From the Department of Pharmacology and Toxicology, the University of Kansas, Lawrence, Kansas 66045-7582
Received for publication, November 5, 2002, and in revised form, January 21, 2003
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ABSTRACT |
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2'(3')-O-(N-Methylanthraniloyl)-(MANT)-substituted
nucleotides are fluorescent and widely used for the kinetic
analysis of enzymes and signaling proteins. We studied the effects of
MANT-guanosine 5'-[ G-proteins are heterotrimeric (-thio]triphosphate (MANT-GTP
S) and
MANT-guanosine 5'-[
,
-imido]triphosphate (MANT-GppNHp) on
G
s- and G
i-protein-mediated signaling.
MANT-GTP
S/MANT-GppNHp had lower affinities for G
s and
G
i than GTP
S/GppNHp as assessed by inhibition of GTP
hydrolysis of receptor-G
fusion proteins. MANT-GTP
S was much less
effective than GTP
S at disrupting the ternary complex between the
formyl peptide receptor and G
i2. MANT-GTP
S/MANT-GppNHp non-competitively inhibited GTP
S/GppNHp-, AlF
2-adrenoceptor plus GTP-,
cholera toxin plus GTP-, and forskolin-stimulated adenylyl cyclase (AC) in G
s-expressing Sf9 insect cell membranes and
S49 wild-type lymphoma cell membranes. AC inhibition by
MANT-GTP
S/MANT-GppNHp was not due to G
s inhibition
because it was also observed in G
s-deficient S49
cyc
lymphoma cell membranes. Mn2+
blocked AC inhibition by GTP
S/GppNHp in S49
cyc
membranes but enhanced the potency of
MANT-GTP
S/MANT-GppNHp at inhibiting AC by ~4-8-fold. MANT-GTP
S
and MANT-GppNHp competitively inhibited
forskolin/Mn2+-stimulated AC in S49
cyc
membranes with Ki
values of 53 and 160 nM, respectively. The
Ki value for MANT-GppNHp at insect cell AC was 155 nM. Collectively, MANT-GTP
S/MANT-GppNHp bind to
G
s- and G
i-proteins with low affinity and
are ineffective at activating G
. Instead, MANT-GTP
S/MANT-GppNHp
constitute a novel class of potent competitive AC inhibitors.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-structure) and
serve as signal transducers between agonist-occupied
GPCRs1 and effector systems
(1, 2). GPCR promotes GDP dissociation from G
. GDP dissociation is
the rate-limiting step of the G-protein cycle. Agonist-occupied GPCR
then forms a ternary complex with guanine nucleotide-free G-protein.
Thereafter, GPCR catalyzes GTP binding to G
. G
GTP
dissociates from GPCR, thereby disrupting the ternary complex. In
addition, G
GTP and
dissociate from each other,
and both G
GTP and
regulate the activity of
effector systems. G-proteins are deactivated by the GTPase of G
that
cleaves GTP into GDP and Pi. The GTP hydrolysis-resistant
GTP analogs GTP
S and GppNHp (Fig. 1)
induce persistent G-protein activation as does AlF
S (Fig. 1) is a partial G-protein activator (5,
6).
View larger version (30K):
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Fig. 1.
Structures of GTP S,
GppNHp, MANT-GTP
S, MANT-GppNHp, and
GDP
S. The
-thiophosphate group in
GTP
S/MANT-GTP
S, the
-thiophosphate group in GDP
S, and the
,
-imido group in GppNHp/MANT-GppNHp render the nucleotides
hydrolysis-resistant (1-3, 5). Introduction of a MANT group at the
2'(3')-O-position of the ribosyl residue confers fluorescent
properties to the nucleotides (7). There is spontaneous isomerization
of the MANT group between the 2'- and 3'-O-position of the
ribosyl residue in MANT-GTP
S and MANT-GppNHp (but not in
2'-deoxy-3'-MANT-GppNHp).
Nucleotides substituted with a MANT group at the
2'(3')-O-position of the ribosyl residue are fluorescent and
widely used for the kinetic analysis of enzymes and signaling proteins
(7). However, only few studies with MANT-nucleotides and G-proteins have been conducted so far, and the data are controversial.
MANT-GTPS and MANT-GppNHp (Fig. 1) bind to purified
Go-proteins with higher affinity than to
Gi-proteins, and the MANT group does not have an effect on
the affinity of GTP
S for purified G
i1 (8, 9). The
maximum fluorescence of Gi/Go-proteins induced
by MANT-GTP
S is higher than the maximum fluorescence induced by
MANT-GppNHp, suggesting that the two nucleotides stabilize different
conformations in G-proteins (8, 10). Moreover, like GTP
S/GppNHp,
MANT-GTP
S/MANT-GppNHp confer protease protection to
Gi/Go-proteins (8). In contrast to the
observations made with Gi/Go-proteins, the MANT
group substantially reduces the affinity of GTP for the retinal
G-protein transducin, and MANT-GTP is ineffective at activating the
effector of transducin, cGMP-degrading phosphodiesterase (11). To the
best of our knowledge, the effects of MANT-nucleotides on
Gs-proteins have not yet been studied.
The goal of our study was to learn more about the functional effects of
MANT-GTPS/MANT-GppNHp on Gs- and
Gi-protein-mediated signaling. As models we used fusion
proteins and co-expression systems of the
2AR with the
G
s-proteins, G
sL, G
sS, or
G
olf (12-14), fusion proteins, and co-expression
systems of the FPR with the G
i-proteins,
G
i1, G
i2, or G
i3 (15, 16),
and individually expressed G
sS. As physiologically
relevant systems, we studied S49 wt lymphoma cell membranes, a standard
model for the analysis of G
s-proteins (17) and S49
cyc
cell membranes, a
G
s-deficient S49 mutant cell line serving as model for
the analysis of Gi-proteins (6, 18). G
s
activates, and G
i inhibits, the effector AC (19, 20).
Surprisingly, we found that MANT-GTP
S/MANT-GppNHp constitute a novel
class of potent competitive AC inhibitors.
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EXPERIMENTAL PROCEDURES |
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Materials--
Initially, MANT-GTPS, MANT-GppNHp, and
MANT-GTP were provided by Drs. R. Sportsman and M. Helms (LJL
Biosystems Inc., Sunnyvale, CA) who obtained the compounds as custom
synthesis products from Marker Gene Technologies (Eugene, OR). Later,
MANT-GppNHp and MANT-GTP were purchased from Molecular Probes (Eugene,
OR). In the last phase of the project, MANT-GTP
S and MANT-GppNHp
were obtained from Jena Bioscience (Jena, Germany).
2'-Deoxy-3'-MANT-GppNHp was also from Jena Bioscience. Nucleotides
obtained from various batches of the different suppliers gave very
consistent results. Stock solutions of MANT-nucleotides (0.5-1
mM) were stored at
20 °C for periods up to 2 years
(longer times were not studied) without loss of potency and efficacy.
FMLP, (
)-isoproterenol, salbutamol, NaF, AlCl3,
MnCl2, forskolin, and cholera toxin were from Sigma. GTP,
GTP
S, GppNHp, GDP
S, ATP (special quality <0.01% (w/w) GTP as
assessed by high performance liquid chromatography), and AMPPNP were
obtained from Roche Molecular Biochemicals. Recombinant baculoviruses
encoding G
sS and G
i2 were kindly provided
by Drs. A. G. Gilman and R. Sunahara (Department of Pharmacology,
University of Texas Southwestern Medical Center, Dallas). Recombinant
baculovirus encoding the
1
2 complex was
donated Dr. P. Gierschik (Department of Pharmacology and Toxicology,
University of Ulm, Germany). S49 wt and S49
cyc
lymphoma cells were obtained from the Cell
Culture Facility of the University of California, San Francisco. The
construction of baculoviruses encoding
2AR-G
s- and FPR-G
i fusion
proteins,
2AR, and FPR have been described elsewhere
(12, 13, 15, 16, 21). [3H]Dihydroalprenolol (85-90
Ci/mmol), [3H]FMLP (56 Ci/mmol),
[
-32P]ATP (3,000 Ci/mmol), and
[
-32P]GTP (6,000 Ci/mmol) were obtained from
PerkinElmer Life Sciences. All other reagents were of the highest
purity available and obtained from Sigma or Fisher.
Cell Culture and Membrane Preparation--
Sf9 cells were
cultured and infected with 1:100 dilutions of high titer virus stocks
as described (22). Sf9 membranes were prepared as described (12)
and stored at 80 °C until use. S49 wt and S49
cyc
cells were cultured under the conditions
described recently (23). S49 wt cells were treated with cholera toxin
(1 µg/ml) for 24 h before membrane preparation. S49 membranes
were prepared as Sf9 membranes except that S49 cells were
disintegrated by nitrogen cavitation at 4 °C and 7000 kPa for 30 min
using a nitrogen cavitation chamber (Parr Instruments, Moline, IL) in a
buffer consisting of 50 mM KH2PO4,
100 mM NaCl, and 0.5 mM EDTA, pH 7.0.
[3H]Dihydroalprenolol and [3H]FMLP
Binding Assays--
Membranes were thawed and sedimented by a 15-min
centrifugation at 4 °C and 15,000 × g to remove
residual endogenous guanine nucleotides as far as possible and were
resuspended in binding buffer (12.5 mM MgCl2, 1 mM EDTA, and 75 mM Tris/HCl, pH 7.4). Expression levels of 2AR-G
s fusion
proteins and
2AR in Sf9 membranes (10-30 µg of
protein/tube) were determined in the presence of 10 nM
[3H]dihydroalprenolol. Nonspecific binding was determined
in the presence of 10 µM (±)-alprenolol. Expression
levels of FPR-G
i fusion proteins and FPR in Sf9
membranes (30-50 µg of protein/tube) were determined in the presence
of 30 nM [3H]FMLP. Nonspecific binding was
determined in the presence of 10 µM FMLP. The total
volume of the binding reactions was 500 µl. Incubations were
performed for 90 min at 25 °C and shaking at 250 rpm. Bound
radioactivity was separated from free radioactivity by rapid filtration
through GF/C filters, followed by three washes with 2 ml of binding
buffer (4 °C). Filter-bound radioactivity was determined by liquid
scintillation counting. The experimental conditions chosen ensured that
not more than 10% of the total amount of radioactivity added to
binding tubes was bound to filters. For studying the effects of
nucleotides on ternary complex formation, reaction mixtures contained
Sf9 membranes expressing
2AR-G
sS (20-25 µg of protein/tube), 1 nM
[3H]dihydroalprenolol, 1 µM salbutamol, and
guanine nucleotides at increasing concentrations. Alternatively,
reaction mixtures contained Sf9 membranes expressing FPR + G
i2 +
1
2 (30-50 µg of
protein/tube), 10 nM [3H]FMLP, and guanine
nucleotides at increasing concentrations.
Steady-state GTPase Assay--
Membranes were thawed and
sedimented by a 15-min centrifugation at 4 °C and 15,000 × g to remove residual endogenous guanine nucleotides as far
as possible and resuspended in 10 mM Tris/HCl, pH 7.4. GTP
hydrolysis was determined as described (14). Assay tubes contained
Sf9 membranes expressing fusion proteins (10 µg of
protein/tube), 1.0 mM MgCl2, 0.1 mM
EDTA, 100 nM to 1.5 µM unlabeled GTP, 0.1 mM ATP, 1 mM AppNHp, 5 mM creatine
phosphate, 40 µg of creatine kinase, and 0.2% (w/v) bovine serum
albumin in 50 mM Tris/HCl, pH 7.4. Tubes additionally
contained various guanine nucleotides at increasing concentrations and
10 µM ()-isoproterenol (
2AR-G
s fusion proteins) or 10 µM FMLP (FPR-G
i fusion proteins). Reaction
mixtures (80 µl) were incubated for 3 min at 25 °C before the
addition of 20 µl of [
-32P]GTP (0.2-0.5
µCi/tube). All stock and work dilutions of [
-32P]GTP
were prepared in 20 mM Tris/HCl, pH 7.4, because
[
-32P]GTP solutions prepared in distilled water were
unstable. Reactions were conducted for 20 min at 25 °C. Reactions
were terminated by the addition of 900 µl of slurry consisting of 5%
(w/v) activated charcoal and 50 mM
NaH2PO4, pH 2.0. Charcoal-quenched reaction mixtures were centrifuged for 15 min at room temperature at 15,000 × g. Seven hundred µl of the supernatant fluid of
reaction mixtures were removed, and 32Pi was
determined by liquid scintillation counting. Non-enzymatic [
-32P]GTP degradation was determined in the presence
of 1 mM unlabeled GTP and was <1% of the total amount of
radioactivity added.
AC Assay--
Membranes were thawed and sedimented by a 15-min
centrifugation at 4 °C and 15,000 × g to remove
residual endogenous guanine nucleotides as far as possible and
resuspended in binding buffer (12.5 mM MgCl2, 1 mM EDTA and 75 mM Tris/HCl, pH 7.4). AC assays were performed as described (14). Briefly, tubes contained various membranes (15-50 µg of protein/tube), 5 mM
MgCl2, 0.4 mM EDTA, 30 mM Tris/HCl,
pH 7.4, and guanine nucleotides at various concentrations without or
with ()-isoproterenol. In some experiments, reaction mixtures
contained NaF at increasing concentrations plus 10 µM AlCl3, forskolin at increasing concentrations, and
MnCl2 (10 mM). Assay tubes containing membranes
and various additions in a total volume of 30 µl were incubated for 3 min at 37 °C before starting reactions by adding 20 µl of reaction
mixture containing (final) [
-32P]ATP (1.0-1.5
µCi/tube) plus 40 µM unlabeled ATP, 2.7 mM
mono(cyclohexyl)ammonium phosphoenolpyruvate, 0.125 IU of pyruvate
kinase, 1 IU of myokinase, and 0.1 mM cAMP. For
determination of the Km value of AC for ATP,
reaction mixtures contained 10 µM to 1 mM
unlabeled ATP/Mn2+ plus 10 mM
MnCl2. Reactions were conducted for 20 min at 37 °C. Reactions were terminated by the addition of 20 µl of 2.2 N HCl. Denatured protein was sedimented by a 1-min
centrifugation at 25 °C and 15,000 × g. Sixty five
µl of the supernatant fluid were applied onto disposable columns
filled with 1.3 g of neutral alumina (Sigma A-1522, super I,
WN-6). [32P]cAMP was separated from
[
-32P]ATP by elution of [32P]cAMP with 4 ml of 0.1 M ammonium acetate, pH 7.0 (24). Recovery of
[32P]cAMP was ~80%. Blank values were routinely
~0.01% of the total amount of [
-32P]ATP added. The
extremely low blank values allowed for the precise determination of
even very low AC activities (such as those observed in Sf9
membranes expressing G
sS in the presence of
MANT-GTP
S/MANT-GppNHp) or high isotopic dilution of
[
-32P]ATP (such as those in the presence of 1 mM unlabeled ATP). [32P]cAMP was determined
by liquid scintillation counting.
Miscellaneous--
Protein was determined using the Bio-Rad DC
protein assay kit (Bio-Rad). Data were analyzed using the Prism 3.02 software (GraphPad, San Diego, CA).
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RESULTS |
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MANT-GTPS/MANT-GppNHp Bind to G
s- and
G
i-proteins with Low Affinity--
We determined the
affinities of GTP
S and GppNHp and their MANT-derivatives for
G
s- and G
i-proteins by measuring
agonist-stimulated steady-state GTP hydrolysis of
2AR-G
s and FPR-G
i fusion
proteins expressed in Sf9 insect cell membranes (13, 14, 16,
23). The affinity profiles of nucleotides were similar for
G
s-proteins (G
sL, G
sS, and
G
olf) (Fig. 2,
A-C) and G
i-proteins (G
i1, G
i2, and G
i3) (Fig. 2, D-F).
GTP
S inhibited GTP hydrolysis with Ki values
ranging from 3.6 (
2AR-G
olf) to 8.9 nM (FPR-G
i3). As expected (1, 2), GppNHp
inhibited GTP hydrolysis with considerably lower potencies
(~20-140-fold) than GTP
S. The Ki values for
GppNHp ranged from 170 (FPR-G
i2) to 520 nM
(
2AR-G
olf). The introduction of the MANT
group at the 2'(3')-O-position of the ribosyl group reduced
the affinity of GTP
S for G
s- and G
i-proteins by ~30-300-fold, i.e. the
Ki values for MANT-GTP
S ranged from 250 (FPR-G
i2) to 1100 nM
(
2AR-G
olf). The affinities of MANT-GppNHp
for G
s- and G
i-proteins were 4.5-11-fold lower than those of GppNHp, i.e. the Ki
values for MANT-GppNHp ranged from 1.2 (FPR-G
i2) to 5.8 µM (
2AR-G
olf).
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We also analyzed GTPases with GTP at increasing concentrations in the
presence of MANT-GTPS at various fixed concentrations and plotted
the data double-reciprocally according to Lineweaver-Burk (Fig.
3). Based on GTPase competition studies
with various G-proteins and nucleotides (25, 26), we expected that the
linear regression lines intersect in the y axis, reflecting
competitive interaction of GTP with MANT-GTP
S. In fact, MANT-GTP
S
exhibited competitive interaction with GTP at G
s (Fig.
3A) and G
i (Fig. 3B).
Collectively, our data show that MANT-GTP
S and MANT-GppNHp bind to
G
s- and G
i-proteins, although with low
affinity.
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Effects of MANT-GTPS on Ternary Complex Formation--
In case
of the
2AR/G
s couple, ternary complex
formation is assessed indirectly by measuring binding of radioligand
antagonist in the presence of unlabeled agonist. Binding assay mixtures
contained [3H]dihydroalprenolol and salbutamol at fixed
concentrations (see "Experimental Procedures"). Guanine nucleotides
reduce agonist affinity of the
2AR and, thereby,
increase [3H]dihydroalprenolol binding (13,
14, 27). As reported before (14), GTP
S potently (IC50,
0.7 nM; CI (CI, 95% confidence interval), 0.2-2.1
nM) and efficaciously disrupted the ternary complex in membranes expressing
2AR-G
sS (Fig.
4A). MANT-GTP
S (1 µM) reduced ternary complex formation in
2AR-G
sS by ~50%, but at higher concentrations, the effect of MANT-GTP
S was reverted. MANT-GTP
S at 10 µM apparently increased ternary complex formation
by 40% above control. However, because ternary complex formation was assessed indirectly through radioligand antagonist binding, we had to
exclude the possibility that MANT-GTP
S inhibited
[3H]dihydroalprenolol binding to the
2AR.
We examined the effect of MANT-GTP
S (10 µM) on binding
of [3H]dihydroalprenolol in Sf9 membranes
expressing the
2AR alone, i.e. a system in
which ternary complex formation is not detected (12). In fact,
MANT-GTP
S (10 µM) inhibited binding of
[3H]dihydroalprenolol (1 nM) by 40%. Thus,
because of interference of MANT-GTP
S with ligand binding to the
2AR, we could not answer the question whether GTP
S
and MANT-GTP
S exhibit similar efficacies at disrupting the ternary
complex in
2AR-G
sS.
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In case of the FPR/Gi2 couple, ternary complex
disruption is measured directly by guanine nucleotide-induced reduction
of high affinity agonist ([3H]FMLP) binding (15, 28). As
reported before (15, 28), GTP
S potently (IC50, 26 nM; CI, 15-43 nM) and efficaciously disrupted the ternary complex of the FPR/G
i2 couple (Fig.
4B). Considering the Ki values of
MANT-GTP
S for G
i-proteins (250-500 nM)
(Fig. 2, D-F), we would have expected maximal disruption of the ternary complex with MANT-GTP
S at 10 µM. However,
MANT-GTP
S (10 µM) decreased [3H]FMLP
binding by not more than 25%. Thus, MANT-GTP
S is rather inefficient
at stabilizing the conformation in G
i that is required for ternary complex disruption. Similarly, IDP, XDP, XTP, UTP, and CTP
are less efficacious than GTP at disrupting the ternary complex between
the
2AR and G
s (23, 27).
MANT-GTPS/MANT-GppNHp Are Potent Inhibitors of
G
s-stimulated AC; AC Inhibition Is Not Due to
Stabilization of an Inhibitory G
s Conformation--
We
analyzed the effects of GTP analogs on AC activity in Sf9
membranes expressing G
sS. GTP
S and GppNHp increased
basal AC activity with EC50 values of 6.2 (CI, 5.1-7.4
nM) and 87 nM (CI, 67-110 nM),
respectively. GTP
S and GppNHp were similarly efficacious at
activating AC. In agreement with previous results (5), GDP
S was less
efficacious at activating AC than GTP
S/GppNHp, i.e. GDP
S acted as a partial G
s activator. MANT-GTP
S
and MANT-GppNHp abolished basal AC activity in Sf9 membranes
expressing G
sS (reflecting the activity of
G
sS bound to GDP (13, 29)) with IC50 values of 7.4 (CI, 3.6-15 µM) and 34 µM (CI,
5.9-110 µM), respectively (Fig.
5A).
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There is evidence to support the concept of multiple G states
(4, 5, 8, 23, 26, 27). Based on the literature and our present data,
the hypothesis evolved that MANT-GTP
S/MANT-GppNHp could stabilize an
inhibitory G
s conformation. To test this hypothesis, we
examined the interactions of MANT-GTP
S/MANT-GppNHp with GTP, GTP
S, GppNHp, GDP
S and AlF
and
stabilize distinct conformations (4, 5, 8, 30). If
MANT-GTP
S/MANT-GppNHp inhibited AC via G
sS, they
should competitively block the stimulatory effects of GTP
S, GppNHp,
GTP, GDP
S, and AlF
sS activator,
GTP
S, with the partial G
sS activator, GDP
S, on AC
(Fig. 5A) (5). Competitive interaction of MANT-GTP
S with
GTP was already observed in the GTPase studies (Fig. 3). GDP
S
shifted the concentration/response curves for GTP
S to the right
without reducing the maximal AC activity obtained with GTP
S (Fig.
5B). These data confirm the competitive interaction of
guanine nucleotides at the nucleotide-binding site of G
s
(31).
MANT-GTPS abolished AC activity stimulated by GTP
S at a
saturating concentration (10 µM) with an IC50
of 1.5 µM (CI, 1.1-2.0 µM) (Fig.
5C). Based on the GTPase competition studies (Fig.
2B), we would have expected that MANT-GTP
S at an
~100-fold molar excess relative to GTP
S would have half-maximally
blocked GTP
S-stimulated AC activity. Thus, in the AC assay,
MANT-GTP
S was almost 3 orders of magnitude more potent than
predicted from the GTPase competition experiments. These findings
raised doubts whether inhibition of AC by MANT-GTP
S involves binding
of the inhibitor to the nucleotide-binding pocket of
G
sS.
In Sf9 membranes co-expressing the 2AR and
G
sS, MANT-GTP (1-100 µM) failed to
support AC activation (data not shown). In contrast, GTP per
se moderately increased basal AC activity in this system (Fig.
5D), reflecting the ability of the constitutively active
2AR at promoting GDP/GTP exchange at G
s
(32). The
2AR agonist (
)-isoproterenol further
increased GTP-dependent AC activity. MANT-GTP
S (3 µM) almost abolished AC activation by GTP and GTP plus
agonist. Even GTP at a 33-fold molar excess relative to MANT-GTP
S failed to overcome this inhibition. Similarly, MANT-GTP
S inhibited AC activation by GTP
S (Fig. 5E), GppNHp (Fig.
5F), and AlF
s and, thereby, blocks its GTPase (33). As a result,
GTP acquires GTP
S/GppNHp-like properties. In agreement with the data
obtained for insect cell AC (Fig. 5, A-G), MANT-GppNHp
reduced GTP-stimulated AC activity in membranes from cholera
toxin-treated S49 wt cells non-competitively (Fig. 6A). The
IC50 of MANT-GppNHp in S49 membranes on GTP-stimulated AC
activity was 2.5 µM (CI, 0.7-8.7 µM) (Fig.
6B).
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The inhibitory effects of MANT-GTPS and MANT-GppNHp on AC were fully
reversible, i.e. pre-treatment of Sf9 membranes
expressing G
sS with MANT-GTP
S or MANT- GppNHp at
10-100 µM for 10 min at 25 °C and subsequent washing
of membranes by centrifugation and suspension in MANT-nucleotide-free
buffer had no inhibitory effect on subsequently determined basal and
GTP
S-stimulated AC activities compared with activities of
solvent-treated membranes (data not shown). These data argue against
the hypothesis that MANT-nucleotides inhibited G
sS
through disulfide bridge formation between the
-thiophosphate and a
cysteinyl residue in the G-protein. Moreover, MANT-GTP
S (10 µM) completely blocked AC in Sf9 membranes
expressing G
sS in the presence of GTP
S (10 µM) within 15 s, i.e. the earliest time
point studied. This result indicates that GTP
S binds to its target
rapidly. In contrast, the onset of AC inhibition by GDP
S in
membranes from cholera toxin-treated turkey erythrocytes requires
several minutes to be complete, reflecting slow dissociation of GTP
from G
s (34).
Taken together, the differences in the interactions of
MANT-GTPS/MANT-GppNHp with GTP
S/GppNHp/GTP/AlF
S with GTP
S/GTP argue
against the hypothesis that MANT-GTP
S/MANT-GppNHp bind to
G
sS to stabilize an inhibitory conformation. Rather, the
noncompetitive interactions of MANT-GTP
S/MANT-GppNHp with
GTP
S/ GppNHp/GTP/AlF
sS.
The Inhibitory Effects of MANT-GTPS/MANT-GppNHp on AC
Are Not Due to G
i Activation--
We addressed the
hypothesis that MANT-GTP
S/MANT-GppNHp inhibit AC via
G
i activation by studying AC regulation in membranes of
the G
s-deficient cell line S49
cyc
. In agreement with previous data (6, 18),
GTP
S and GppNHp inhibited forskolin-stimulated AC in S49
cyc
membranes in the presence of
Mg2+ by 45-50% (Fig.
7A). As expected (6, 18),
GDP
S was less efficacious at activating G
i than
GTP
S/GppNHp (Fig. 7A). The IC50 values of
GTP
S and GppNHp for AC inhibition were 0.4 (CI, 0.05-2.4
nM) and 3.7 nM (CI, 1.2-12 nM),
respectively. We explain the ~20-60-fold higher potencies of
GTP
S/GppNHp in the AC inhibition assay relative to the GTPase
competition assay (compare Fig. 2, D-F, with Fig.
7A) by a model in which only a small fraction of the
G
i molecules has to be activated for AC inhibition,
whereas the GTPase assay assesses the entire population of
G
i molecules.
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In contrast to GTPS, GppNHp, and GDP
S, MANT-GTP
S and
MANT-GppNHp abolished AC activity in S49 cyc
membranes (IC50 MANT-GTP
S, 320 nM (CI,
120-900 nM); IC50 MANT-GppNHp, 1.6 µM (CI, 0.7-3.9 µM)). We did not observe
increases in potency of MANT-GTP
S/MANT-GppNHp in the AC inhibition
assay relative to the GTPase competition assay (Fig. 2,
D-F, and Fig. 7A). The differences in the
effects of GTP
S/GppNHp/GDP
S versus
MANT-GTP
S/MANT- GppNHp on AC activity in S49
cyc
membranes and in the GTPase competition
assay indicate that MANT-nucleotides did not inhibit AC via
G
i activation but through a different mechanism.
MANT-GTPS/MANT-GppNHp Are Competitive AC
Inhibitors--
We then addressed the hypothesis that MANT-nucleotides
inhibit AC directly. AC possesses a catalytic site for the substrate, ATP, and a regulatory site for the stimulatory diterpene, forskolin (19, 20, 35). In Sf9 membranes expressing G
sS,
forskolin increased AC activity with an EC50 of 3.5 µM (CI, 1.2-8.8 µM) (Fig. 5H).
MANT-GTP
S inhibited this AC activation non-competitively. This
finding indicates that MANT-GTP
S does not interact with the
forskolin-binding site of AC.
AC inhibitors are divided into two classes, i.e. competitive inhibitors that bind to the empty catalytic site and noncompetitive inhibitors that bind to the AC-PPi conformation (36-38). Typically, noncompetitive AC inhibitors are substituted with a (poly)phosphate at the 3'-O-position of the ribosyl group (39), whereas certain nucleotides with a polyphosphate at the 5'-O-position of the ribosyl group are competitive inhibitors of mammalian membranous ACs and the soluble catalytic subunits of these enzymes (40, 41).
To test the hypothesis that MANT-GTPS/MANT-GppNHp inhibit AC
directly, we wished to study AC regulation independently of G
s and G
i. We took advantage of the fact
that Mn2+ (10 mM) blocks G
i
inhibition of AC in S49 cyc
membranes (6, 18).
The exchange of Mg2+ against Mn2+ increased AC
activity by almost 5-fold (compare Fig. 7, A and B) (6, 18). As predicted (6, 18), GTP
S, GppNHp, and GDP
S did not inhibit AC in S49 cyc
membranes in the presence of Mn2+ (Fig. 7B). In
marked contrast, MANT-GTP
S and MANT- GppNHp abolished AC activity
in S49 cyc
membranes in the presence of
Mn2+ with IC50 values of 84 (CI, 62-110
nM) and 210 nM (CI, 150-300 nM),
respectively. The exchange of Mg2+ against Mn2+
increased the potencies of MANT-GTP
S and MANT-GppNHp at inhibiting AC by ~4- and ~8-fold, respectively (Fig. 7, A and
B). These data indicate that, indeed,
MANT-GTP
S/MANT-GppNHp inhibit AC through interaction with the
catalytic site of AC. The slopes of the concentration/response curves
for AC inhibition in the presence of Mn2+ were steeper than
in the presence of Mg2+. This difference may reflect
differences in the interactions of MANT-nucleotides with AC in the
presence of Mg2+ and Mn2+.
To answer the question whether AC inhibition by MANT-nucleotides is
competitive or noncompetitive, we determined AC activity in S49
cyc membranes in the presence of
ATP/Mn2+ at various concentrations plus MnCl2
(10 mM) with MANT-GTP
S at various fixed concentrations.
The data were plotted double-reciprocally according to Lineweaver-Burk
(Fig. 8A). The linear
regression lines intersected in the y axis, indicative for
competitive interaction of MANT-GTP
S with ATP. Non-linear regression
analysis showed that under these conditions, the Km
of AC was 132 µM (CI, 81-190 µM), and the
Vmax was 297 pmol/mg/min (CI, 262-332 pmol/mg/min). The Ki of MANT-GTP
S for S49
cyc
AC was 53 nM (CI, 44-75
nM). MANT-GppNHp competitively inhibited S49
cyc
AC with a Ki of 161 nM (CI, 113-230 nM).
|
The MANT group spontaneously isomerizes between the 2'- and
3'-O-position of the ribosyl group (Fig. 1) (7). At neutral pH, i.e. the pH conditions used in our study (see
"Experimental Procedures"), isomerization occurs quite rapidly
(t1/2, 7 min) (7). Thus, we actually examined a
mixture of 2'- and 3'-MANT-GTPS/2'- and 3'-MANT-GppNHp. To answer
the question which isomer is biologically active, one has to examine
MANT-nucleotides in which the fluorophore cannot isomerize. This goal
can be achieved by comparing 2'-deoxy-3'-MANT-nucleotides with
3'-deoxy-2'-MANT-nucleotides (7). 2'-Deoxy-3'-MANT-GppNHp was used to
analyze the crystal structure of p21H-ras (42) and is
commercially available (see "Experimental Procedures"). In S49
cyc
membranes, 2'-deoxy-3'-MANT- GppNHp
inhibited Mn2+/forskolin-stimulated AC with a
Ki of 880 nM (CI, 673-1160 nM). Unfortunately, 3'-deoxy-2'-MANT- GppNHp was not
available to us.
Finally, we determined the mechanism by which MANT-nucleotides inhibit
Sf9 membrane AC. In the presence of MnCl2 (10 mM), the insect cell enzyme had a Km of
144 µM (CI, 82-205 µM) and a
Vmax of 282 pmol/mg/min (CI, 251-323
pmol/mg/min) as assessed by non-linear regression analysis. The linear
regression lines of the double-reciprocal plot of AC activities in the
presence of MANT-GppNHp at various fixed concentrations intersected in the y axis (Fig. 8B), indicative of competitive
antagonism. The Ki value of MANT-GppNHp for insect
cell membrane AC was 155 nM (CI, 110-220
nM).
![]() |
DISCUSSION |
---|
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---|
MANT-GTPS/MANT-GppNHp Bind to G
s- and
G
i-proteins with Low Affinity and Are Inefficient G
Activators--
A MANT group at the 2'(3')-O-position of
the ribosyl group reduces the affinity of GTP
S and GppNHp for
G
s and G
i (Fig. 2). Our data fit to the
low affinity of MANT-GTP for transducin (11) and support the notion
that the 2'- and 3'-OH groups of the ribosyl residue of GTP/GTP
analogs, blocked by the MANT group (Fig. 1), are important for hydrogen
bonding with G
(43). It is also conceivable that there are steric
hindrances to accommodate the bulky MANT group in the tight
nucleotide-binding pocket of G
(4, 43).
The MANT group reduces the affinity of GTPS for G
to a greater
extent than the affinity of GppNHp (Fig. 1). These data indicate that
GTP
S and GppNHp bind to G
in non-identical manners. In fact, the
-thiophosphate of GTP
S is bulkier than the
,
-imidophosphate of GppNHp, resulting in different crystal structures of the catalytic sites of G
-GTP
S and G
- GppNHp (30). Through differentially propagated conformational changes from the catalytic site of G
to
the ribosyl residue-binding domain, G
-GppNHp could accommodate the
additional MANT group more readily than G
-GTP
S.
Even at high concentrations that fully saturate G with
MANT-GTP
S/MANT-GppNHp (10-100 µM) (Fig. 2), these
nucleotides are inefficient at stimulating AC via G
s
(Fig. 5A), inhibiting AC via G
i (Fig.
7A), and disrupting the ternary complex between FPR and
G
i2 (Fig. 4B). In addition, MANT-GTP is
inefficient at activating transducin (11) and G
sS. Thus,
MANT modification of guanine nucleotides is also associated with a loss
of efficacy at activating G
. These results corroborate the concept
of multiple G
states that are differentially stabilized by various
nucleotides (4, 5, 8, 23, 26, 27).
In marked contrast to the data obtained with GPCR-G fusion proteins
(Fig. 1) and transducin examined in native membranes (11), the MANT
group had no adverse effect on nucleotide affinities of purified
G
i1 (9). We cannot explain the molecular mechanism for
these discrepancies, but differences in the guanine nucleotide-binding properties of purified G-proteins and G-proteins in native membranes were observed earlier (44).
MANT-GTPS/MANT-GppNHp Are Potent Competitive AC
Inhibitors; Comparison with the Literature--
The most prominent
effects of MANT-GTP
S/MANT-GppNHp on G
s- and
G
i-mediated signaling were evident at the AC level.
Under all conditions studied, MANT-GTP
S/MANT-GppNHp abolished AC
activity (Figs. 5-8). We ruled out G
s,
G
i, and the forskolin-binding site of AC as targets of
action of MANT-GTP
S/MANT-GppNHp. Instead, MANT-GTP
S/MANT-GppNHp
are competitive AC inhibitors (Fig. 8).
-L-2'3'-Dideoxyadenosine 5'-triphosphate is the most
potent competitive AC inhibitor known so far (IC50 for rat
brain AC with 100 µM ATP in the presence of
Mn2+/forskolin, 24 nM) (40). MANT-GTP
S is
structurally quite different from
-L-2'3'-dideoxyadenosine 5'-triphosphate and ~3-fold
less potent under comparable experimental conditions (IC50
of MANT-GTP
S for S49 cyc
membrane AC with
100 µM ATP in the presence of Mn2+/forskolin,
78 nM; CI, 59-101 nM). However, we must be
cautious with the comparison of potencies because we do not know
whether the AC isoenzymes in S49 cyc
membranes
(AC6 and AC7 (45)) and brain (mostly AC1, AC2 and AC8 (19)) are
similarly sensitive to blockade by competitive inhibitors.
2'-Deoxy-3'-anthraniloyl-ATP exhibits rather low potency for inhibition
of Bordetella pertussis and Bacillus anthracis AC
toxins (Ki, ~10 µM) (46). It remains
to be determined inasmuch as the differences in Ki
values of 2'-deoxy-3'-anthraniloyl-ATP versus
2'-deoxy-3'-MANT-GppNHp (Ki ~880 nM)
reflect differences in the structure/activity relationships of AC
inhibitors at various AC subtypes.
Most studies regarding AC inhibitors focused on adenosine derivatives and adenine nucleotides (36, 38-40, 47, 48). However, 2'-deoxyguanosine and 2'-deoxyguanosine 3'-monophosphate inhibit rat brain AC non-competitively with Ki values of >300 µM (48). In addition, GTP non-competitively inhibits soluble AC from Sf9 cells with a Ki of 1 mM (49). Moreover, GTP competitively inhibits particulate AC from Escherichia coli (50). Based on these data, it is not totally unexpected that GTP analogs are competitive inhibitors of membranous ACs.
The exchange of Mg2+ against Mn2+ increased the
potencies of MANT-GTPS and MANT-GppNHp at inhibiting
forskolin-stimulated AC in S49 cyc
membranes
by ~4- and ~8-fold, respectively (Fig. 7). Similar observations
were made for the competitive AC inhibitor ApCHpp at the purified
catalytic subunits C1 of AC5 and C2 of AC2 (41). Future studies will
have to clarify the molecular basis for the increase in affinity of
competitive AC inhibitors by Mn2+.
Molecular Basis for the High Affinity Interaction of
MANT-GTPS/MANT-GppNHp with AC--
At concentrations up to 100 µM, GTP
S/GppNHp does not directly inhibit AC (Fig.
7B). Therefore, the most intriguing question is why the MANT
group at the 2'(3')-O-position of the ribosyl residue (Fig.
1) increases the affinity of GTP
S/GppNHp for AC so dramatically
(Fig. 7B). The crystal structure of the soluble catalytic
subunits of AC and molecular modeling (C1-subunit of AC5 and C2-subunit
of AC2) revealed that Leu-438 (AC5), Ile-940 (AC2), and Phe-889 (AC-2)
form a hydrophobic pocket in the catalytic site that faces toward the
2'-OH ribosyl group of ATP (35, 51). In contrast, the 3'-OH ribosyl
group of ATP points toward more hydrophilic amino acids. Thus, strong
hydrophobic interactions of the MANT group attached to the
2'-O-ribosyl group of GTP
S/GppNHp with Leu-438 (AC5),
Ile-940 (AC2), and Phe-889 (AC-2), overcoming the non-ideal fit of the
guanine ring into the catalytic site of AC (51), could explain the
dramatic increase in affinity of GTP
S/GppNHp for AC. Accordingly,
substitution of the 3'-O-ribosyl group with MANT should
reduce GTP
S/GppNHp affinity for AC because the hydrophobic MANT
faces a hydrophilic environment. Thus, the expected order of affinity
of nucleotides for AC is 3'-deoxy-2'-MANT-GTP
S/GppNHp > 2'(3')-MANT-GTP
S/GppNHp > 2'-deoxy-3'-MANT-GTP
S/GppNHp. In agreement with this expectation, 2'-deoxy-3'-MANT-GppNHp was a 5.5-fold
less potent AC inhibitor than 2'(3')-MANT-GppNHp, but we could not
examine 3'-deoxy-2'-MANT-GppNHp. The hydrophobic amino acids mentioned
above are highly conserved among membranous ACs and soluble guanylyl
cyclase (20). In accordance with the conserved pocket is our finding
that MANT-GppNHp is a similarly potent AC inhibitor in S49
cyc
membranes and Sf9 insect cell membranes.
Future Studies--
The results of this study open several
novel avenues of research on AC. The analysis of the structure/activity
relationships of MANT-nucleotides with respect to the base, phosphate
chain, position of MANT substitution, and hydroxyl groups of the
ribosyl residue in conjunction with molecular modeling and
crystallographic studies will answer the question why MANT-GTPS and
MANT-GppNHp are such potent AC inhibitors. The systematic analysis of
structure/activity relationships of
2'(3')-O-ribosyl-substituted nucleotides including the
exchange of the MANT group against other substituents will probably
generate compounds with even higher specificity for AC relative to
G-proteins than MANT-GTP
S and MANT-GppNHp. Additionally, MANT-nucleotides may provide a starting point to design selective inhibitors for particular AC isoenzymes and soluble guanylyl cyclase. Furthermore, MANT-nucleotides could be used as fluorescent ligands for
AC, allowing for the analysis of AC kinetics with high temporal resolution. Finally, compared with their parent molecules, MANT-GTP
S and MANT-GppNHp are more lipophilic. Thus, it may be possible to
develop cell-permeable fluorescent AC inhibitors with broad applications as experimental tools and therapeutic use in diseases associated with elevated AC activity such as cholera. Among the several
possible research avenues, our laboratory currently focuses on the
structure/activity relationships of nucleotides for inhibition of recombinant/purified AC isoforms and soluble guanylyl cyclase.
![]() |
ACKNOWLEDGEMENTS |
---|
We thank Dr. S. R. Sprang (Howard Hughes
Medical Institute, University of Texas Southwestern Medical Center,
Dallas, TX) and Dr. R. A. Johnson (Department of Physiology and
Biophysics, State University, Stony Brook, NY) for stimulating
discussions and the reviewers of this paper for their most valuable
critique. We also thank Drs. R. Sportsman and M. Helms (LJL Biosystems,
Sunnyvale, CA) for providing initial batches of MANT-GTPS and
MANT-GppNHp and helpful discussions.
![]() |
FOOTNOTES |
---|
* This work was supported by Army Research Office Grant DAAD19-00-1-0069, The J. R. and Inez Jay Biomedical Research Award of the University of Kansas (to R. S.), and a predoctoral fellowship from the Studienstiftung des Deutschen Volkes (to A. G.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
To whom correspondence should be addressed: Dept. of Pharmacology
and Toxicology, the University of Kansas, 1251 Wescoe Hall Dr., Malott
Hall, Rm. 5064, Lawrence, KS 66045-7582. Tel.: 785-864-3525; Fax:
785-864-5219; E-mail: rseifert@ku.edu.
Published, JBC Papers in Press, February 3, 2003, DOI 10.1074/jbc.M211292200
![]() |
ABBREVIATIONS |
---|
The abbreviations used are:
GPCR, G-protein-coupled receptor;
AC, adenylyl cyclase;
2AR,
2-adrenoceptor;
2AR-G
olf, fusion protein consisting of the
2-adrenoceptor and
G
olf;
2AR-G
sL, fusion
protein consisting of the
2-adrenoceptor and the long
splice variant of G
s;
2AR-G
sS, fusion protein consisting of the
2-adrenoceptor and the short splice variant of
G
s;
FMLP, N-formyl-L-methionyl-L-leucyl-L-phenylalanine;
FPR, formyl peptide receptor;
FPR-G
i1, 2,3, fusion
protein consisting of the formyl peptide receptor and
G
i1, G
i2, or G
i3;
G
, non-specified G-protein
-subunit;
G
i, family of three
G-protein
-subunits (G
i1, G
i2, and
G
i3) that mediates adenylyl cyclase inhibition;
G
s, family of three G-protein
-subunits
(G
sL, G
sS, and G
olf) that
mediates adenylyl cyclase activation;
G
olf, olfactory
G
s-protein;
G
sL, long splice variant of
G
s;
G
sS, short splice variant of
G
s;
GDP
S, guanosine 5'-[
-thio]diphosphate;
GppNHp, guanosine 5'-[
,
-imido]-triphosphate;
GTP
S, guanosine 5'-[
-thio]triphosphate;
MANT-GppNHp, 2'(3')-O-(N-methylanthraniloyl)-guanosine
5'-[
,
-imido]-triphosphate;
MANT-GTP
S, 2'(3')-O-(N-methylanthraniloyl)-guanosine
5'-[
-thio]triphosphate;
S49 wt cells, S49 wild-type lymphoma
cells;
S49 cyc
cells, G
s-deficient S49 lymphoma cells;
AppNHp, adenosine
5'-[
,
-imido]triphosphate;
ApCHpp, adenosine
5'-[
,
-methylene]triphosphate.
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