pγ-24-45 binding site on the Pα subunit, we have expressed a region linking a second noncatalytic cGMP-binding site with the catalytic domain of Pα as a GST fusion protein in E. coli. This selection was made for the following reasons; this region: (a) appears to be unique for photoreceptor PDEs; (b) contains several patches of acidic amino acid residues (Fig. 6B), and (c) has unknown function. The choice is consistent with the unique ability of photoreceptor PDE to be inhibited by Pγ and with the polycationic nature of the Pγ-24-45 region. The Pα-461-553 polypeptide, purified after cleavage of the GST fusion protein, blocks inhibition of PDE activity by Pγ. In addition, Pα-461-553 binds to fluorescently labeled Pγ-24-45BC and induces a maximal increase in fluorescence, similar to that of taPDE binding with Pγ-24-45BC.Synthetic peptides were used to localize the Pγ-24-45 binding to residues 517-541 of Pα. Peptide Pα-517-541 effectively suppressed inhibition of PDE activity by Pγ and bound to Pγ-24-45BC in the fluorescent assay. Despite the fact that Pα-517-541 contains 4 acidic and 3 basic residues and Pα-467-491 contains 8 acidic and 3 basic residues, the latter was able to interfere with the Pγ inhibition only at high concentrations and did not show binding to Pγ-24-45BC. This implies that interactions other than electrostatic ones are very important for the Pγ-24-45 binding to Pαβ. It also eliminates a concern one might have regarding the specificity of the initially observed interaction between the overall negatively charged region Pα-461-553 and the polycationic region Pγ-24-45. Nevertheless, Pα-467-491 may represent an additional, weaker interaction site between Pα and Pγ-24-45. Results of the study by Oppert et al. (27Oppert B. Cunnick J.M. Hurt D. Takemoto D.J. J. Biol. Chem. 1991; 266: 16607-16613Abstract Full Text PDF PubMed Google Scholar) indicated that a weak interaction between region Pα-453-563 and Pγ may complement major interactions that involve the N-terminal regions of Pα, Pα-16-30, and Pα-78-90. Our data suggest that the Pα-517-541 domain is the major site of Pα-Pγ interaction that binds to Pγ-24-45. Different methods used in this study and the study by Oppert et al. (27Oppert B. Cunnick J.M. Hurt D. Takemoto D.J. J. Biol. Chem. 1991; 266: 16607-16613Abstract Full Text PDF PubMed Google Scholar) could in part account for the different conclusions. Also, peptides Pα-16-30 and Pα-78-90 are basic and may at high concentrations bind to Pαβ and compete with Pγ by a mechanism similar to known nonspecific effects of histones and protamines on PDE (Miki et al., 25Miki N. Baraban J.M. Keirns J.J. Boyce J.J. Bitensky M.W. J. Biol. Chem. 1975; 250: 6320-6327Abstract Full Text PDF PubMed Google Scholar). The conclusion that highly dissimilar regions of Pα and Pβ interact with Pγ (Oppert et al., 27Oppert B. Cunnick J.M. Hurt D. Takemoto D.J. J. Biol. Chem. 1991; 266: 16607-16613Abstract Full Text PDF PubMed Google Scholar, Oppert and Takemoto, 26Oppert B. Takemoto D.J. Biochem. Biophys. Res. Commun. 1991; 178: 474-479Crossref PubMed Scopus (9) Google Scholar) appear to be inconsistent with the evidence that both Pα and Pβ interact with identical sites on Pγ with high affinity (Wensel and Stryer, 39Wensel T.G. Stryer L. Biochemistry. 1990; 29: 2155-2161Crossref PubMed Scopus (69) Google Scholar; Artemyev and Hamm, 1Artemyev N.O. Hamm H.E. Biochem. J. 1992; 283: 273-279Crossref PubMed Scopus (72) Google Scholar; Brown, 8Brown R.L. Biochemistry. 1992; 31: 5918-5925Crossref PubMed Scopus (61) Google Scholar).Analysis of the sequences from the regions of Pβ and cone Pα′ that correspond to Pα-517-541 indicates that Pα and Pβ are more than 80% identical in this region, whereas Pα′ has only ∼40% homology to Pα or Pβ (Fig. 6A). Most likely, Pβ-515-539 binds Pγ-24-45, as is seen with Pα-517-541. The lower homology between rod PDE and cone PDE in this region may explain why rod Pγ inhibits rod PDE more effectively (Ki, 80 pM) than cone PDE (Ki, 600 pM) (Hamilton et al., 14Hamilton S.E. Prusti R.K. Bentley J.K. Beavo J.A. Hurley J.B. FEBS Lett. 1993; 318: 157-161Crossref PubMed Scopus (30) Google Scholar). The high level of homology between the Pα and Pβ sites that bind the Pγ C terminus (Artemyev et al., 4Artemyev 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) and Pγ-24-45 favors a model with similar, although not necessarily identical, affinities for the Pγ-Pα and Pγ-Pβ binding sites (Wensel and Stryer, 39Wensel T.G. Stryer L. Biochemistry. 1990; 29: 2155-2161Crossref PubMed Scopus (69) Google Scholar). Fig. 6C shows a linear model for the Pγ interactions with Pα and Pβ based on our results.Previous studies have demonstrated that the Pγ-24-45 site is involved in the interaction with GtαGTP (Lipkin et al., 20Lipkin 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; Artemyev et al., 2Artemyev N.O. Rarick H.M. Mills J.S. Skiba N.P. Hamm H.E. J. Biol. Chem. 1992; 267: 25067-25072Abstract Full Text PDF PubMed Google Scholar; Takemoto et al., 36Takemoto D.J. Hurt D. Oppert B. Cunnick J. Biochem. J. 1992; 281: 637-643Crossref PubMed Scopus (42) Google Scholar). Here we show that Pα-517-541 effectively competes with GtαGTPγS for the binding to PγLY. The question still to be answered is whether this competition is functionally relevant. A very tight binding of Pγ to Pαβ with a Kd < 50 pM (Wensel and Stryer, 39Wensel T.G. Stryer L. Biochemistry. 1990; 29: 2155-2161Crossref PubMed Scopus (69) Google Scholar) does not allow for Pγ dissociation from Pαβ during the time of photoresponse (0.2 s). However, noncompetitive binding of GtαGTP to the Pγ-63-76 region near the major Pγ inhibitory domain Pγ-77-87 (Skiba et al., 31Skiba 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) may induce a conformational change of Pγ, resulting in the availability of the Pγ-24-45 region for the interaction with GtαGTP. This would increase the affinity of GtαGTP-Pγ interaction in the active complex GtαGTP-Pγ-Pαβ-Pγ-GtαGTP.Current classification of cyclic nucleotide phosphodiesterases separates all known mammalian PDEs into seven families (Beavo et al., 6Beavo J.A. Conti M. Heaslip R.J. Mol. Pharmacol. 1994; 46: 399-405PubMed Google Scholar). All PDEs contain a highly conserved catalytic domain within the C-terminal part of the enzyme. Photoreceptor PDEs have similar general domain organization with two PDE families: cGMP-stimulated PDE and cGMP-binding, cGMP-specific PDE (cGB-PDE). These PDEs have two internally homologous repeats for noncatalytic cGMP binding (Li et al., 18Li T. Volpp K. Applebury M.L. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 293-297Crossref PubMed Scopus (98) Google Scholar; Lipkin et al., 21Lipkin V.M. Khramtsov N.V. Vasilevskaya I.A. Atabekova N.V. Muradov K.G. Gubanov V.V. Li T. Johnston J.P. Volpp K.J. Applebury M.L. J. Biol. Chem. 1990; 265: 12955-12959Abstract Full Text PDF PubMed Google Scholar; Trong et al., 38Trong H.L. Beier N. Sonnenburg W.K. Stroop S.D. Walsh K.A. Beavo J.A. Charbonneau H. Biochemistry. 1990; 29: 10280-10288Crossref PubMed Scopus (51) Google Scholar; McAllister-Lucas et al., 23McAllister-Lucas L.M. Sonnenburg W.K. Kadlecek A. Seger D. Trong H.L. Colbran J.L. Thomas M.K. Walsh K.A. Francis S.H. Corbin J.D. Beavo J.A. J. Biol. Chem. 1993; 268: 22863-22873Abstract Full Text PDF PubMed Google Scholar, 24McAllister-Lucas L.M. Haik T.L. Colbran J.L. Sonnenburg W.K. Seger D. Turko I.V. Beavo J.A. Francis S.H. Corbin J.D. J. Biol. Chem. 1995; 270: 30671-30679Abstract Full Text Full Text PDF PubMed Scopus (83) Google Scholar). Direct binding studies have shown that only two molecules of cGMP are bound to the dimeric PDE molecule, which has four cGMP binding segments (Gillespie and Beavo, 12Gillespie P.G. Beavo J.A. Proc. Natl. Acad. Sci. U. S. A. 1989; 86: 4311-4315Crossref PubMed Scopus (81) Google Scholar; Thomas et al., 37Thomas M.K. Francis S.H. Corbin J.D. J. Biol. Chem. 1990; 265: 14964-14970Abstract Full Text PDF PubMed Google Scholar; Stroop and Beavo, 34Stroop S.D. Beavo J.A. J. Biol. Chem. 1991; 266: 23802-23809Abstract Full Text PDF PubMed Google Scholar). Perhaps, each cGMP binding site is formed by two analogous motifs from both catalytic PDE subunits (McAllister-Lucas et al., 24McAllister-Lucas L.M. Haik T.L. Colbran J.L. Sonnenburg W.K. Seger D. Turko I.V. Beavo J.A. Francis S.H. Corbin J.D. J. Biol. Chem. 1995; 270: 30671-30679Abstract Full Text Full Text PDF PubMed Scopus (83) Google Scholar). A segment of approximately 60-70 amino acid residues connects the second noncatalytic cGMP binding site with the PDE catalytic domain. The functional role of this domain for different cGMP-binding PDEs is not known. We performed a local alignment search (BLAST) to compare sequences from bovine photoreceptor PDEs corresponding to this "linker" region against protein sequence data bases. This search revealed 65-70% homology between the linker regions of Pα and Pβ from different species and 45-50% homology between Pα or Pβ and cone Pα′. Interestingly, the cGB-PDE also has a significant level of homology to photoreceptor PDEs within this region. The sequence corresponding to residues 524-583 of bovine cGB-PDE is 35-37% identical (51-55% similar) to the Pα-481-540 and Pβ-479-538 regions (Fig. 6B). Thus far, no protein modulators of activity of cGB- and cGMP-stimulated PDEs have been identified. The universal Ca2+-binding protein modulator calmodulin stimulates activity of calmodulin-dependent PDEs through binding to two sites located N-terminally to the catalytic domain (Sonnenburg et al., 33Sonnenburg W.K. Seger D. Kwak K.S. Huang J. Charbonneau H. Beavo J.A. J. Biol. Chem. 1995; 270: 30989-31000Abstract Full Text Full Text PDF PubMed Scopus (105) Google Scholar). It is tempting to speculate that the linker regions in PDEs with noncatalytic cGMP binding sites could represent potential sites for PDE regulation by other proteins. INTRODUCTIONIn vertebrate photoreceptor cells, light-activated rhodopsin catalyzes GDP-GTP exchange on the rod G-protein α-subunit (Gtα), 1The abbreviations used are: Gtαα subunit of the photoreceptor G-protein transducinPDErod outer segment cGMP phosphodiesterasePα, Pβ, and Pγα, β, and γ subunits of PDEtaPDEtrypsin-activated PDEBC3-(bromoacetyl)-7-diethylaminocoumarinLYlucifer yellow vinyl sulfoneHPLChigh performance liquid chromatographyGSTglutathione S-transferaseGTPγSguanosine 5′-O-(thiotriphosphate)PBSphosphate-buffered salinecGB-PDEcGMP-binding, cGMP-specific PDE. which in turn activates cGMP phosphodiesterase (PDE). PDE activation leads to the rapid hydrolysis of cytoplasmic cGMP and closure of sodium channels, resulting in hyperpolarization of the rod and cone cells (for review, see Chabre and Deterre (10Chabre M. Deterre P. Eur. J. Biochem. 1989; 179: 255-266Crossref PubMed Scopus (219) Google Scholar) and Stryer (35Stryer L. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 557-559Crossref PubMed Scopus (46) Google Scholar)). Rod photoreceptor PDE is composed of two large homologous catalytic α and β subunits (Pα and Pβ) of nearly identical size (Mr 99,261 and 98,308, respectively) and two copies of an inhibitory γ subunit (Pγ, Mr 9700) (Ovchinnikov et al., 28Ovchinnikov Y.A. Lipkin V.M. Kumarev V.P. Gubanov V.V. Khramtsov N.V. Akhmedov N.B. Zagranichny V.E. Muradov K.G. FEBS Lett. 1986; 204: 288-292Crossref PubMed Scopus (89) Google Scholar, 29Ovchinnikov Y.A. Gubanov V.V. Khramtsov K.A. Ischenko K.A. Zagranichny V.E. Muradov K.G. Shuvaeva T.M. Lipkin V.M. FEBS Lett. 1987; 223: 169-173Crossref PubMed Scopus (78) Google Scholar; Deterre et al., 11Deterre P. Bigay J. Forquet F. Robert M. Chabre M. Proc. Natl. Acad. Sci. U. S. A. 1988; 85: 2424-2428Crossref PubMed Scopus (166) Google Scholar; Lipkin et al., 21Lipkin V.M. Khramtsov N.V. Vasilevskaya I.A. Atabekova N.V. Muradov K.G. Gubanov V.V. Li T. Johnston J.P. Volpp K.J. Applebury M.L. J. Biol. Chem. 1990; 265: 12955-12959Abstract Full Text PDF PubMed Google Scholar). The primary structures of Pα, Pβ, and cone-specific Pα′ subunits (Li et al., 18Li T. Volpp K. Applebury M.L. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 293-297Crossref PubMed Scopus (98) Google Scholar) revealed that these PDEs constitute one family, that of PDE6 (Beavo et al., 6Beavo J.A. Conti M. Heaslip R.J. Mol. Pharmacol. 1994; 46: 399-405PubMed Google Scholar). The photoreceptor PDEs belong to a broader group of cGMP-binding PDEs, which contain two noncatalytic cGMP binding sites located N-terminally to the conserved PDE catalytic domain (Yamazaki et al., 40Yamazaki A. Sen I. Bitensky M.W. Casnellie J. Greengard P. J. Biol. Chem. 1980; 255: 11619-11624Abstract Full Text PDF PubMed Google Scholar; Gillespie and Beavo, 12Gillespie P.G. Beavo J.A. Proc. Natl. Acad. Sci. U. S. A. 1989; 86: 4311-4315Crossref PubMed Scopus (81) Google Scholar; Lipkin et al., 21Lipkin V.M. Khramtsov N.V. Vasilevskaya I.A. Atabekova N.V. Muradov K.G. Gubanov V.V. Li T. Johnston J.P. Volpp K.J. Applebury M.L. J. Biol. Chem. 1990; 265: 12955-12959Abstract Full Text PDF PubMed Google Scholar; Trong et al., 38Trong H.L. Beier N. Sonnenburg W.K. Stroop S.D. Walsh K.A. Beavo J.A. Charbonneau H. Biochemistry. 1990; 29: 10280-10288Crossref PubMed Scopus (51) Google Scholar; McAllister-Lucas et al., 23McAllister-Lucas L.M. Sonnenburg W.K. Kadlecek A. Seger D. Trong H.L. 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Cunnick J. Biochem. J. 1992; 281: 637-643Crossref PubMed Scopus (42) Google Scholar). Both of these Pγ domains bind to Pαβ, allowing effective inhibition of PDE activity by the Pγ C terminus. Initial studies indicated that the major sites of Pα and Pβ interaction with Pγ are different and located in the N-terminal regions (Pα, 16-30 and 78-90; Pβ, 91-110 and 211-230) in areas with a high level of dissimilarity between catalytic subunits (Oppert et al., 27Oppert B. Cunnick J.M. Hurt D. Takemoto D.J. J. Biol. Chem. 1991; 266: 16607-16613Abstract Full Text PDF PubMed Google Scholar; Oppert and Takemoto, 26Oppert B. Takemoto D.J. Biochem. Biophys. Res. Commun. 1991; 178: 474-479Crossref PubMed Scopus (9) Google Scholar).More recently, using a cross-linking approach we have demonstrated that the C terminus of Pγ interacts with region Pα-751-763 located within the PDE catalytic domain (Artemyev et al., 4Artemyev 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). In this study, to identify a Pα region for binding to Pγ-24-45, we have expressed several large domains of Pα as GST fusion proteins in Escherichia coli. One of these fusion proteins contained a region unique for photoreceptor PDEs that links a second noncatalytic cGMP binding site with the catalytic domain. This protein was used to obtain a polypeptide, Pα-461-553, which blocks inhibition of PDE activity by Pγ and binds to Pγ-24-45. Characterization of the Pα-461-553 region using a set of synthetic peptides revealed that the Pα-517-541 site is primarily responsible for Pα binding to the polycationic region of Pγ.