2'(3')-O-(N-Methylanthraniloyl)-substituted
GTP Analogs: A Novel Class of Potent Competitive Adenylyl Cyclase
Inhibitors*
Andreas
Gille and
Roland
Seifert
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 |
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'-[
-thio]triphosphate (MANT-GTPS) and
MANT-guanosine 5'-[
,-imido]triphosphate (MANT-GppNHp) on
G
s- and Gi-protein-mediated signaling.
MANT-GTPS/MANT-GppNHp had lower affinities for Gs and
Gi than GTPS/GppNHp as assessed by inhibition of GTP
hydrolysis of receptor-G fusion proteins. MANT-GTPS was much less
effective than GTPS at disrupting the ternary complex between the
formyl peptide receptor and Gi2. MANT-GTPS/MANT-GppNHp non-competitively inhibited GTPS/GppNHp-, AlF
-, 2-adrenoceptor plus GTP-,
cholera toxin plus GTP-, and forskolin-stimulated adenylyl cyclase (AC) in Gs-expressing Sf9 insect cell membranes and
S49 wild-type lymphoma cell membranes. AC inhibition by
MANT-GTPS/MANT-GppNHp was not due to Gs inhibition
because it was also observed in Gs-deficient S49
cyc
lymphoma cell membranes. Mn2+
blocked AC inhibition by GTPS/GppNHp in S49
cyc membranes but enhanced the potency of
MANT-GTPS/MANT-GppNHp at inhibiting AC by ~4-8-fold. MANT-GTPS
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-GTPS/MANT-GppNHp bind to
Gs- and Gi-proteins with low affinity and
are ineffective at activating G. Instead, MANT-GTPS/MANT-GppNHp
constitute a novel class of potent competitive AC inhibitors.
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INTRODUCTION |
G-proteins are heterotrimeric (-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. GGTP
dissociates from GPCR, thereby disrupting the ternary complex. In
addition, GGTP and dissociate from each other,
and both GGTP 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 GTPS and GppNHp (Fig. 1)
induce persistent G-protein activation as does AlF,
the latter mimicking the transition state of GTP hydrolysis as
GDP-AlF complex (1, 3, 4). The hydrolysis-resistant
GDP analog GDPS (Fig. 1) is a partial G-protein activator (5,
6).
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Fig. 1.
Structures of GTPS,
GppNHp, MANT-GTPS, MANT-GppNHp, and
GDPS. The -thiophosphate group in
GTPS/MANT-GTPS, the -thiophosphate group in GDPS, 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-GTPS and MANT-GppNHp (but not in
2'-deoxy-3'-MANT-GppNHp).
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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 GTPS for purified Gi1 (8, 9). The
maximum fluorescence of Gi/Go-proteins induced
by MANT-GTPS 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 GTPS/GppNHp,
MANT-GTPS/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
Gs-proteins, GsL, GsS, or
Golf (12-14), fusion proteins, and co-expression
systems of the FPR with the Gi-proteins,
Gi1, Gi2, or Gi3 (15, 16),
and individually expressed GsS. As physiologically
relevant systems, we studied S49 wt lymphoma cell membranes, a standard
model for the analysis of Gs-proteins (17) and S49
cyc cell membranes, a
Gs-deficient S49 mutant cell line serving as model for
the analysis of Gi-proteins (6, 18). Gs
activates, and Gi inhibits, the effector AC (19, 20).
Surprisingly, we found that MANT-GTPS/MANT-GppNHp constitute a novel
class of potent competitive AC inhibitors.
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EXPERIMENTAL PROCEDURES |
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-GTPS 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,
GTPS, GppNHp, GDPS, 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 GsS and Gi2 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 12 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-Gs- and FPR-Gi 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-Gs 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-Gi 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-GsS (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 + Gi2 + 12 (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-Gs fusion proteins) or 10 µM FMLP (FPR-Gi 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 GsS in the presence of
MANT-GTPS/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 |
MANT-GTPS/MANT-GppNHp Bind to Gs- and
Gi-proteins with Low Affinity--
We determined the
affinities of GTPS and GppNHp and their MANT-derivatives for
Gs- and Gi-proteins by measuring
agonist-stimulated steady-state GTP hydrolysis of
2AR-Gs and FPR-Gi fusion
proteins expressed in Sf9 insect cell membranes (13, 14, 16,
23). The affinity profiles of nucleotides were similar for
Gs-proteins (GsL, GsS, and
Golf) (Fig. 2,
A-C) and Gi-proteins (Gi1, Gi2, and Gi3) (Fig. 2, D-F).
GTPS inhibited GTP hydrolysis with Ki values
ranging from 3.6 (2AR-Golf) to 8.9 nM (FPR-Gi3). As expected (1, 2), GppNHp
inhibited GTP hydrolysis with considerably lower potencies
(~20-140-fold) than GTPS. The Ki values for
GppNHp ranged from 170 (FPR-Gi2) to 520 nM
(2AR-Golf). The introduction of the MANT
group at the 2'(3')-O-position of the ribosyl group reduced
the affinity of GTPS for Gs- and Gi-proteins by ~30-300-fold, i.e. the
Ki values for MANT-GTPS ranged from 250 (FPR-Gi2) to 1100 nM
(2AR-Golf). The affinities of MANT-GppNHp
for Gs- and Gi-proteins were 4.5-11-fold lower than those of GppNHp, i.e. the Ki
values for MANT-GppNHp ranged from 1.2 (FPR-Gi2) to 5.8 µM (2AR-Golf).
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Fig. 2.
Competition of
[-32P]GTP hydrolysis in
Sf9 membranes expressing
2AR-Gs-
and FPR-Gi fusion proteins by
GTPS, GppNHp,
MANT-GTPS, and MANT-GppNHp. GTPase
activity in Sf9 membranes was determined as described under
"Experimental Procedures." Reaction mixtures contained Sf9
membranes (10 µg of protein/tube) expressing various fusion proteins,
100 nM GTP, [-32P]GTP (0.2-0.5
µCi/tube), and unlabeled GTPS, GppNHp, MANT-GTPS, or
MANT-GppNHp at increasing concentrations. Reaction mixtures
additionally contained 10 µM ()-isoproterenol
(2AR-Gs fusion proteins) or 10 µM FMLP (FPR-Gi fusion proteins). GTPase
activities in the absence of competitor (control) were as follows.
2AR-Golf (expressed at 13.7 pmol/mg,
[3H]dihydroalprenolol saturation binding), 8.5 ± 0.4 pmol/mg/min; 2AR-GsS (expressed at
4.0 pmol/mg), 3.3 ± 0.3 pmol/mg/min;
2AR-GsL (expressed at 6.0 pmol/mg),
5.2 ± 0.6 pmol/mg/min; FPR-Gi1 (expressed at 0.77 pmol/mg, [3H]FMLP saturation binding), 17.2 pmol/mg/min;
and FPR-Gi2 (0.47 pmol/mg, 11.1 pmol/mg/min),
FPR-Gi3 (0.98 pmol/mg, 14.7 pmol/mg/min). These GTPase
activities were defined as 100% (control). Competition curves were
extended until complete inhibition of GTP hydrolysis in Sf9
membranes. This point was defined as 0%. All other data points
referred to those calibration points. The Km values
of agonist-stimulated GTP hydrolysis of fusion proteins were reported
earlier (14, 16, 27) and were used to calculate Ki
values from IC50 values. Competition isotherms were
obtained by non-linear regression analysis. Data shown are the
means ± S.D. of 3 independent experiments performed in
duplicate.
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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-GTPS. In fact, MANT-GTPS
exhibited competitive interaction with GTP at Gs (Fig.
3A) and Gi (Fig. 3B).
Collectively, our data show that MANT-GTPS and MANT-GppNHp bind to
Gs- and Gi-proteins, although with low
affinity.
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Fig. 3.
Lineweaver-Burk analysis of the interaction
of MANT-GTPS with GTP at the GTPase of
GsS and
Gi2. GTPase activity in
Sf9 membranes was determined as described under "Experimental
Procedures." Reaction mixtures contained Sf9 membranes (10 µg of protein/tube) expressing 2AR-GsS
(4.0 pmol/mg, [3H]dihydroalprenolol saturation binding)
or FPR (0.5 pmol/mg, [3H]FMLP saturation binding) + Gi2 (300 pmol/mg as assessed by quantitative
immunoblotting using the 2AR-Gi2 fusion
protein as standard) + 12, 100 nM to 1.5 µM unlabeled GTP plus
[-32P]GTP (0.2-0.5 µCi/tube), and unlabeled
MANT-GTPS at the concentrations indicated in the graph. Reaction
mixtures additionally contained 10 µM ()-isoproterenol
(2AR-GsS) or 10 µM FMLP
(FPR). Data were plotted double-reciprocally and analyzed by linear
regression according to Lineweaver-Burk. The r2
values of the regression lines were 0.98-0.99. Shown are the results
of a representative experiment. Similar results were obtained in three
independent experiments.
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Effects of MANT-GTPS on Ternary Complex Formation--
In case
of the 2AR/Gs 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), GTPS 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-GsS (Fig.
4A). MANT-GTPS (1 µM) reduced ternary complex formation in
2AR-GsS by ~50%, but at higher concentrations, the effect of MANT-GTPS was reverted. MANT-GTPS 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-GTPS inhibited
[3H]dihydroalprenolol binding to the 2AR.
We examined the effect of MANT-GTPS (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-GTPS (10 µM) inhibited binding of
[3H]dihydroalprenolol (1 nM) by 40%. Thus,
because of interference of MANT-GTPS with ligand binding to the
2AR, we could not answer the question whether GTPS
and MANT-GTPS exhibit similar efficacies at disrupting the ternary
complex in 2AR-GsS.
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Fig. 4.
Effects of GTPS and
MANT-GTPS on ternary complex formation in
the
2AR/GsS
couple and the FPR/Gi2
couple. Ternary complex formation in Sf9 membranes was
determined as described under "Experimental Procedures."
A, for ternary complex formation with the
2AR/GsS couple, reaction mixtures
contained Sf9 membranes expressing
2AR-GsS (3.5-5.0 pmol/mg) (20-25 µg
of protein/tube), 1 nM [3H]dihydroalprenolol,
1 µM salbutamol and guanine nucleotides at increasing
concentrations. The [3H]dihydroalprenolol binding
observed in the absence of added guanine nucleotides was defined as
100% ternary complex formation, and the
[3H]dihydroalprenolol binding observed in the presence of
1 µM GTPS was defined as 0% ternary complex
formation. All other data referred to those calibration points.
B, for ternary complex formation with the
FPR/Gi2 couple, reaction mixtures contained Sf9
membranes expressing FPR (0.5-1.0 pmol/mg) + Gi2 (300 pmol/mg) + 12 (30-50 µg of
protein/tube), 10 nM [3H]FMLP, and guanine
nucleotides at increasing concentrations. The [3H]FMLP
binding observed in the absence of added guanine nucleotide was defined
as 100% ternary complex formation, and the [3H]FMLP
binding observed in the presence of 10 µM GTPS was
defined as 0% ternary complex formation. All other data referred to
those calibration points. Competition isotherms were obtained by
non-linear regression analysis. Data shown are the means of 3-4
independent experiments performed in triplicate.
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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), GTPS potently (IC50, 26 nM; CI, 15-43 nM) and efficaciously disrupted the ternary complex of the FPR/Gi2 couple (Fig.
4B). Considering the Ki values of
MANT-GTPS for Gi-proteins (250-500 nM)
(Fig. 2, D-F), we would have expected maximal disruption of the ternary complex with MANT-GTPS at 10 µM. However,
MANT-GTPS (10 µM) decreased [3H]FMLP
binding by not more than 25%. Thus, MANT-GTPS is rather inefficient
at stabilizing the conformation in Gi 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 Gs (23, 27).
MANT-GTPS/MANT-GppNHp Are Potent Inhibitors of
Gs-stimulated AC; AC Inhibition Is Not Due to
Stabilization of an Inhibitory Gs Conformation--
We
analyzed the effects of GTP analogs on AC activity in Sf9
membranes expressing GsS. GTPS 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. GTPS and GppNHp were similarly efficacious at
activating AC. In agreement with previous results (5), GDPS was less
efficacious at activating AC than GTPS/GppNHp, i.e. GDPS acted as a partial Gs activator. MANT-GTPS
and MANT-GppNHp abolished basal AC activity in Sf9 membranes
expressing GsS (reflecting the activity of
GsS 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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Fig. 5.
Regulation of AC activity in Sf9
membranes by guanine nucleotides,
2AR, NaF, and forskolin. AC
activity in Sf9 membranes (15-30 µg of protein/tube) was
determined as described under "Experimental Procedures." AC
activity was determined in Sf9 membranes expressing
GsS (5.9 ± 0.3 pmol/mg as assessed by quantitative
immunoblotting using the 2AR-GsS fusion
protein as standard) (A-C and E-H) or
Sf9 membranes expressing 2AR (9.5 pmol/mg) + GsS (6.4 pmol/mg) (D). A, reaction
mixtures contained GTPS, GppNHp, GDPS, MANT-GTPS, or
MANT-GppNHp at increasing concentrations. B, reaction
mixtures contained GTPS at increasing concentrations in the absence
or presence of GDPS at various fixed concentrations. C,
reaction mixtures contained 10 µM GTPS plus
MANT-GTPS at increasing concentrations. D, reaction
mixtures contained GTP at increasing concentrations in the absence of
()-isoproterenol (ISO) or in the presence of 10 µM ()-isoproterenol (+ISO). Reaction
mixtures additionally contained distilled water (control) or 3 µM MANT-GTPS. E, reaction mixtures
contained GTPS at increasing concentrations in the absence or
presence of MANT-GTPS at various fixed concentrations. F,
reaction mixtures contained GppNHp at increasing concentrations in the
absence or presence of MANT-GTPS (10 µM).
G, reaction mixtures contained NaF at increasing
concentrations and 10 µM AlCl3 in the absence
or presence of MANT-GTPS at various fixed concentrations.
H, reaction mixtures contained forskolin at increasing
concentrations in the absence or presence of MANT-GTPS (3 µM). Data were analyzed by non-linear regression. Data
shown are the means ± S.D. of 2-4 independent experiments
performed in duplicate.
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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-GTPS/MANT-GppNHp could stabilize an
inhibitory Gs conformation. To test this hypothesis, we
examined the interactions of MANT-GTPS/MANT-GppNHp with GTP, GTPS, GppNHp, GDPS and AlF, i.e. substances that all bind to the nucleotide-binding pocket of G and
stabilize distinct conformations (4, 5, 8, 30). If
MANT-GTPS/MANT-GppNHp inhibited AC via GsS, they
should competitively block the stimulatory effects of GTPS, GppNHp,
GTP, GDPS, and AlF on AC. As positive control, we studied the interaction of the full GsS activator,
GTPS, with the partial GsS activator, GDPS, on AC
(Fig. 5A) (5). Competitive interaction of MANT-GTPS with
GTP was already observed in the GTPase studies (Fig. 3). GDPS
shifted the concentration/response curves for GTPS to the right
without reducing the maximal AC activity obtained with GTPS (Fig.
5B). These data confirm the competitive interaction of
guanine nucleotides at the nucleotide-binding site of Gs
(31).
MANT-GTPS abolished AC activity stimulated by GTPS 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-GTPS at an
~100-fold molar excess relative to GTPS would have half-maximally
blocked GTPS-stimulated AC activity. Thus, in the AC assay,
MANT-GTPS 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-GTPS involves binding
of the inhibitor to the nucleotide-binding pocket of
GsS.
In Sf9 membranes co-expressing the 2AR and
GsS, 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 Gs
(32). The 2AR agonist ()-isoproterenol further
increased GTP-dependent AC activity. MANT-GTPS (3 µM) almost abolished AC activation by GTP and GTP plus
agonist. Even GTP at a 33-fold molar excess relative to MANT-GTPS failed to overcome this inhibition. Similarly, MANT-GTPS inhibited AC activation by GTPS (Fig. 5E), GppNHp (Fig.
5F), and AlF (Fig. 5G)
non-competitively. Fig. 6 depicts the
effect of MANT-GppNHp on AC activity in membranes from cholera
toxin-treated S49 wt lymphoma cells. Cholera toxin ADP-ribosylates
Gs and, thereby, blocks its GTPase (33). As a result,
GTP acquires GTPS/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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Fig. 6.
Regulation of AC activity by GTP and
MANT-GppNHp in membranes from cholera toxin-treated S49 wt lymphoma
cells. Prior to membrane preparation, S49 wt cells were treated
with 1 µg/ml cholera toxin for 24 h. AC activity in S49 wt cell
membranes (50 µg of protein/tube) was determined as described under
"Experimental Procedures." A, reaction mixtures
contained GTP at increasing concentrations in the absence or presence
of MANT-GppNHp (5 µM). B, reaction mixtures
contained 10 µM GTP in the presence of MANT-GppNHp at
increasing concentrations. Data were analyzed by non-linear regression.
Data shown are the means ± S.D. of 3 independent experiments
performed in duplicate.
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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 GsS with MANT-GTPS 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
GTPS-stimulated AC activities compared with activities of
solvent-treated membranes (data not shown). These data argue against
the hypothesis that MANT-nucleotides inhibited GsS
through disulfide bridge formation between the -thiophosphate and a
cysteinyl residue in the G-protein. Moreover, MANT-GTPS (10 µM) completely blocked AC in Sf9 membranes
expressing GsS in the presence of GTPS (10 µM) within 15 s, i.e. the earliest time
point studied. This result indicates that GTPS binds to its target
rapidly. In contrast, the onset of AC inhibition by GDPS in
membranes from cholera toxin-treated turkey erythrocytes requires
several minutes to be complete, reflecting slow dissociation of GTP
from Gs (34).
Taken together, the differences in the interactions of
MANT-GTPS/MANT-GppNHp with GTPS/GppNHp/GTP/AlF versus the interactions of GDPS with GTPS/GTP argue
against the hypothesis that MANT-GTPS/MANT-GppNHp bind to
GsS to stabilize an inhibitory conformation. Rather, the
noncompetitive interactions of MANT-GTPS/MANT-GppNHp with
GTPS/ GppNHp/GTP/AlF regarding AC inhibition
point to a site of action of MANT-nucleotides that is distinct from
GsS.
The Inhibitory Effects of MANT-GTPS/MANT-GppNHp on AC
Are Not Due to Gi Activation--
We addressed the
hypothesis that MANT-GTPS/MANT-GppNHp inhibit AC via
Gi activation by studying AC regulation in membranes of
the Gs-deficient cell line S49
cyc. In agreement with previous data (6, 18),
GTPS and GppNHp inhibited forskolin-stimulated AC in S49
cyc membranes in the presence of
Mg2+ by 45-50% (Fig.
7A). As expected (6, 18),
GDPS was less efficacious at activating Gi than
GTPS/GppNHp (Fig. 7A). The IC50 values of
GTPS 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
GTPS/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
Gi molecules has to be activated for AC inhibition,
whereas the GTPase assay assesses the entire population of
Gi molecules.
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Fig. 7.
Regulation of AC activity by guanine
nucleotides in membranes from S49 cyc
lymphoma cells. AC activity in S49 cyc
cell membranes (50 µg of protein/tube) was determined as described
under "Experimental Procedures." A, reaction mixtures
contained 5 mM MgCl2, 100 µM
forskolin, and guanine nucleotides at increasing concentrations.
B, reaction mixtures contained 10 mM
MnCl2, 100 µM forskolin, and guanine
nucleotides at increasing concentrations. Note the different scales of
the y axes in A and B. Data were
analyzed by non-linear regression. Data shown are the means ± S.D. of 3-4 independent experiments performed in duplicate.
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In contrast to GTPS, GppNHp, and GDPS, MANT-GTPS and
MANT-GppNHp abolished AC activity in S49 cyc
membranes (IC50 MANT-GTPS, 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-GTPS/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 GTPS/GppNHp/GDPS versus
MANT-GTPS/MANT- GppNHp on AC activity in S49
cyc membranes and in the GTPase competition
assay indicate that MANT-nucleotides did not inhibit AC via
Gi 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 GsS,
forskolin increased AC activity with an EC50 of 3.5 µM (CI, 1.2-8.8 µM) (Fig. 5H).
MANT-GTPS inhibited this AC activation non-competitively. This
finding indicates that MANT-GTPS 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 Gs and Gi. We took advantage of the fact
that Mn2+ (10 mM) blocks Gi
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), GTPS, GppNHp, and GDPS did not inhibit AC in S49 cyc
membranes in the presence of Mn2+ (Fig. 7B). In
marked contrast, MANT-GTPS 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-GTPS and MANT-GppNHp at inhibiting AC by ~4- and ~8-fold, respectively (Fig. 7, A and
B). These data indicate that, indeed,
MANT-GTPS/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-GTPS 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-GTPS 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-GTPS 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).
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Fig. 8.
Lineweaver-Burk analysis of the interaction
of MANT-GTPS/MANT-GppNHp with ATP at AC in S49
cyc cell membranes and membranes from
uninfected Sf9 cells. AC activity in S49
cyc cell membranes and membranes from
uninfected Sf9 cells (to exclude interference of
GsS with MANT-GppNHp) were determined as described under
"Experimental Procedures." Reaction mixtures contained membranes
(50 µg of protein/tube), 10 mM MnCl2, 100 µM forskolin, and 10 µM-1 mM
unlabeled ATP/Mn2+ plus 1.5 µCi of
[-32P]ATP and MANT-GTPS/MANT-GppNHp at the
concentrations indicated on the graph. Data were plotted
double-reciprocally and analyzed by linear regression according to
Lineweaver-Burk. The r2 values of the regression
lines were 0.88-0.99. Shown are the results of a representative
experiment. Similar results were obtained in 3 independent
experiments.
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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).
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DISCUSSION |
MANT-GTPS/MANT-GppNHp Bind to Gs- and
Gi-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 GTPS and GppNHp for
Gs and Gi (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
GTPS and GppNHp bind to G in non-identical manners. In fact, the
-thiophosphate of GTPS is bulkier than the ,-imidophosphate of GppNHp, resulting in different crystal structures of the catalytic sites of G-GTPS 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-GTPS.
Even at high concentrations that fully saturate G with
MANT-GTPS/MANT-GppNHp (10-100 µM) (Fig. 2), these
nucleotides are inefficient at stimulating AC via Gs
(Fig. 5A), inhibiting AC via Gi (Fig.
7A), and disrupting the ternary complex between FPR and
Gi2 (Fig. 4B). In addition, MANT-GTP is
inefficient at activating transducin (11) and GsS. 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
Gi1 (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-GTPS/MANT-GppNHp on Gs- and
Gi-mediated signaling were evident at the AC level.
Under all conditions studied, MANT-GTPS/MANT-GppNHp abolished AC
activity (Figs. 5-8). We ruled out Gs,
Gi, and the forskolin-binding site of AC as targets of
action of MANT-GTPS/MANT-GppNHp. Instead, MANT-GTPS/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-GTPS is
structurally quite different from
-L-2'3'-dideoxyadenosine 5'-triphosphate and ~3-fold
less potent under comparable experimental conditions (IC50
of MANT-GTPS 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 </
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ABSTRACT
INTRODUCTION
