Báo cáo khoa học: Binding of cGMP to the transducin-activated cGMP phosphodiesterase, PDE6, initiates a large conformational change involved in its deactivation ppt

Binding of cGMP to the transducin-activated cGMP phosphodiesterase, PDE6, initiates a large conformational change involved in its deactivation Akio Yamazaki 1,2,3 , Fumio Hayashi 4 , Isao Matsuura 5 and Vladimir A. Bondarenko 6 1 Kresge Eye Institute, Wayne State University, Detroit, MI, USA 2 Department of Ophthalmology, Wayne State University, Detroit, MI, USA 3 Department of Pharmacology, Wayne State University, Detroit, MI, USA 4 Department of Biology, Kobe University, Japan 5 Division of Molecular and Genomic Medicine, National Health Research Institutes, Zhunan Town, Taiwan 6 College of Osteopathic Medicine, Touro University, Henderson, NV, USA Keywords cGMP binding; cGMP-binding-dependent protein conformational change; GAF domains; G-protein-mediated signal transduction; PDE Correspondence V. A. Bondarenko, College of Osteopathic Medicine, Touro University, Henderson, NV 89014, USA Fax: +1 702 777 1799 Tel: +1 702 777 1806 E-mail: vladimir.bondarenko@tun.touro.edu (Received 30 January 2011, revised 17 March 2011, accepted 22 March 2011) doi:10.1111/j.1742-4658.2011.08104.x Retinal photoreceptor phosphodiesterase (PDE6), a key enzyme for photo- transduction, consists of a catalytic subunit complex (Pab) and two inhibi- tory subunits (Pcs). Pab has two noncatalytic cGMP-binding sites. Here, using bovine PDE preparations, we show the role of these cGMP-binding sites in PDE regulation. Pabcc and its transducin-activated form, Pabc, contain two and one cGMP, respectively. Only Pabc shows [ 3 H]cGMP binding with a K d  50 nM and Pc inhibits the [ 3 H]cGMP binding. Binding of cGMP to Pabc is suppressed during its formation, implying that cGMP binding is not involved in Pabcc activation. Once bound to Pabc, [ 3 H]cGMP is not dissociated even in the presence of a 1000-fold excess of unlabeled cGMP, binding of cGMP changes the apparent Stokes’ radius of Pabc, and the amount of [ 3 H]cGMP-bound Pabc trapped by a filter is spontaneously increased during its incubation. These results suggest that Pabc slowly changes its conformation after cGMP binding, i.e. after for- mation of Pabc containing two cGMPs. Binding of Pc greatly shortens the time to detect the increase in the filter-trapped level of [ 3 H]cGMP-bound Pabc, but alters neither the level nor its Stokes’ radius. These results sug- gest that Pc accelerates the conformational change, but does not add another change. These observations are consistent with the view that Pabc changes its conformation during its deactivation and that the binding of cGMP and Pc is crucial for this change. These observations also imply that Pabcc changes its conformation during its activation and that release of Pc and cGMP is essential for this change. Structured digital abstract l PDE6 alpha, PDE6 beta and PDE6 gamma physically interact by molecular sieving (View interaction) Abbreviations GAF, a domain derived from cGMP-regulated cyclic nucleotide phosphodiesterases, certain adenylyl cyclases, the bacterial transcription factor FhlA; GTPcS, guanosine 5¢-O-(3-thiotriphosphate); IBMX, 1-methyl-3-isobutylxanthine; OS, outer segments of retinal photoreceptors; PDE, cGMP phosphodiesterase; PMSF, phenylmethylsulfonyl fluoride; Pa and Pb, rod PDE catalytic subunits; Pa¢, cone PDE catalytic subunit; Pab ⁄ Pc,Pab complexes having an unknown number of Pc;Pd, a prenyl-binding protein; Pc, rod PDE inhibitory subunit; Pc¢, cone PDE inhibitory subunit; T, transducin. 1854 FEBS Journal 278 (2011) 1854–1872 ª 2011 The Authors Journal compilation ª 2011 FEBS Introduction Cyclic GMP phosphodiesterase (EC 3.1.4.17), classified as PDE6 in the PDE family, is one of the key enzymes for phototransduction in the outer segments (OS) of retinal photoreceptors. Its activation is G-protein-med- iated: illuminated rhodopsin stimulates GTP ⁄ GDP exchange on transducin (T)a, followed by dissociation of GTP–Ta from Tbc. The GTP–Ta activates PDE, resulting in a decrease in the cytoplasmic [cGMP], clo- sure of cGMP-gated channels and hyperpolarization of plasma membranes [1–3]. The inactive form of rod PDE is composed of a cat- alytic subunit complex, Pab, and two inhibitory subun- its, Pcs, i.e. Pabcc [4–10]. A study using electron microscopy and image analysis of single particles [11] shows that bovine Pabcc, 150 · 108 · 60 A ˚ , has the shape of a flattened bell with a handle-like protrusion ( 30 A ˚ ) and that the structure is divided into three distinct substructures by two holes. Except for the pro- trusion, the structure also appears to consist of two homologous structures arranged side by side. These characteristics are consistent with a model in which Pabcc’s structure is determined by a dimer of homolo- gous catalytic subunits consisting of two GAF (a domain derived from cGMP-regulated cyclic nucleo- tide phosphodiesterases, certain adenylyl cyclases, the bacterial transcription factor FhlA) regions and one catalytic region. Indeed, bovine Pabcc contains two cGMPs and these bind tightly to substructures formed by GAF regions [12]. These two substructures, called the noncatalytic cGMP-binding sites, are similar, but not identical, in shape and size [11]. This implies that the manner of cGMP binding to each site and ⁄ or the role of cGMP binding to each site in PDE regulation, if present, may be different. The current predominant model for PDE regulation is simple [13]. For activation, GTP–Ta interacts with Pc in Pabcc, and the GTP–TaÆPabcc complex, with- out altering the firm interaction between Pab and Pc, expresses a high cGMP hydrolytic activity. For deacti- vation, GTP in the GTP–TaÆPabcc complex is hydro- lyzed with the help of RGS9 and accessory proteins, i.e. the GTP is hydrolyzed after formation of a huge complex, and Pabcc is recovered after dissociation of various proteins, including GDP-bound Ta (GDP– Ta). This model conveniently explains the rapid acti- vation and deactivation of PDE; however, there is no clear evidence to show a firm and continuous interac- tion between GTP–Ta and Pabcc during Pabcc acti- vation, as would be shown by the isolation of a complex of Pabcc with Ta containing a hydrolysis- resistant GTP analogue such as guanosine 5¢ -O-(3- thiotriphosphate) (GTPcS). In addition, there is no definitive evidence to prove the formation of a GTP– TaÆPabcc complex containing RGS9 and accessory proteins and its decomposition during deactivation of GTP–Ta-activated PDE. Binding of cGMP to the noncatalytic site in Pab is believed to be involved in PDE regulation. Two mod- els, the cGMP-regulated Pab-Pc interaction model [14–18] and the cGMP-binding direct regulation model [19], have been proposed to explain the role of cGMP- binding sites in PDE regulation. In the former model, the interaction between Pab and Pc is dependent upon the presence of cGMP at the noncatalytic site. When the noncatalytic sites of Pabcc are saturated with cGMP, GTP–Ta activates Pabcc without changing the tight interaction between Pab and Pc, i.e. a GTP–TaÆ- Pabcc complex is formed and the complex expresses a high PDE activity. However, when the noncatalytic sites are not saturated, GTP–Ta activates Pabcc through dissociation of Pc complexed with GTP–Ta, i.e. a Pc-depleted PDE(s) is produced. Pc in the GTP– Ta complex enhances the GTPase activity of Ta; the resulting GDP–Ta instantly releases Pc, and the released Pc deactivates the GTP–Ta-activated PDE. In the latter model, binding of cGMP to the noncatalytic sites directly regulates PDE catalytic activity. These two models appear to explain some observations of cGMP binding to noncatalytic sites. However, as dis- cussed later, these models have many ambiguous and controversial points. Thus, it is difficult to integrate these concepts smoothly into a coherent model for PDE regulation. We have recently challenged the dominant model for PDE regulation by proposing a new and comprehen- sive model [11,13,20] in which GTP–Ta activates Pabcc by forming a complex with a Pc, thereby disso- ciating the PcÆGTP–Ta complex. This occurs on mem- branes and is independent of the cytoplasmic [cGMP]. A significant portion of the PcÆGTP–Ta complex is then released into the soluble fraction. Thus, Pabc is the GTP–Ta-activated PDE. After hydrolysis of GTP, both soluble and membranous PcÆGDP–Ta complexes deactivate Pabc without liberating Pc. These PcÆGDP– Ta complexes appear to have a preferential order in deactivating Pabc. This new model is based on the fol- lowing observations: (a) Pabc, but not Pab, is isolated only when OS homogenates are incubated with GTPcS; (b) the ratio of Pc ⁄ Pab in Pabcc and Pabc is 2 : 1; (c) the enzymatic activity of Pabc is  12 times higher than that of Pabcc and is inhibited by 30 nm Pc; (d) the basic structure of these PDE species is not A. Yamazaki et al. Roles of cGMP binding in PDE6 regulation FEBS Journal 278 (2011) 1854–1872 ª 2011 The Authors Journal compilation ª 2011 FEBS 1855 changed when Pabcc is shifted to Pabc; (e) PcÆGTPcS–Ta is isolated from membranous and solu- ble fractions; (f) both membranous and soluble PcÆGDP–Ta complexes deactivate Pabc without liber- ating Pc; (g) the membranous PcÆGDP–Ta complex appears to be consumed earlier than the soluble PcÆGDP–Ta complex; and (h) PDE regulatory mecha- nisms similar to this model are also found in mamma- lian and amphibian photoreceptors, as well as in rods and cones. During these studies, we have also shown that: (a) the interaction between Pabcc and GTPcS– Ta is short-lived, indicating that GTP–TaÆPabcc is an intermediate, but not GTP–Ta-activated PDE; (b) free Pc is not detected in any preparations, implying that Pc always forms complexes with other proteins; (c) Pabccd and Pabcdd are formed when Pabcc and Pabc are solubilized with Pd, a prenyl-binding protein; (d) the stoichiometry of Pabccd suggests that only one lipid moiety may be involved in the interaction of Pabcc with membranes; and (e) the stoichiometry of Pabcdd suggests that a lipid moiety in Pab is also affected by Pc dissociation. In this study, we extend our model by integrating the role of cGMP binding to the noncatalytic site. We demonstrate that Pabcc and Pabc contain two and one cGMP, respectively, that only Pabc expresses [ 3 H]cGMP-binding activity and that Pc inhibits [ 3 H]cGMP binding to Pabc. We also show that the cGMP binding to Pabc is suppressed during Pabcc activation, i.e. cGMP binding is not involved in Pabcc activation. We also suggest that cGMP binding to Pabc slowly changes its conformation and that binding of Pc accelerates the conformational change. Based on these studies, we propose that binding of cGMP to Pabc is the first step in PDE deactivation. Results Binding of [ 3 H]cGMP to OS membranes Bovine OS membranes contain a [ 3 H]cGMP-binding site(s) ( Fig. 1A). Both GTPcS-treated and nontreated membranes showed [ 3 H]cGMP-binding activities; how- ever, the activity in GTPcS-treated membranes was much higher than in GTPcS-nontreated membranes, indicating that GTPcS–Ta somehow enhances the [ 3 H]cGMP-binding activity. By contrast, the soluble fraction, whether obtained from GTPcS-treated or nontreated OS homogenates, showed only negligible [ 3 H]cGMP-binding activity (data not shown). This sug- gests that no protein in the soluble fraction contains the [ 3 H]cGMP-binding site and ⁄ or expresses [ 3 H]cGMP- binding activity under our experimental conditions. Solubilization and isolation of membranous proteins showed that a [ 3 H]cGMP-binding activity (Fig. 1B) was detected only in the fraction containing a protein- doublet (m  88 kDa) (Fig. 1C) and that the activity appeared to be proportional to the level of the pro- tein-doublet. These fractions also contained a PDE activity that was proportional to the level of the pro- tein-doublet (data not shown). The protein-doublet has been identified as Pab and 70–80% of Pab is extracted from membranes under these conditions [13,20]. These results suggest that the [ 3 H]cGMP-binding activity in membranes is due to a Pab complex(s). This implies that cone PDEs, Pa¢a¢⁄Pc¢ complexes, are also present and that a Pa¢a¢⁄Pc¢ complex(s) expresses [ 3 H]cGMP- Fig. 1. Binding of [ 3 H]cGMP to membranous PDE. (A) Levels of [ 3 H]cGMP binding to OS membranes treated with or without GTPcS. OS homogenates (27.5 mg protein) were suspended in 18.4 mL of buffer A and divided into two portions. After incubation of a portion with 50 l M GTPcS overnight on ice, its membranes were washed twice with 5 mL buffer A supplemented with 50 l M GTPcS, twice with 5 mL buffer A and suspended in 5 mL buffer A. The other portion was treated in the same way but without GTPcS. Binding of [ 3 H]cGMP to these suspensions (10 lL) was assayed using 1 l M [ 3 H]cGMP. (B,C) [ 3 H]cGMP binding to proteins extracted from OS membranes treated with or without GTPcS. OS homogen- ates (27.7 mg protein) were suspended in 18 mL of buffer A, divided into two portions and treated with or without GTPcS. Pro- teins were extracted from membranes with 3 mL buffer B (·7), concentrated to  0.5 mL and applied to Bio-Gel A 0.5-m column. [ 3 H]cGMP-binding activity (B) and PDE activity (not shown) were assayed using 60 and 5 lL of the fraction, respectively. Protein pro- files in the fraction (90 lL) were analyzed by SDS ⁄ PAGE and stain- ing with Coomassie Brilliant Blue (C). The left end lane shows the molecular mass of standard proteins, 94, 67 and 43 kDa. Roles of cGMP binding in PDE6 regulation A. Yamazaki et al. 1856 FEBS Journal 278 (2011) 1854–1872 ª 2011 The Authors Journal compilation ª 2011 FEBS binding activity. However, neither Pa¢ nor its [ 3 H]cGMP-binding activity could be identified. These failures, we believe, are because of its small abundance in OS. The soluble fraction also contained a Pab ⁄ Pc complex (peak b in [13]); however, the complex showed only negligible [ 3 H]cGMP-binding activity (data not shown). This is consistent with the above-mentioned conclusion that [ 3 H]cGMP-binding activity was not detected in the soluble fraction. Interestingly, the [ 3 H]cGMP-binding activity in GTPcS-treated PDE was higher than in GTPcS-non- treated PDE (Fig. 1B). When OS homogenates are incubated with GTPcS, the Pab content in membranes is increased 20–30% by binding of the Pab ⁄ Pc com- plex existing in the soluble fraction [13]. Therefore, binding of the Pab ⁄ Pc complex to membranes and the resulting expression of a [ 3 H]cGMP-binding activity could increase the activity in membranes. However, the increase in the activity by GTPcS was much higher,  2.4 times (Fig. 1B). In addition, Pab in the Pab ⁄ Pc complex has two cGMP-binding sites at most [12]. Therefore, we conclude that even if the Pab ⁄ Pc complex could express [ 3 H]cGMP-binding activity, the greater part of the increase is due to an increase in the activity of a Pab ⁄ Pc complex(s) located on mem- branes. This is unexpected because previous studies using frog PDE ⁄ membranes [21,22] showed that their [ 3 H]cGMP-binding activity in GTP-nontreated PDE was much higher than that in GTP-treated PDE. We also note that this result, with the observation shown in Fig. 1A, implies that [ 3 H]cGMP binding to solubi- lized PDE species is similar to binding to membranous PDE species, i.e. the properties of cGMP binding to membranous PDE species may be estimated by study- ing cGMP binding to solubilized PDE species. Identification of PDE species expressing [ 3 H]cGMP-binding activity GTPcS-nontreated membranes contain Pabcc, and GTPcS-treated membranes have Pabcc and Pabc as major species and a Pab ⁄ Pc complex as a minor species [20]. These PDE species were extracted using a hypo- tonic buffer (Fig. 2A) or Pd in an isotonic buffer (Fig. 2C) and their [ 3 H]cGMP-binding activities were measured after isolation. The use of Pd in an isotonic buffer may exclude a possible artifact(s) caused by the hypotonic extraction. OS homogenates were also treated with GTPcS in the presence of cGMP (GTPcS+ cGMP), and after isolation of Pab ⁄ Pc complexes, their [ 3 H]cGMP-binding activities were measured (Fig. 2B). The result is compared with the results in Fig. 2A, as shown later. Pabcc extracted by a hypotonic buffer Pabcc was obtained from GTPcS-nontreated mem- branes (Fig. 2A, upper) and GTPcS-treated membranes (Fig. 2A, lower). In the former preparation, the [ 3 H]cGMP-binding activity appeared to be proportional to the level of Pab, implying that Pabcc may express [ 3 H]cGMP-binding activity. However, the molecular ratio of [ 3 H]cGMP to Pab was < 0.01, indicating that only a negligible portion of the Pabcc expresses this activity. In the latter preparation, a small [ 3 H] radio- activity was detected in the fraction close to the Pabcc peak. However, the level of [ 3 H] radioactivity was not proportional to that of Pab in the Pabcc fraction, indi- cating that the [ 3 H] radioactivity is not attributable to [ 3 H]cGMP bound to the Pabcc, i.e. the Pabcc does not show [ 3 H]cGMP-binding activity and⁄or the Pabcc, when it exists with GTP–Ta, appears to lose a portion that may express [ 3 H]cGMP-binding activity (Fig. 2A, upper). Pabcc extracted with Pd in an isotonic buffer The Pabccd preparation was obtained from GTPcS- nontreated membranes (data not shown) and GTPcS- treated membranes (Fig. 2C). In the former prepara- tion, the [ 3 H]cGMP-binding activity appeared to be proportional to the level of Pab; however, the molecu- lar ratio of [ 3 H]cGMP to Pab in the Pabccd was < 0.01. These observations are identical to those for Pabcc extracted with a hypotonic buffer (Fig. 2A, upper). In the latter preparation, Pabccd appeared to show a small [ 3 H]cGMP-binding activity (Fig. 2C, upper). However, the amount of binding was not exactly proportional to the Pab level in the fraction, indicating that the [ 3 H] radioactivity was not due to [ 3 H]cGMP bound to the Pabccd. As shown later (Fig. 7), Pabcc can be trapped by a Millipore filter with a high efficiency, implying that the lack of [ 3 H]cGMP-binding activity and ⁄ or the negligi- ble level of [ 3 H]cGMP-binding activity in Pabcc prepa- rations are not due to the failure to trap [ 3 H]cGMP- bound Pabcc. Taken together, our results strongly suggest that Pabcc does not express [ 3 H]cGMP-bind- ing activity and that negligible activities occasionally detected in fractions containing Pabcc may be artifacts caused by experimental procedures. The level of [ 3 H] radioactivity was not proportional to the level of Ta (Fig. 2C). This confirms that Ta has no cGMP-binding site [23]. The amino acid sequence of Ta also supports this notion. This is specifically noted here because we use this information in a later discussion. A. Yamazaki et al. Roles of cGMP binding in PDE6 regulation FEBS Journal 278 (2011) 1854–1872 ª 2011 The Authors Journal compilation ª 2011 FEBS 1857 Pabc and Pab ⁄ Pc Whether extracted with the hypotonic buffer (Fig. 2A, lower) or with Pd in the isotonic buffer (Fig. 2C), frac- tions containing these PDE species clearly showed [ 3 H]cGMP-binding activities. In addition, the level of Pab was proportional to that of [ 3 H]cGMP-binding activity in these fractions. These results indicate that both Pabc and Pab ⁄ Pc express [ 3 H]cGMP-binding activity. We emphasize that [ 3 H]cGMP-binding activity in the fraction containing Pabcdd (Fig. 2C, upper) was similar to that in the fraction containing Pabc (Fig. 2A, lower), although these activities were apparently different due to the use of different amounts of OS homogenates and different volumes of the fraction in the assay. We con- firmed this observation by comparing the [ 3 H]cGMP- binding activity of Pabc with that of Pabcdd (data not shown). These results indicate that Pd binding to the lipid moiety of Pab does not affect the level of [ 3 H]cGMP-binding activity in Pabc, implying that mem- brane binding of Pabc may not affect its cGMP-binding activity. This implication also supports our above-men- tioned view that properties of cGMP binding to mem- branous PDE species may be estimated by studying the cGMP binding to solubilized PDE species. We also note that the NaCl gradient in the study (shown in Fig. 2C) was modified to collect both rod and cone PDEs with fraction numbers similar to those for rod PDEs (Fig. 2A). Therefore, their elution profile was slightly different from that shown in Fig. 2A. We have already shown that the elution profile of PDE species containing Fig. 2. Binding of [ 3 H]cGMP to PDE species extracted from OS membranes. (A,B) PDE species extracted with a hypotonic buffer. Details of the procedure are given in Experimental procedures. OS homogenates (50.4 mg protein) were suspended in 20 mL buffer A and divided into three portions. After incubation with cGMP (A, upper), GTPcS (A, lower) or cGMP + GTPcS (B), proteins were extracted with buffer B (a hypotonic buffer), applied to a TSK–DEAE 5PW column and eluted. Fractions containing PDE species were determined by SDS ⁄ PAGE and assaying PDE activity. Elution profiles of the 88-kDa protein, Pab, are shown in each panel. The elution profile of other proteins is detailed elsewhere [20]. PDE species were identified as described previously [20]. Binding of [ 3 H]cGMP to the fraction (60 lL) was measured with 0.5 l M [ 3 H]cGMP. (C) PDE species extracted with Pd in an isotonic buffer. OS homogenates (12.4 mg) were suspended in 13 mL of buf- fer A and divided into two portions. After incubation of a portion with GTPcS (50 l M) for 1 h on ice, membranes were washed with 2 mL of buffer A containing GTPcS (50 l M) and 2 mL of buffer A. The other portion was treated in the same way but without GTPcS. These mem- branes were suspended in 2.5 mL of buffer D, incubated with Pd (final 3 l M) overnight on ice, and washed twice with 2 mL of buffer D. All supernatants were collected and applied to a TSK–DEAE 5PW column. Rod and cone PDE species and their stoichiometry and transducin subunits were identified as described previously [20]. Binding of [ 3 H]cGMP to the fraction (50 lL) was measured with 0.5 lM [ 3 H]cGMP (upper). Protein profiles in fractions (40 lL) were analyzed by SDS ⁄ PAGE and staining with Coomassie Brilliant Blue (lower). Owing to the limited space, only results from GTPcS-treated membranes are shown. Profiles of PDE species from GTPcS-nontreated membranes are given in Yamazaki et al. [20]. Roles of cGMP binding in PDE6 regulation A. Yamazaki et al. 1858 FEBS Journal 278 (2011) 1854–1872 ª 2011 The Authors Journal compilation ª 2011 FEBS Pd is identical to that of PDE species without Pd when the same NaCl gradient was used [20]. Comparison of the [ 3 H]cGMP-binding activity of cone PDE with that of Pabc is discussed later. Contents of cGMP in Pabcc and Pabc Pabcc and Pabc were purified from GTPcS-treated OS homogenates (Fig. 3A). These PDE species were clearly separated and characterization of these species includ- ing their specific activity and Pc-sensitivity verified the clear separation [20]. We also note that the level of pro- tein staining with Coomassie Brilliant Blue is propor- tional to the molecular mass calculated based on its amino acid sequence under our staining conditions, i.e. the Pc ⁄ Pab ratios also showed the clear separation [20]. Molecular sieve chromatography of these PDE species also showed that the Pc ⁄ Pab ratio in these PDE species was not changed during their storage. We found that 3.0 pmol of the Pabcc contained  6.5 pmol of cGMP (Fig. 3B). This indicates that Pabcc contains two cGMPs. Pabcc isolated from GTPcS-nontreated OS homogenates also contained two cGMPs (data not shown). These results indicate that noncatalytic sites of Pabcc, whether located with or without GTP–Ta, are saturated by cGMP. These results also suggest that saturation is a reason for the lack of [ 3 H]cGMP-binding activity in Pabcc. These Pabcc preparations had been exposed to cGMP-free conditions for at least 1 week. This suggests that these cGMPs bind tightly to Pabcc, confirming previous observations [12]. Pabc, 6.0 pmol, contained  6.1 pmol of cGMP (Fig. 3B). This indicates that Pabc contains one cGMP, i.e. one of the noncatalytic sites in Pabc is empty. The possibility that cGMP existing in Pabc can be exchanged by [ 3 H]cGMP during the assay of [ 3 H]cGMP binding is quite low, as discussed later. Therefore, we conclude that the [ 3 H]cGMP-binding activity in Pabc we observed is due to the binding of [ 3 H]cGMP to the empty site, i.e. [ 3 H]cGMP-bound Pabc contains one original cGMP and one [ 3 H]cGMP. These results also indicate that GTP–Ta dissociates not only a single Pc, but also one cGMP from Pabcc during its activation. In other words, PDE activation is the mechanism by which Pabcc having two cGMPs changes to Pabc having one cGMP, and PDE deacti- vation is the mechanism by which Pabc having one cGMP shifts to P abcc having two cGMPs. Pab ⁄ Pc (Fig. 2A lower and C upper) is a minor species that is difficult to purify [20]. Therefore, the content of cGMP in Pab ⁄ Pc could not be measured. Pabc was exposed to cGMP-free conditions for > 3 days. Under these conditions, the molecular ratio of cGMP to Pab in Pabc is always  1.0 (Fig. 3B). This observation suggests that the affinity for cGMP is clearly different in Pabcc’s two noncatalytic sites and that GTPcS–Ta (GTP–Ta) releases cGMP only from the same one site in Pabcc during its activation. This also implies that GTP–Ta dissociates Pc from the same site in Pabcc during its activation. Characterization of [ 3 H]cGMP binding to Pabc Purified Pabc showed a [ 3 H]cGMP-binding activity (Fig. 4A). The level of [ 3 H]cGMP binding reached a plateau as the [ 3 H]cGMP concentration increased. Scatchard plotting of this saturable [ 3 H]cGMP binding (Fig. 4A, insert) indicates that Pabc has one type of cGMP-binding site with K d  50 nm. This is consistent with the above-mentioned view that [ 3 H]cGMP binds to the same site in Pabc. The level of bound [ 3 H]cGMP reached a plateau in < 2 min under these conditions (Fig. 4B). Unlabeled cGMP, but not cAMP, competitively inhibited [ 3 H]cGMP binding (Fig. 4C). This indicates that the [ 3 H]cGMP-binding site in Pabc is cGMP-specific. Trapping of [ 3 H]cGMP-bound Pabc to a Millipore filter After incubation with [ 3 H]cGMP, Pabc was applied to a molecular sieve column and the amount of [ 3 H]cGMP Fig. 3. Levels of cGMP contained in Pabcc and Pabc.Pabcc (6.50 lgÆ20 lL )1 ) and Pabc (4.75 lgÆ50 lL )1 ) were purified from GTPcS-treated OS homogenates. (A) Purity of these PDE prepara- tions. Preparations of Pabcc (10 lL) and Pabc (25 lL) were applied to SDS ⁄ PAGE followed by staining with Coomassie Brilliant Blue. (B) Levels of cGMP contained in these PDE species. Contents of cGMP were measured using a cGMP immunoassay kit. A. Yamazaki et al. Roles of cGMP binding in PDE6 regulation FEBS Journal 278 (2011) 1854–1872 ª 2011 The Authors Journal compilation ª 2011 FEBS 1859 bound to Pabc was calculated based on the [ 3 H] radio- activity in the Pabc fraction (Fig. 4D). We found that 70 lL of fraction 15, the peak fraction, contained 3.2 lg Pabc (15.5 pmol) and 13.1 pmol of [ 3 H]cGMP, i.e.  83% of Pabc in the fraction was occupied by [ 3 H]cGMP. The average level of the occupation was  86% in three experiments. These results indicate that  100% of Pabc binds [ 3 H]cGMP under these condi- tions. The result is also confirmed later (Fig. 5). How- ever, only  17% of the activity was detected when 70 lL of the fraction was applied to the filter and the [ 3 H]cGMP-binding activity was obtained based on the [ 3 H] radioactivity trapped by the filter (Fig. 4D). This shows that the Millipore filter traps  17% of the [ 3 H]cGMP-bound Pabc existing in the assay mixture. We could not get a result showing that 100% of Pabc expressed [ 3 H]cGMP-binding activity. We believe that this is resulted from an artifact caused by our experi- mental procedures, because the Pabc preparation we obtained appears to contain one type of Pabc [20], and [ 3 H]cGMP, once bound to Pabc, is not dissociated even in the presence of a 1000-fold excess of unlabeled cGMP (Fig. 5). In Fig. 4D, fraction 15 apparently shows that 100% of Pabc binds [ 3 H]cGMP. This is due to our intention to show the ratio of [ 3 H]cGMP-binding activ- ity measured by the filter. It should be noted that  18.2% of the [ 3 H]cGMP-bound Pabc in the assay mixture was trapped by the filter in the studies shown in Fig. 4A, however this low rate does not affect the prop- erties shown in Fig. 4A–C, because these properties are not affected by the low efficiency of the filter to trap [ 3 H]cGMP-bound Pabc. Fig. 4. Binding of [ 3 H]cGMP to Pabc. (A) Concentration of [ 3 H]cGMP. [ 3 H]cGMP binding to Pabc (1.92 lg) was measured with the indicated concentrations of [ 3 H]cGMP. The [ 3 H]cGMP-binding activity was analyzed by Scatchard plotting (insert). (B) Time-course. Pabc (17.3 lg) was incubated in 55 m M Tris ⁄ HCl, (pH 7.5) containing 4.4 mM EDTA and 1.1 mM IBMX (final volume, 720 lL) on ice for 10 min. The [ 3 H]cGMP binding was initiated by adding 80 lLof10l M [ 3 H]cGMP. After incubation for the indicated periods, an aliquot (80 lL) was taken and applied to a Millipore filter. (C) The cyclic nucleotide specificity. After incubation of Pabc (1.92 lg) with the indicated concentration of unlabeled cGMP ( • ) or cAMP (s) on ice for 10 min, [ 3 H]cGMP binding was measured with 1 lM [ 3 H]cGMP. The 100% activity indicates that 1.46 pmol [ 3 H]cGMP bound to Pabc in tubes. (D) Levels of [ 3 H]cGMP-bound Pabc trapped by the filter. OS homogenates (18.9 mg protein) were suspended in 9.7 mL of buffer A. After isolation by the TSK–DEAE 5PW column chromatography and concentration to 0.3 mL, the Pabc preparation ( 80 lg) was incubated with 1 l M [ 3 H]cGMP for 30 min on ice and applied to a TSK 250 column that had been equili- brated with buffer D. The level of [ 3 H]cGMP bound to Pabc was calculated based on the [ 3 H] radioactivity in 70 lL of the fraction ( • ). The fraction (70 lL) was also applied to a Millipore filter and the [ 3 H] radioactivity on the filter was measured (h). Only fractions containing Pabc are shown. (Insert) The rate of [ 3 H] radioactivity on the filter per the level of [ 3 H] radioactivity in the fraction. The 100% radioactivity indicates the [ 3 H] radioactivity detected in fraction 15. Fraction 15 (70 lL) contained 3.2 lgPabc (15.5 pmol) and 13.1 pmol of [ 3 H]cGMP. Roles of cGMP binding in PDE6 regulation A. Yamazaki et al. 1860 FEBS Journal 278 (2011) 1854–1872 ª 2011 The Authors Journal compilation ª 2011 FEBS GTPcS–Ta-activated and Pd-extracted cone PDE, Pa¢a¢c¢dd [20], expressed [ 3 H]cGMP-binding activity (Fig. 2C, upper). We note that all Pa¢a¢c¢c¢ complexes present were activated to Pa¢a¢c¢ under our conditions [20]. Interestingly, the level of [ 3 H]cGMP-binding activity in fraction 13 was approximatley five times higher than that of fraction 27 (Fig. 2C, upper). A similar observation was also obtained when these PDEs were extracted with a hypotonic buffer (data not shown). These results indicate that  85% of [ 3 H]cGMP-bound Pa¢a¢c¢ was trapped by the Millipore filter under the following assumptions: (a) the content of Pab and Pa¢a¢ in these fractions are similar, (b) [ 3 H]cGMP binds to all Pa¢a¢c¢ complexes, (c) Pa¢a¢c¢ has one cGMP binding site, (d) Pd binding does not affect the level of [ 3 H]cGMP-binding activity in Pa¢a¢c¢, and (e)  17% of [ 3 H]cGMP-bound Pabc is trapped by the Millipore filter. We note that the level of protein staining with Coomassie Brilliant Blue is proportional to the molcular mass calculated based on its amino acid sequence under our staining conditions [20]. Thus, amounts of Pab and Pa¢a¢ can be compared by comparing their staining levels in the same gel. We found that the stained level of Pab was similar to that of Pa¢a¢ (Fig. 2C, lower). This indicates that levels of Pab and Pa¢a¢ are similar, i.e. assumption (a) was pro- ven. As described, we found that the [ 3 H]cGMP-bind- ing activity of Pa¢a¢c¢dd was similar to that of Pa¢a¢c¢, i.e. assumption (d) was proven. Assumption (e) was also proven, as described above. Assumptions (b) and (c) are not yet proven; however, these assumptions are reasonable if characteristics of the [ 3 H]cGMP binding to Pabc are taken into consideration. Therefore, we conclude that the low trapping rate is specific to [ 3 H]cGMP-bound Pabc. Conformational change of Pabc by cGMP binding After incubation with [ 3 H]cGMP for 30 min (i.e. after binding of [ 3 H]cGMP to  100% of Pabc), dissocia- tion of [ 3 H]cGMP bound to Pabc was followed with or without 1 mm unlabeled cGMP (Fig. 5A). We found that the level of [ 3 H]cGMP binding to Pabc was not changed even in the presence of 1 mm unlabeled cGMP, at least for the first 5 min. Under similar con- ditions, [ 3 H]cGMP binding to Pabc reached a maxi- mum in < 2 min (Fig. 4B), indicating that the 5-min incubation was enough to chase [ 3 H]cGMP bound to Pabc, if indeed [ 3 H]cGMP could be chased. Therefore, this observation indicates that [ 3 H]cGMP, once bound Fig. 5. Change of Pabc’s characteristics by cGMP binding. (A) Dissociation of [ 3 H]cGMP bound to Pabc. Purified Pabc (16.0 lg) suspended in 640 lL of 55.5 m M Tris ⁄ HCl (pH 7.5) containing 4.44 mM EDTA and 1.11 mM IBMX, and [ 3 H]cGMP binding was initiated by adding 80 lL of 9 l M [ 3 H]cGMP. After incubation for 30 min on ice, an aliquot (72 lL) was withdrawn, applied to a Millipore filter, and its radioactivity was designated as the level at time 0. Simultaneously, 72 lLof10m M unlabeled cGMP ( • ) or water (s) was added to the assay mixture. After incubation for 0.25, 0.5, 0.75, 1, 2, 5, 10 and 20 min, an aliquot (80 lL) was withdrawn, applied to a Millipore filter, and its [ 3 H] radioactivity was measured. The arrow indicates the addition of cGMP or water. The 100% activity indicates that 1.32 pmol of [ 3 H]cGMP was detected in 1.6 lgofPabc (7.72 pmol). (B) Elution profile of Pabc from a gel-filtration column. Purified Pabc (70 lg) was incubated with (black) or without (red) unlabeled cGMP (0.5 m M) in 0.5 mL of 25 mM Tris ⁄ HCl, pH 7.5, 0.1 mM EDTA and 1 mM IBMX for 30 min on ice and applied to a Superdex 200 HR column that had been equilibrated with buffer E. Detailed conditions for this elution are in the Experimental proce- dures. PDE activity was assayed using 5 lL of the fraction ( • ). The 100% PDE activity indicates that 12.5 nmol cGMP was hydrolyzed per min per tube. [ 3 H]cGMP binding activity was measured using 50 lL of the fraction (h). The 100% activity indicates that 1.50 pmol of [ 3 H]cGMP was detected in the assay mixture. A. Yamazaki et al. Roles of cGMP binding in PDE6 regulation FEBS Journal 278 (2011) 1854–1872 ª 2011 The Authors Journal compilation ª 2011 FEBS 1861 to Pabc, cannot be dissociated. This strongly suggests that Pabc, after binding of [ 3 H]cGMP, changes its conformation, particularly that of its noncatalytic site and ⁄ or a region(s) near the noncatalytic site, and that Pabc, after changing its conformation, firmly holds the [ 3 H]cGMP. A large conformational change initiated by cGMP binding has been reported in one of GAF domains in cone PDE [24]. A similar conformational change may occur when [ 3 H]cGMP binds to Pabc, i.e. when Pabc having one cGMP is shifted to Pabc hav- ing two cGMPs. To further prove that binding of cGMP changes the conformation of Pabc , we directly compared the rela- tive compactness (Stokes’ radius) of cGMP-treated Pabc with that of cGMP-nontreated Pabc (Fig. 5B). This method has been used to show a conformational change by cGMP binding in PDE5 [25–27]. After incu- bation of Pabc with or without cGMP for 30 min on ice, these Pabcs were applied to a gel-filtration column and PDE activity was measured to identify the fraction containing Pabc. As expected, the cGMP-nontreated Pabc was eluted as a single peak with the peak activity in fraction 38. [ 3 H]cGMP-binding activity was also observed in these fractions. However, cGMP-treated Pabc was eluted as two peaks, the major peak in frac- tion 34 and the minor peak in fraction 38, and only Pabc in fraction 38 showed [ 3 H]cGMP-binding activ- ity. These observations indicate that the apparent Stokes’ radius of cGMP-treated Pabc was 4–7 A ˚ larger than that of cGMP-nontreated Pabc, i.e. the Stokes’ radius of Pabc appears to be increased when Pabc having one cGMP is shifted to Pabc having two cGMPs. We note that the difference in the Stokes’ radius was observed in Tris buffer; however, the differ- ence was less clear in a phosphate buffer (data not shown). This may be because of a tendency of Pabc to change its structure in Tris buffer [11]. We also note that 50 lL of the peak fraction of the cGMP-nontreat- ed Pabc contained 2.4 lgPabc (11.6 pmol of Pabc) and bound 9.90 pmol [ 3 H]cGMP. This indicates that  85% of the Pabc expressed [ 3 H]cGMP-binding activity, confirming that almost all Pabc complexes show [ 3 H]cGMP-binding activity (Fig. 4D). We also note that the major peak of the cGMP-treated Pabc showed no ability to bind [ 3 H]cGMP, confirming that cGMP, once bound to Pabc, is not dissociated (Fig. 5A). Rate of the conformational change in Pabc The level of [ 3 H]cGMP-binding increased abruptly after a 10-min incubation (Fig. 5A). The level was increased approximately three times the level at time 0 after 20 min (Fig. 5A) and approximately four times after 40 min (data not shown). Because  100% of Pabc present bound [ 3 H]cGMP during preincubation, these observations indicate that the amount of [ 3 H]cGMP-bound Pabc trapped by the filter increased abruptly during incubation. Incubation of [ 3 H]cGMP-bound Pabc was initiated by the addition of unlabeled cGMP or water (Fig. 5A). An increase in the trapped level of [ 3 H]cGMP-bound Pabc was observed after addition of 1 mm unlabeled cGMP, indicating that the increase is not due to new binding of [ 3 H]cGMP to Pabc. The increase was also detected after addition of water, implying that the unlabeled cGMP is not involved in this increase. Addi- tion of unlabeled cGMP or water slightly diluted the mixture, by  10%; however, it is unlikely that such a small dilution could cause this increase. Modification of the Pabc during incubation could also be ignored because the Pabc was pure (Fig. 3A) and the incu- bation was carried out on ice. Taken together, these observations deny the possibility that the increase is attributed to a reaction that occurred during incubation. During preincubation, [ 3 H]cGMP bound to Pabc. As another important change during preincubation, the buffer in the Pabc preparation, a phosphate buffer containing Mg 2+ , was changed to a Tris buffer con- taining 1-methyl-3-isobutylxanthine (IBMX), but not Mg 2+ .Pabc appears to have a tendency to change its structure in a Tris buffer, but not in a phosphate buf- fer [11], and Mg 2+ binds to Pab [28,29]. IBMX may also increase the cGMP affinity of noncatalytic sites, as discussed later. Therefore, these changes might affect the properties of Pabc and this change might increase the level of [ 3 H]cGMP-bound Pabc trapped by the filter. However, this increase was observed with either Pabc stored in the original buffer or in the preincubation buffer, a Tris buffer without Mg 2+ (data not shown). The increase was also detected with or without IBMX (data not shown). Therefore, these explanations may be disregarded. Modification of Pabc during preincubation could also be ignored, as described above. Taken together, these observations strongly suggest that [ 3 H]cGMP binding to Pabc dur- ing preincubation is the sole reason for the increase in the filter-trapping level of [ 3 H]cGMP-bound Pabc, i.e. the increase appears to be caused by a conformational change in Pabc upon binding of [ 3 H]cGMP. This increase in the filter-trapping level of [ 3 H]cGMP-bound Pabc was observed only after  10- min incubation, i.e.  40 min appeared to be required to detect the increase (Fig. 5A). Why is the increase detected after such a long incubation if it is due to Roles of cGMP binding in PDE6 regulation A. Yamazaki et al. 1862 FEBS Journal 278 (2011) 1854–1872 ª 2011 The Authors Journal compilation ª 2011 FEBS [ 3 H]cGMP binding? We believe that the conforma- tional change caused by [ 3 H]cGMP binding progresses consistently, but slowly, and that the increase is detected only after the Pabc with an altered conforma- tion accumulates to a certain level. In other words, there is a threshold to trap the [ 3 H]cGMP-bound Pabc. We emphasize that a mechanism to accelerate the conformational change should be present if this conformational change is indeed involved in PDE regulation. Suppression of cGMP binding during activation of Pabcc Two possible stages for cGMP binding to Pabc are expected in PDE regulation: during Pabcc activation to Pabc and during Pabc deactivation to Pabcc. First, we investigate whether cGMP binds to Pabc during the activation of Pabcc to Pabc. After incuba- tion of OS homogenates with GTPcS in the presence (Fig. 2B) or absence (Fig. 2A, lower) of cGMP, PDE species were extracted with buffer B and applied to a TSK–DEAE column, and their [ 3 H]cGMP-binding activities were measured. Both OS homogenates were incubated in the presence of IBMX and > 20% of added cGMP remained in the cGMP-added homoge- nate when membranes were isolated. We found that the [ 3 H]cGMP binding activity of cGMP-treated Pabc appeared to be slightly higher than that of cGMP- nontreated Pabc. However, the difference was not clear in another two studies. Therefore, we conclude that cGMP-incubated P abc has the ability to bind [ 3 H]cGMP similar to that seen in Pabc obtained with- out cGMP. The same result was obtained when Pabc was extracted with Pd in a isotonic buffer (data not shown). Pabc, once it binds cGMP, holds the cGMP and cannot accept [ 3 H]cGMP (Fig. 5B). Therefore, the [ 3 H]cGMP-binding activity we observed (Fig. 2B) indicates that Pabc cannot bind cGMP during activa- tion of Pabcc to Pabc. Pab ⁄ Pc, the minor GTPcS–Ta-activated PDE (Fig. 2), lost its [ 3 H]cGMP-binding activity when the fraction containing Pab ⁄ Pc was pretreated with cGMP (data not shown). However, Pab ⁄ Pc obtained from cGMP-treated OS homogenates showed a [ 3 H]cGMP- binding activity (Fig. 2B) similar to that of Pab ⁄ Pc obtained from cGMP-nontreated homogenates (Fig. 2A, lower). This suggests that binding of cGMP to Pab ⁄ Pc is suppressed during its formation. Together, our observations indicate that the cGMP- binding activity of GTP–Ta-activated PDE species is suppressed during its formation. This, we believe, is a critical finding to identify the function of cGMP bind- ing in PDE regulation. We note that the Pab ⁄ Pc was eluted slightly earlier when OS homogenates were incu- bated with cGMP, as previously shown [20]. The pres- ence of cGMP may be crucial for the early elution; however, the real reason is unknown. Binding of cGMP during deactivation of Pabc Next, we investigated whether cGMP binds to Pabc during deactivation of Pabc to Pabcc. Binding of cGMP may be involved in Pabc deactivation in two ways: after interaction with Pc and before interaction with Pc. First, we studied whether cGMP binds to Pabc after Pc binding to Pabc. We assayed [ 3 H]cGMP-binding activity of Pabc after incubation of Pabc with Pc or its mutants (Fig. 6). Here, these P abc complexes are termed PabcÆPc or PabcÆPc-mutant to emphasize that [ 3 H]cGMP-binding activity is assayed after formation of these complexes. PcÆGDP–Ta, instead of Pc, should be used, because PcÆGDP–Ta, but not free Pc, is the endogenous inhibitor of Pabc [13,20]. However, it is not known whether the Pc mutants we used form a complex with GDP–Ta. Therefore, free Pc was used in this study. [ 3 H]cGMP binding to Pabc (control) The level of [ 3 H]cGMP binding to Pabc reached a pla- teau in < 2 min and was not changed during the incu- bation period of at least 40 min (Figs 4B and 6B). After reaching the plateau,  100% of Pabc bound [ 3 H]cGMP in the mixture. However, the plateau indi- cates the level of [ 3 H]cGMP-bound Pabc trapped by the filter. In this case, the filter trapped  16% of the Pabc existing in the mixture. [ 3 H]cGMP binding to PabcÆPc The level of bound [ 3 H]cGMP was reduced when PabcÆPc was formed (Fig. 6A). A reason for the reduc- tion is that binding of [ 3 H]cGMP to PabcÆPc was slow and, even after 30 min incubation, did not reach the level that Pabc could reach in 2 min (Fig. 6B). The K d for cGMP in Pabc ⁄ Pc is  0.33 lm (Fig. 6C), indicat- ing that the binding of Pc to Pabc reduces its affinity for cGMP by  6.5 times. This reduction may be a rea- son for the slow binding of [ 3 H]cGMP to PabcÆPc. She efficiency of a Millipore filter for trapping [ 3 H]cGMP- bound Pabc is increased when Pc binds to [ 3 H]cGMP- bound Pabc, as shown below (Fig. 7A). Therefore, the reduction in the level of [ 3 H]cGMP binding to PabcÆPc is not due to a reduction in the Millipore filter’s ability to trap the [ 3 H]cGMP-bound PabcÆPc. A. Yamazaki et al. Roles of cGMP binding in PDE6 regulation FEBS Journal 278 (2011) 1854–1872 ª 2011 The Authors Journal compilation ª 2011 FEBS 1863 [...]... zinc binding sites in retinal rod cGMP phosphodiesterase, PDE6alpha beta J Biol Chem 275, 20572–20577 Yamazaki M, Li N, Bondarenko VA, Yamazaki RK, Baehr W & Yamazaki A (2002) Binding of cGMP to GAF domains in amphibian rod photoreceptor cGMP phosphodiesterase (PDE) Identification of GAF domains in PDE alphabeta subunits and distinct domains in the PDE gamma subunit involved in stimulation of cGMP binding. .. [3H ]cGMP to Pabc implies that cGMP binding may be involved in the activation of Pabcc to Pabc and ⁄ or in the deactivation of Pabc to Pabcc Here, we show that the ability of Pabc to bind cGMP is suppressed during the formation of Pabc by GTPcS–Ta in OS homogenates (Fig 2) This indicates that cGMP binding is not involved in the activation of Pabcc to Pabc It is not known now how this property of Pabc... this Pabc’s conformational change is slow (Fig 5A) ; however, binding of Pc to Pabc having two cGMPs accelerates its conformational change (Fig 7) Overall, these findings indicate that cGMP binds to Pabc first and then Pc binds to the Pabc in Pabc deactivation (Fig 8) In this scheme, the residual [cGMP] is crucial for the rate of Pabc deactivation The residual [cGMP] in OS is dependent upon the level of. .. of cGMP binding in PDE6 regulation A Yamazaki et al result strongly suggests that binding of Pc accelerates cGMP- dependent conformational change in Pabc The rapid increase by Pc in the level of [3H]cGMPbound Pabc trapped by the filter (Fig 7A) might be due to a change by Pc in the surface of [3H]cGMPbound Pabc and ⁄ or in the total charge of Pabc In such cases, a slow increase (Fig 5A) could also be... that Pabc rapidly changes its conformation during deactivation and that binding of cGMP and Pc play crucial roles in this change (Fig 8) These findings also imply that Pabcc rapidly changes its conformation during its activation and that the release of Pc and cGMP play important roles in this change (Fig 8) To the best of our knowledge, this is the first model in which the role of noncatalytic binding. .. Yamazaki A, Yu H, Yamazaki M, Honkawa H, Matsuura I, Usukura J & Yamazaki RK (2003) A critical role for ATP in the stimulation of retinal guanylyl cyclase by guanylyl cyclase-activating proteins J Biol Chem 278, 33150–33160 Yamazaki A, Yamazaki M, Yamazaki RK & Usukura J (2006) Illuminated rhodopsin is required for strong activation of retinal guanylate cyclase by guanylate cyclase-activating proteins...Roles of cGMP binding in PDE6 regulation A Yamazaki et al Fig 6 Effects of Pc and its mutants on [3H ]cGMP binding to Pabc (A) Effect on the level of [3H ]cGMP binding After incubation of Pabc (1.92 lg) with various concentrations of Pc or its mutants, the [3H ]cGMP -binding activity was measured The 100% activity indicates that 1.46 pmol [3H ]cGMP bound to Pabc in tubes Following Pc and its mutants were... hydrolysis of [3H ]cGMP We found that both the amount of [3H ]cGMP bound to Pabc and the affinity of Pabc for [3H ]cGMP were not notably changed in the presence or absence of 5 mm MgCl2 (data not shown) Thus, we believe that the lack of Mg2+ in the assay mixture does not affect the affinity of Pabc for cGMP Roles of cGMP binding in PDE6 regulation Binding of cGMP to Pabc is not involved PDE activation Binding of. .. between amino acids 19–22 in the Pc sequence may be crucial for the acceleration In conclusion, these results strongly suggest that Pc binding accelerates the cGMP -binding- initiated conformational change in Pabc Together with the result showing that Pc inhibits cGMP -binding to Pabc (Fig 6), these results imply that the scheme by which cGMP binds to Pabc first and Pc then binds to the Pabc is appropriate... the inactive form, and Pabc, the GTP–Ta-activated form, contain two and one cGMP, respectively, and that only Pabc shows [3H ]cGMP binding We also show that the ability of Pabc to bind cGMP is suppressed during formation of Pabc We also strongly suggest that cGMP binding slowly changes the conformation of Pabc and that Pc binding accelerates this change These findings are consistent with the view that . Binding of cGMP to the transducin-activated cGMP phosphodiesterase, PDE6, initiates a large conformational change involved in its deactivation Akio Yamazaki 1,2,3 ,. investigated whether cGMP binds to Pabc during deactivation of Pabc to Pabcc. Binding of cGMP may be involved in Pabc deactivation in two ways: after interaction