Binding of cGMP to Pabc is suppressed during its formation, implying that cGMP binding is not involved in Pabcc activation.. Once bound to Pabc, [3H]cGMP is not dissociated even in the p
Trang 1phosphodiesterase, PDE6, initiates a large conformational change involved in its deactivation
Akio Yamazaki1,2,3, Fumio Hayashi4, Isao Matsuura5and Vladimir A Bondarenko6
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 [3H]cGMP binding with a Kd 50 nMand Pc inhibits the [3H]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, [3H]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 [3H]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 [3H]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.
Trang 2Cyclic 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 deactiacti-vation 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
Trang 3changed 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
[3H]cGMP-binding activity and that Pc inhibits
[3H]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 [3H]cGMP to OS membranes
Bovine OS membranes contain a [3H]cGMP-binding
site(s) (Fig 1A) Both GTPcS-treated and nontreated
membranes showed [3H]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
[3H]cGMP-binding activity By contrast, the soluble
fraction, whether obtained from GTPcS-treated or
nontreated OS homogenates, showed only negligible
[3H]cGMP-binding activity (data not shown) This
sug-gests that no protein in the soluble fraction contains the
[3H]cGMP-binding site and⁄ or expresses [3
H]cGMP-binding activity under our experimental conditions
Solubilization and isolation of membranous proteins showed that a [3H]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 propro-tein-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 [3H]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 [3H]cGMP (B,C) [3H]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.
Trang 4binding activity However, neither Pa¢ nor its
[3H]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 [3H]cGMP-binding activity (data not
shown) This is consistent with the above-mentioned
conclusion that [3H]cGMP-binding activity was not
detected in the soluble fraction
Interestingly, the [3H]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 [3H]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 [3H]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
[3H]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 [3H]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 bindstudy-ing to solubilized PDE species
Identification of PDE species expressing
[3H]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 [3H]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
[3H]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 memmem-branes (Fig 2A, lower) In the former preparation, the [3H]cGMP-binding activity appeared to be proportional
to the level of Pab, implying that Pabcc may express [3H]cGMP-binding activity However, the molecular ratio of [3H]cGMP to Pab was < 0.01, indicating that only a negligible portion of the Pabcc expresses this activity In the latter preparation, a small [3H] radio-activity was detected in the fraction close to the Pabcc peak However, the level of [3H] radioactivity was not proportional to that of Pab in the Pabcc fraction, indi-cating that the [3H] radioactivity is not attributable to [3H]cGMP bound to the Pabcc, i.e the Pabcc does not show [3H]cGMP-binding activity and⁄ or the Pabcc, when it exists with GTP–Ta, appears to lose a portion that may express [3H]cGMP-binding activity (Fig 2A, upper)
Pabcc extracted with Pd in an isotonic buffer The Pabccd preparation was obtained from nontreated membranes (data not shown) and GTPcS-treated membranes (Fig 2C) In the former prepara-tion, the [3H]cGMP-binding activity appeared to be proportional to the level of Pab; however, the molecu-lar ratio of [3H]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 [3H]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 [3H] radioactivity was not due to [3H]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 [3H]cGMP-binding activity and⁄ or the negligi-ble level of [3H]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 [3H] 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
Trang 5Pabc 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
[3H]cGMP-binding activities In addition, the level of
Pab was proportional to that of [3H]cGMP-binding
activity in these fractions These results indicate that
both Pabc and Pab⁄ Pc express [3H]cGMP-binding
activity
We emphasize that [3H]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 [3H]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 [3H]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 l M [ 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].
Trang 6Pd is identical to that of PDE species without Pd when
the same NaCl gradient was used [20] Comparison of
the [3H]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
[3H]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 [3H]cGMP during the assay of [3H]cGMP binding is quite low, as discussed later Therefore, we conclude that the [3H]cGMP-binding activity in Pabc we observed is due to the binding of [3H]cGMP to the empty site, i.e [3H]cGMP-bound Pabc contains one original cGMP and one [3H]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 Pabcc 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 [3H]cGMP binding to Pabc Purified Pabc showed a [3H]cGMP-binding activity (Fig 4A) The level of [3H]cGMP binding reached a plateau as the [3H]cGMP concentration increased Scatchard plotting of this saturable [3H]cGMP binding (Fig 4A, insert) indicates that Pabc has one type of cGMP-binding site with Kd 50 nm This is consistent with the above-mentioned view that [3H]cGMP binds
to the same site in Pabc The level of bound [3H]cGMP reached a plateau in < 2 min under these conditions (Fig 4B) Unlabeled cGMP, but not cAMP, competitively inhibited [3H]cGMP binding (Fig 4C) This indicates that the [3H]cGMP-binding site in Pabc is cGMP-specific
Trapping of [3H]cGMP-bound Pabc to a Millipore filter
After incubation with [3H]cGMP, Pabc was applied to a molecular sieve column and the amount of [3H]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.
Trang 7bound to Pabc was calculated based on the [3H]
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 [3H]cGMP, i.e
83% of Pabc in the fraction was occupied by
[3H]cGMP The average level of the occupation was
86% in three experiments These results indicate that
100% of Pabc binds [3H]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
[3H]cGMP-binding activity was obtained based on the
[3H] radioactivity trapped by the filter (Fig 4D) This
shows that the Millipore filter traps 17% of the
[3H]cGMP-bound Pabc existing in the assay mixture
We could not get a result showing that 100% of Pabc
expressed [3H]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 [3H]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 [3H]cGMP This is due to our intention to show the ratio of [3H]cGMP-binding activ-ity measured by the filter It should be noted that
18.2% of the [3H]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 propprop-erties are not affected by the low efficiency of the filter to trap [3H]cGMP-bound Pabc
Fig 4 Binding of [3H]cGMP to Pabc (A) Concentration of [3H]cGMP [3H]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 m M EDTA and 1.1 m M IBMX (final volume, 720 lL) on ice for 10 min The [ 3 H]cGMP binding was initiated by adding 80 lL of 10 l M [3H]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 l M [ 3 H]cGMP The 100% activity indicates that 1.46 pmol [3H]cGMP bound to Pabc in tubes (D) Levels of [3H]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 [3H]cGMP bound to Pabc was calculated based on the [3H] 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 lg Pabc (15.5 pmol) and 13.1 pmol of [ 3 H]cGMP.
Trang 8GTPcS–Ta-activated and Pd-extracted cone PDE,
Pa¢a¢c¢dd [20], expressed [3H]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 [3H]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
[3H]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)
[3H]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 [3H]cGMP-binding activity in
Pa¢a¢c¢, and (e) 17% of [3H]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 [3H]cGMP binding
to Pabc are taken into consideration Therefore, we conclude that the low trapping rate is specific to [3H]cGMP-bound Pabc
Conformational change of Pabc by cGMP binding After incubation with [3H]cGMP for 30 min (i.e after binding of [3H]cGMP to 100% of Pabc), dissocia-tion of [3H]cGMP bound to Pabc was followed with
or without 1 mm unlabeled cGMP (Fig 5A) We found that the level of [3H]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, [3H]cGMP binding to Pabc reached a maxi-mum in < 2 min (Fig 4B), indicating that the 5-min incubation was enough to chase [3H]cGMP bound to Pabc, if indeed [3H]cGMP could be chased Therefore, this observation indicates that [3H]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 m M EDTA and 1.11 m M IBMX, and [ 3 H]cGMP binding was initiated by adding 80 lL
of 9 l M [3H]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 lL of 10 m 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 [3H]cGMP was detected
in 1.6 lg of Pabc (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 m M Tris ⁄ HCl, pH 7.5, 0.1 m M EDTA and 1 m M 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.
Trang 9to Pabc, cannot be dissociated This strongly suggests
that Pabc, after binding of [3H]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
[3H]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 [3H]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 [3H]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 fracfrac-tion 38, and only
Pabc in fraction 38 showed [3H]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 containcGMP-nontreat-ed 2.4 lg Pabc (11.6 pmol of Pabc)
and bound 9.90 pmol [3H]cGMP This indicates that
85% of the Pabc expressed [3H]cGMP-binding
activity, confirming that almost all Pabc complexes
show [3H]cGMP-binding activity (Fig 4D) We also
note that the major peak of the cGMP-treated Pabc
showed no ability to bind [3H]cGMP, confirming that
cGMP, once bound to Pabc, is not dissociated
(Fig 5A)
Rate of the conformational change in Pabc
The level of [3H]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 [3H]cGMP during preincubation, these observations indicate that the amount of [3H]cGMP-bound Pabc trapped by the filter increased abruptly during incubation
Incubation of [3H]cGMP-bound Pabc was initiated
by the addition of unlabeled cGMP or water (Fig 5A)
An increase in the trapped level of [3H]cGMP-bound Pabc was observed after addition of 1 mm unlabeled cGMP, indicating that the increase is not due to new binding of [3H]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, [3H]cGMP bound to Pabc
As another important change during preincubation, the buffer in the Pabc preparation, a phosphate buffer containing Mg2+, was changed to a Tris buffer con-taining 1-methyl-3-isobutylxanthine (IBMX), but not
Mg2+ Pabc appears to have a tendency to change its structure in a Tris buffer, but not in a phosphate buf-fer [11], and Mg2+ 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 [3H]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 Mg2+ (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 [3H]cGMP binding to Pabc dur-ing preincubation is the sole reason for the increase in the filter-trapping level of [3H]cGMP-bound Pabc, i.e the increase appears to be caused by a conformational change in Pabc upon binding of [3H]cGMP
This increase in the filter-trapping level of [3H]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
Trang 10[3H]cGMP binding? We believe that the
conforma-tional change caused by [3H]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 [3H]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 [3H]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 [3H]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 Pabc has the ability to bind
[3H]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 [3H]cGMP (Fig 5B) Therefore,
the [3H]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 [3H]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 [3H]cGMP-binding activity of Pabc after incubation of Pabc with Pc or its mutants (Fig 6) Here, these Pabc complexes are termed PabcÆPc or PabcÆPc-mutant to emphasize that [3H]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
[3H]cGMP binding to Pabc (control) The level of [3H]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 [3H]cGMP in the mixture However, the plateau indi-cates the level of [3H]cGMP-bound Pabc trapped by the filter In this case, the filter trapped 16% of the Pabc existing in the mixture
[3H]cGMP binding to PabcÆPc The level of bound [3H]cGMP was reduced when PabcÆPc was formed (Fig 6A) A reason for the reduc-tion is that binding of [3H]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 Kd for cGMP in Pabc⁄ Pc is 0.33 lm (Fig 6C), indicat-ing that the bindindicat-ing 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 [3H]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 [3H]cGMP binding to PabcÆPc
is not due to a reduction in the Millipore filter’s ability
to trap the [3H]cGMP-bound PabcÆPc