2'(3')-O-(N-Methylanthraniloyl)-substituted GTP Analogs: A Novel Class of Potent Competitive Adenylyl Cyclase Inhibitors*

Andreas Gille and Roland SeifertDagger

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

    ABSTRACT
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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'-[gamma -thio]triphosphate (MANT-GTPgamma S) and MANT-guanosine 5'-[beta ,gamma -imido]triphosphate (MANT-GppNHp) on Galpha s- and Galpha i-protein-mediated signaling. MANT-GTPgamma S/MANT-GppNHp had lower affinities for Galpha s and Galpha i than GTPgamma S/GppNHp as assessed by inhibition of GTP hydrolysis of receptor-Galpha fusion proteins. MANT-GTPgamma S was much less effective than GTPgamma S at disrupting the ternary complex between the formyl peptide receptor and Galpha i2. MANT-GTPgamma S/MANT-GppNHp non-competitively inhibited GTPgamma S/GppNHp-, AlF<UP><SUB>4</SUB><SUP>−</SUP></UP>-, beta 2-adrenoceptor plus GTP-, cholera toxin plus GTP-, and forskolin-stimulated adenylyl cyclase (AC) in Galpha s-expressing Sf9 insect cell membranes and S49 wild-type lymphoma cell membranes. AC inhibition by MANT-GTPgamma S/MANT-GppNHp was not due to Galpha s inhibition because it was also observed in Galpha s-deficient S49 cyc- lymphoma cell membranes. Mn2+ blocked AC inhibition by GTPgamma S/GppNHp in S49 cyc- membranes but enhanced the potency of MANT-GTPgamma S/MANT-GppNHp at inhibiting AC by ~4-8-fold. MANT-GTPgamma 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-GTPgamma S/MANT-GppNHp bind to Galpha s- and Galpha i-proteins with low affinity and are ineffective at activating Galpha . Instead, MANT-GTPgamma S/MANT-GppNHp constitute a novel class of potent competitive AC inhibitors.

    INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

G-proteins are heterotrimeric (alpha beta gamma -structure) and serve as signal transducers between agonist-occupied GPCRs1 and effector systems (1, 2). GPCR promotes GDP dissociation from Galpha . 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 Galpha . Galpha GTP dissociates from GPCR, thereby disrupting the ternary complex. In addition, Galpha GTP and beta gamma dissociate from each other, and both Galpha GTP and beta gamma regulate the activity of effector systems. G-proteins are deactivated by the GTPase of Galpha that cleaves GTP into GDP and Pi. The GTP hydrolysis-resistant GTP analogs GTPgamma S and GppNHp (Fig. 1) induce persistent G-protein activation as does AlF<UP><SUB>4</SUB><SUP>−</SUP></UP>, the latter mimicking the transition state of GTP hydrolysis as GDP-AlF<UP><SUB>4</SUB><SUP>−</SUP></UP> complex (1, 3, 4). The hydrolysis-resistant GDP analog GDPbeta S (Fig. 1) is a partial G-protein activator (5, 6).


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Fig. 1.   Structures of GTPgamma S, GppNHp, MANT-GTPgamma S, MANT-GppNHp, and GDPbeta S. The gamma -thiophosphate group in GTPgamma S/MANT-GTPgamma S, the beta -thiophosphate group in GDPbeta S, and the beta ,gamma -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-GTPgamma 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-GTPgamma S 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 GTPgamma S for purified Galpha i1 (8, 9). The maximum fluorescence of Gi/Go-proteins induced by MANT-GTPgamma 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 GTPgamma S/GppNHp, MANT-GTPgamma 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-GTPgamma S/MANT-GppNHp on Gs- and Gi-protein-mediated signaling. As models we used fusion proteins and co-expression systems of the beta 2AR with the Galpha s-proteins, Galpha sL, Galpha sS, or Galpha olf (12-14), fusion proteins, and co-expression systems of the FPR with the Galpha i-proteins, Galpha i1, Galpha i2, or Galpha i3 (15, 16), and individually expressed Galpha sS. As physiologically relevant systems, we studied S49 wt lymphoma cell membranes, a standard model for the analysis of Galpha s-proteins (17) and S49 cyc- cell membranes, a Galpha s-deficient S49 mutant cell line serving as model for the analysis of Gi-proteins (6, 18). Galpha s activates, and Galpha i inhibits, the effector AC (19, 20). Surprisingly, we found that MANT-GTPgamma S/MANT-GppNHp constitute a novel class of potent competitive AC inhibitors.

    EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
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Materials-- Initially, MANT-GTPgamma S, 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-GTPgamma 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, GTPgamma S, GppNHp, GDPbeta 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 Galpha sS and Galpha 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 beta 1gamma 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 beta 2AR-Galpha s- and FPR-Galpha i fusion proteins, beta 2AR, and FPR have been described elsewhere (12, 13, 15, 16, 21). [3H]Dihydroalprenolol (85-90 Ci/mmol), [3H]FMLP (56 Ci/mmol), [alpha -32P]ATP (3,000 Ci/mmol), and [gamma -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 beta 2AR-Galpha s fusion proteins and beta 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-Galpha 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 beta 2AR-Galpha 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 + Galpha i2 + beta 1gamma 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 (beta 2AR-Galpha s fusion proteins) or 10 µM FMLP (FPR-Galpha i fusion proteins). Reaction mixtures (80 µl) were incubated for 3 min at 25 °C before the addition of 20 µl of [gamma -32P]GTP (0.2-0.5 µCi/tube). All stock and work dilutions of [gamma -32P]GTP were prepared in 20 mM Tris/HCl, pH 7.4, because [gamma -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 [gamma -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) [alpha -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 [alpha -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 [alpha -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 Galpha sS in the presence of MANT-GTPgamma S/MANT-GppNHp) or high isotopic dilution of [alpha -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).

    RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

MANT-GTPgamma S/MANT-GppNHp Bind to Galpha s- and Galpha i-proteins with Low Affinity-- We determined the affinities of GTPgamma S and GppNHp and their MANT-derivatives for Galpha s- and Galpha i-proteins by measuring agonist-stimulated steady-state GTP hydrolysis of beta 2AR-Galpha s and FPR-Galpha i fusion proteins expressed in Sf9 insect cell membranes (13, 14, 16, 23). The affinity profiles of nucleotides were similar for Galpha s-proteins (Galpha sL, Galpha sS, and Galpha olf) (Fig. 2, A-C) and Galpha i-proteins (Galpha i1, Galpha i2, and Galpha i3) (Fig. 2, D-F). GTPgamma S inhibited GTP hydrolysis with Ki values ranging from 3.6 (beta 2AR-Galpha olf) to 8.9 nM (FPR-Galpha i3). As expected (1, 2), GppNHp inhibited GTP hydrolysis with considerably lower potencies (~20-140-fold) than GTPgamma S. The Ki values for GppNHp ranged from 170 (FPR-Galpha i2) to 520 nM (beta 2AR-Galpha olf). The introduction of the MANT group at the 2'(3')-O-position of the ribosyl group reduced the affinity of GTPgamma S for Galpha s- and Galpha i-proteins by ~30-300-fold, i.e. the Ki values for MANT-GTPgamma S ranged from 250 (FPR-Galpha i2) to 1100 nM (beta 2AR-Galpha olf). The affinities of MANT-GppNHp for Galpha s- and Galpha 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-Galpha i2) to 5.8 µM (beta 2AR-Galpha olf).


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Fig. 2.   Competition of [gamma -32P]GTP hydrolysis in Sf9 membranes expressing beta 2AR-Galpha s- and FPR-Galpha i fusion proteins by GTPgamma S, GppNHp, MANT-GTPgamma S, 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, [gamma -32P]GTP (0.2-0.5 µCi/tube), and unlabeled GTPgamma S, GppNHp, MANT-GTPgamma S, or MANT-GppNHp at increasing concentrations. Reaction mixtures additionally contained 10 µM (-)-isoproterenol (beta 2AR-Galpha s fusion proteins) or 10 µM FMLP (FPR-Galpha i fusion proteins). GTPase activities in the absence of competitor (control) were as follows. beta 2AR-Galpha olf (expressed at 13.7 pmol/mg, [3H]dihydroalprenolol saturation binding), 8.5 ± 0.4 pmol/mg/min; beta 2AR-Galpha sS (expressed at 4.0 pmol/mg), 3.3 ± 0.3 pmol/mg/min; beta 2AR-Galpha sL (expressed at 6.0 pmol/mg), 5.2 ± 0.6 pmol/mg/min; FPR-Galpha i1 (expressed at 0.77 pmol/mg, [3H]FMLP saturation binding), 17.2 pmol/mg/min; and FPR-Galpha i2 (0.47 pmol/mg, 11.1 pmol/mg/min), FPR-Galpha i3 (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.

We also analyzed GTPases with GTP at increasing concentrations in the presence of MANT-GTPgamma S 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-GTPgamma S. In fact, MANT-GTPgamma S exhibited competitive interaction with GTP at Galpha s (Fig. 3A) and Galpha i (Fig. 3B). Collectively, our data show that MANT-GTPgamma S and MANT-GppNHp bind to Galpha s- and Galpha i-proteins, although with low affinity.


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Fig. 3.   Lineweaver-Burk analysis of the interaction of MANT-GTPgamma S with GTP at the GTPase of Galpha sS and Galpha i2. GTPase activity in Sf9 membranes was determined as described under "Experimental Procedures." Reaction mixtures contained Sf9 membranes (10 µg of protein/tube) expressing beta 2AR-Galpha sS (4.0 pmol/mg, [3H]dihydroalprenolol saturation binding) or FPR (0.5 pmol/mg, [3H]FMLP saturation binding) + Galpha i2 (300 pmol/mg as assessed by quantitative immunoblotting using the beta 2AR-Galpha i2 fusion protein as standard) + beta 1gamma 2, 100 nM to 1.5 µM unlabeled GTP plus [gamma -32P]GTP (0.2-0.5 µCi/tube), and unlabeled MANT-GTPgamma S at the concentrations indicated in the graph. Reaction mixtures additionally contained 10 µM (-)-isoproterenol (beta 2AR-Galpha sS) 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.

Effects of MANT-GTPgamma S on Ternary Complex Formation-- In case of the beta 2AR/Galpha 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 beta 2AR and, thereby, increase [3H]dihydroalprenolol binding (13, 14, 27). As reported before (14), GTPgamma 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 beta 2AR-Galpha sS (Fig. 4A). MANT-GTPgamma S (1 µM) reduced ternary complex formation in beta 2AR-Galpha sS by ~50%, but at higher concentrations, the effect of MANT-GTPgamma S was reverted. MANT-GTPgamma 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-GTPgamma S inhibited [3H]dihydroalprenolol binding to the beta 2AR. We examined the effect of MANT-GTPgamma S (10 µM) on binding of [3H]dihydroalprenolol in Sf9 membranes expressing the beta 2AR alone, i.e. a system in which ternary complex formation is not detected (12). In fact, MANT-GTPgamma S (10 µM) inhibited binding of [3H]dihydroalprenolol (1 nM) by 40%. Thus, because of interference of MANT-GTPgamma S with ligand binding to the beta 2AR, we could not answer the question whether GTPgamma S and MANT-GTPgamma S exhibit similar efficacies at disrupting the ternary complex in beta 2AR-Galpha sS.


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Fig. 4.   Effects of GTPgamma S and MANT-GTPgamma S on ternary complex formation in the beta 2AR/Galpha sS couple and the FPR/Galpha i2 couple. Ternary complex formation in Sf9 membranes was determined as described under "Experimental Procedures." A, for ternary complex formation with the beta 2AR/Galpha sS couple, reaction mixtures contained Sf9 membranes expressing beta 2AR-Galpha sS (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 GTPgamma S was defined as 0% ternary complex formation. All other data referred to those calibration points. B, for ternary complex formation with the FPR/Galpha i2 couple, reaction mixtures contained Sf9 membranes expressing FPR (0.5-1.0 pmol/mg) + Galpha i2 (300 pmol/mg) + beta 1gamma 2 (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 GTPgamma S 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.

In case of the FPR/Galpha i2 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), GTPgamma S potently (IC50, 26 nM; CI, 15-43 nM) and efficaciously disrupted the ternary complex of the FPR/Galpha i2 couple (Fig. 4B). Considering the Ki values of MANT-GTPgamma S for Galpha i-proteins (250-500 nM) (Fig. 2, D-F), we would have expected maximal disruption of the ternary complex with MANT-GTPgamma S at 10 µM. However, MANT-GTPgamma S (10 µM) decreased [3H]FMLP binding by not more than 25%. Thus, MANT-GTPgamma S is rather inefficient at stabilizing the conformation in Galpha 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 beta 2AR and Galpha s (23, 27).

MANT-GTPgamma S/MANT-GppNHp Are Potent Inhibitors of Galpha s-stimulated AC; AC Inhibition Is Not Due to Stabilization of an Inhibitory Galpha s Conformation-- We analyzed the effects of GTP analogs on AC activity in Sf9 membranes expressing Galpha sS. GTPgamma 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. GTPgamma S and GppNHp were similarly efficacious at activating AC. In agreement with previous results (5), GDPbeta S was less efficacious at activating AC than GTPgamma S/GppNHp, i.e. GDPbeta S acted as a partial Galpha s activator. MANT-GTPgamma S and MANT-GppNHp abolished basal AC activity in Sf9 membranes expressing Galpha sS (reflecting the activity of Galpha 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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Fig. 5.   Regulation of AC activity in Sf9 membranes by guanine nucleotides, beta 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 Galpha sS (5.9 ± 0.3 pmol/mg as assessed by quantitative immunoblotting using the beta 2AR-Galpha sS fusion protein as standard) (A-C and E-H) or Sf9 membranes expressing beta 2AR (9.5 pmol/mg) + Galpha sS (6.4 pmol/mg) (D). A, reaction mixtures contained GTPgamma S, GppNHp, GDPbeta S, MANT-GTPgamma S, or MANT-GppNHp at increasing concentrations. B, reaction mixtures contained GTPgamma S at increasing concentrations in the absence or presence of GDPbeta S at various fixed concentrations. C, reaction mixtures contained 10 µM GTPgamma S plus MANT-GTPgamma S 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-GTPgamma S. E, reaction mixtures contained GTPgamma S at increasing concentrations in the absence or presence of MANT-GTPgamma S at various fixed concentrations. F, reaction mixtures contained GppNHp at increasing concentrations in the absence or presence of MANT-GTPgamma S (10 µM). G, reaction mixtures contained NaF at increasing concentrations and 10 µM AlCl3 in the absence or presence of MANT-GTPgamma S at various fixed concentrations. H, reaction mixtures contained forskolin at increasing concentrations in the absence or presence of MANT-GTPgamma S (3 µM). Data were analyzed by non-linear regression. Data shown are the means ± S.D. of 2-4 independent experiments performed in duplicate.

There is evidence to support the concept of multiple Galpha states (4, 5, 8, 23, 26, 27). Based on the literature and our present data, the hypothesis evolved that MANT-GTPgamma S/MANT-GppNHp could stabilize an inhibitory Galpha s conformation. To test this hypothesis, we examined the interactions of MANT-GTPgamma S/MANT-GppNHp with GTP, GTPgamma S, GppNHp, GDPbeta S and AlF<UP><SUB>4</SUB><SUP>−</SUP></UP>, i.e. substances that all bind to the nucleotide-binding pocket of Galpha and stabilize distinct conformations (4, 5, 8, 30). If MANT-GTPgamma S/MANT-GppNHp inhibited AC via Galpha sS, they should competitively block the stimulatory effects of GTPgamma S, GppNHp, GTP, GDPbeta S, and AlF<UP><SUB>4</SUB><SUP>−</SUP></UP> on AC. As positive control, we studied the interaction of the full Galpha sS activator, GTPgamma S, with the partial Galpha sS activator, GDPbeta S, on AC (Fig. 5A) (5). Competitive interaction of MANT-GTPgamma S with GTP was already observed in the GTPase studies (Fig. 3). GDPbeta S shifted the concentration/response curves for GTPgamma S to the right without reducing the maximal AC activity obtained with GTPgamma S (Fig. 5B). These data confirm the competitive interaction of guanine nucleotides at the nucleotide-binding site of Galpha s (31).

MANT-GTPgamma S abolished AC activity stimulated by GTPgamma 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-GTPgamma S at an ~100-fold molar excess relative to GTPgamma S would have half-maximally blocked GTPgamma S-stimulated AC activity. Thus, in the AC assay, MANT-GTPgamma 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-GTPgamma S involves binding of the inhibitor to the nucleotide-binding pocket of Galpha sS.

In Sf9 membranes co-expressing the beta 2AR and Galpha 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 beta 2AR at promoting GDP/GTP exchange at Galpha s (32). The beta 2AR agonist (-)-isoproterenol further increased GTP-dependent AC activity. MANT-GTPgamma S (3 µM) almost abolished AC activation by GTP and GTP plus agonist. Even GTP at a 33-fold molar excess relative to MANT-GTPgamma S failed to overcome this inhibition. Similarly, MANT-GTPgamma S inhibited AC activation by GTPgamma S (Fig. 5E), GppNHp (Fig. 5F), and AlF<UP><SUB>4</SUB><SUP>−</SUP></UP> (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 Galpha s and, thereby, blocks its GTPase (33). As a result, GTP acquires GTPgamma 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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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.

The inhibitory effects of MANT-GTPgamma S and MANT-GppNHp on AC were fully reversible, i.e. pre-treatment of Sf9 membranes expressing Galpha sS with MANT-GTPgamma 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 GTPgamma S-stimulated AC activities compared with activities of solvent-treated membranes (data not shown). These data argue against the hypothesis that MANT-nucleotides inhibited Galpha sS through disulfide bridge formation between the gamma -thiophosphate and a cysteinyl residue in the G-protein. Moreover, MANT-GTPgamma S (10 µM) completely blocked AC in Sf9 membranes expressing Galpha sS in the presence of GTPgamma S (10 µM) within 15 s, i.e. the earliest time point studied. This result indicates that GTPgamma S binds to its target rapidly. In contrast, the onset of AC inhibition by GDPbeta S in membranes from cholera toxin-treated turkey erythrocytes requires several minutes to be complete, reflecting slow dissociation of GTP from Galpha s (34).

Taken together, the differences in the interactions of MANT-GTPgamma S/MANT-GppNHp with GTPgamma S/GppNHp/GTP/AlF<UP><SUB>4</SUB><SUP>−</SUP></UP> versus the interactions of GDPbeta S with GTPgamma S/GTP argue against the hypothesis that MANT-GTPgamma S/MANT-GppNHp bind to Galpha sS to stabilize an inhibitory conformation. Rather, the noncompetitive interactions of MANT-GTPgamma S/MANT-GppNHp with GTPgamma S/ GppNHp/GTP/AlF<UP><SUB>4</SUB><SUP>−</SUP></UP> regarding AC inhibition point to a site of action of MANT-nucleotides that is distinct from Galpha sS.

The Inhibitory Effects of MANT-GTPgamma S/MANT-GppNHp on AC Are Not Due to Galpha i Activation-- We addressed the hypothesis that MANT-GTPgamma S/MANT-GppNHp inhibit AC via Galpha i activation by studying AC regulation in membranes of the Galpha s-deficient cell line S49 cyc-. In agreement with previous data (6, 18), GTPgamma 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), GDPbeta S was less efficacious at activating Galpha i than GTPgamma S/GppNHp (Fig. 7A). The IC50 values of GTPgamma 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 GTPgamma 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 Galpha i molecules has to be activated for AC inhibition, whereas the GTPase assay assesses the entire population of Galpha i 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.

In contrast to GTPgamma S, GppNHp, and GDPbeta S, MANT-GTPgamma S and MANT-GppNHp abolished AC activity in S49 cyc- membranes (IC50 MANT-GTPgamma 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-GTPgamma 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 GTPgamma S/GppNHp/GDPbeta S versus MANT-GTPgamma 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 Galpha i activation but through a different mechanism.

MANT-GTPgamma S/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 Galpha sS, forskolin increased AC activity with an EC50 of 3.5 µM (CI, 1.2-8.8 µM) (Fig. 5H). MANT-GTPgamma S inhibited this AC activation non-competitively. This finding indicates that MANT-GTPgamma 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-GTPgamma S/MANT-GppNHp inhibit AC directly, we wished to study AC regulation independently of Galpha s and Galpha i. We took advantage of the fact that Mn2+ (10 mM) blocks Galpha 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), GTPgamma S, GppNHp, and GDPbeta S did not inhibit AC in S49 cyc- membranes in the presence of Mn2+ (Fig. 7B). In marked contrast, MANT-GTPgamma 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-GTPgamma S and MANT-GppNHp at inhibiting AC by ~4- and ~8-fold, respectively (Fig. 7, A and B). These data indicate that, indeed, MANT-GTPgamma 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-GTPgamma 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-GTPgamma 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-GTPgamma 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).


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Fig. 8.   Lineweaver-Burk analysis of the interaction of MANT-GTPgamma S/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 Galpha sS 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 [alpha -32P]ATP and MANT-GTPgamma S/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.

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-GTPgamma S/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
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

MANT-GTPgamma S/MANT-GppNHp Bind to Galpha s- and Galpha i-proteins with Low Affinity and Are Inefficient Galpha Activators-- A MANT group at the 2'(3')-O-position of the ribosyl group reduces the affinity of GTPgamma S and GppNHp for Galpha s and Galpha 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 Galpha (43). It is also conceivable that there are steric hindrances to accommodate the bulky MANT group in the tight nucleotide-binding pocket of Galpha (4, 43).

The MANT group reduces the affinity of GTPgamma S for Galpha to a greater extent than the affinity of GppNHp (Fig. 1). These data indicate that GTPgamma S and GppNHp bind to Galpha in non-identical manners. In fact, the gamma -thiophosphate of GTPgamma S is bulkier than the beta ,gamma -imidophosphate of GppNHp, resulting in different crystal structures of the catalytic sites of Galpha -GTPgamma S and Galpha - GppNHp (30). Through differentially propagated conformational changes from the catalytic site of Galpha to the ribosyl residue-binding domain, Galpha -GppNHp could accommodate the additional MANT group more readily than Galpha -GTPgamma S.

Even at high concentrations that fully saturate Galpha with MANT-GTPgamma S/MANT-GppNHp (10-100 µM) (Fig. 2), these nucleotides are inefficient at stimulating AC via Galpha s (Fig. 5A), inhibiting AC via Galpha i (Fig. 7A), and disrupting the ternary complex between FPR and Galpha i2 (Fig. 4B). In addition, MANT-GTP is inefficient at activating transducin (11) and Galpha sS. Thus, MANT modification of guanine nucleotides is also associated with a loss of efficacy at activating Galpha . These results corroborate the concept of multiple Galpha states that are differentially stabilized by various nucleotides (4, 5, 8, 23, 26, 27).

In marked contrast to the data obtained with GPCR-Galpha fusion proteins (Fig. 1) and transducin examined in native membranes (11), the MANT group had no adverse effect on nucleotide affinities of purified Galpha 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-GTPgamma S/MANT-GppNHp Are Potent Competitive AC Inhibitors; Comparison with the Literature-- The most prominent effects of MANT-GTPgamma S/MANT-GppNHp on Galpha s- and Galpha i-mediated signaling were evident at the AC level. Under all conditions studied, MANT-GTPgamma S/MANT-GppNHp abolished AC activity (Figs. 5-8). We ruled out Galpha s, Galpha i, and the forskolin-binding site of AC as targets of action of MANT-GTPgamma S/MANT-GppNHp. Instead, MANT-GTPgamma S/MANT-GppNHp are competitive AC inhibitors (Fig. 8). beta -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-GTPgamma S is structurally quite different from beta -L-2'3'-dideoxyadenosine 5'-triphosphate and ~3-fold less potent under comparable experimental conditions (IC50 of MANT-GTPgamma 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-GTPgamma S 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-GTPgamma S/MANT-GppNHp with AC-- At concentrations up to 100 µM, GTPgamma 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 GTPgamma 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 GTPgamma 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 GTPgamma S/GppNHp for AC. Accordingly, substitution of the 3'-O-ribosyl group with MANT should reduce GTPgamma 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-GTPgamma S/GppNHp > 2'(3')-MANT-GTPgamma S/GppNHp > 2'-deoxy-3'-MANT-GTPgamma 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-GTPgamma S 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-GTPgamma 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-GTPgamma 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-GTPgamma S 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.

Dagger 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; beta 2AR, beta 2-adrenoceptor; beta 2AR-Galpha olf, fusion protein consisting of the beta 2-adrenoceptor and Galpha olf; beta 2AR-Galpha sL, fusion protein consisting of the beta 2-adrenoceptor and the long splice variant of Galpha s; beta 2AR-Galpha sS, fusion protein consisting of the beta 2-adrenoceptor and the short splice variant of Galpha s; FMLP, N-formyl-L-methionyl-L-leucyl-L-phenylalanine; FPR, formyl peptide receptor; FPR-Galpha i1, 2,3, fusion protein consisting of the formyl peptide receptor and Galpha i1, Galpha i2, or Galpha i3; Galpha , non-specified G-protein alpha -subunit; Galpha i, family of three G-protein alpha -subunits (Galpha i1, Galpha i2, and Galpha i3) that mediates adenylyl cyclase inhibition; Galpha s, family of three G-protein alpha -subunits (Galpha sL, Galpha sS, and Galpha olf) that mediates adenylyl cyclase activation; Galpha olf, olfactory Galpha s-protein; Galpha sL, long splice variant of Galpha s; Galpha sS, short splice variant of Galpha s; GDPbeta S, guanosine 5'-[beta -thio]diphosphate; GppNHp, guanosine 5'-[beta ,gamma -imido]-triphosphate; GTPgamma S, guanosine 5'-[gamma -thio]triphosphate; MANT-GppNHp, 2'(3')-O-(N-methylanthraniloyl)-guanosine 5'-[beta ,gamma -imido]-triphosphate; MANT-GTPgamma S, 2'(3')-O-(N-methylanthraniloyl)-guanosine 5'-[gamma -thio]triphosphate; S49 wt cells, S49 wild-type lymphoma cells; S49 cyc- cells, Galpha s-deficient S49 lymphoma cells; AppNHp, adenosine 5'-[beta ,gamma -imido]triphosphate; ApCHpp, adenosine 5'-[alpha ,beta -methylene]triphosphate.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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