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

2003; Elsevier BV; Volume: 278; Issue: 15 Linguagem: Inglês

10.1074/jbc.m211292200

ISSN

1083-351X

Autores

Andreas Gille, Roland Seifert,

Tópico(s)

Phosphodiesterase function and regulation

Resumo

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′-[γ-thio]triphosphate (MANT-GTPγS) and MANT-guanosine 5′-[β,γ-imido]triphosphate (MANT-GppNHp) on Gαs- and Gαi-protein-mediated signaling. MANT-GTPγS/MANT-GppNHp had lower affinities for Gαs and Gαi than GTPγS/GppNHp as assessed by inhibition of GTP hydrolysis of receptor-Gα fusion proteins. MANT-GTPγS was much less effective than GTPγS at disrupting the ternary complex between the formyl peptide receptor and Gαi2. MANT-GTPγS/MANT-GppNHp non-competitively inhibited GTPγS/GppNHp-, AlF4−-, β2-adrenoceptor plus GTP-, cholera toxin plus GTP-, and forskolin-stimulated adenylyl cyclase (AC) in Gαs-expressing Sf9 insect cell membranes and S49 wild-type lymphoma cell membranes. AC inhibition by MANT-GTPγS/MANT-GppNHp was not due to Gαs inhibition because it was also observed in Gαs-deficient S49cyc− lymphoma cell membranes. Mn2+blocked AC inhibition by GTPγS/GppNHp in S49cyc− membranes but enhanced the potency of MANT-GTPγS/MANT-GppNHp at inhibiting AC by ∼4–8-fold. MANT-GTPγS and MANT-GppNHp competitively inhibited forskolin/Mn2+-stimulated AC in S49cyc− membranes with Kivalues of 53 and 160 nm, respectively. TheKi value for MANT-GppNHp at insect cell AC was 155 nm. Collectively, MANT-GTPγS/MANT-GppNHp bind to Gαs- and Gαi-proteins with low affinity and are ineffective at activating Gα. Instead, MANT-GTPγS/MANT-GppNHp constitute a novel class of potent competitive AC inhibitors. 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′-[γ-thio]triphosphate (MANT-GTPγS) and MANT-guanosine 5′-[β,γ-imido]triphosphate (MANT-GppNHp) on Gαs- and Gαi-protein-mediated signaling. MANT-GTPγS/MANT-GppNHp had lower affinities for Gαs and Gαi than GTPγS/GppNHp as assessed by inhibition of GTP hydrolysis of receptor-Gα fusion proteins. MANT-GTPγS was much less effective than GTPγS at disrupting the ternary complex between the formyl peptide receptor and Gαi2. MANT-GTPγS/MANT-GppNHp non-competitively inhibited GTPγS/GppNHp-, AlF4−-, β2-adrenoceptor plus GTP-, cholera toxin plus GTP-, and forskolin-stimulated adenylyl cyclase (AC) in Gαs-expressing Sf9 insect cell membranes and S49 wild-type lymphoma cell membranes. AC inhibition by MANT-GTPγS/MANT-GppNHp was not due to Gαs inhibition because it was also observed in Gαs-deficient S49cyc− lymphoma cell membranes. Mn2+blocked AC inhibition by GTPγS/GppNHp in S49cyc− membranes but enhanced the potency of MANT-GTPγS/MANT-GppNHp at inhibiting AC by ∼4–8-fold. MANT-GTPγS and MANT-GppNHp competitively inhibited forskolin/Mn2+-stimulated AC in S49cyc− membranes with Kivalues of 53 and 160 nm, respectively. TheKi value for MANT-GppNHp at insect cell AC was 155 nm. Collectively, MANT-GTPγS/MANT-GppNHp bind to Gαs- and Gαi-proteins with low affinity and are ineffective at activating Gα. Instead, MANT-GTPγS/MANT-GppNHp constitute a novel class of potent competitive AC inhibitors. G-protein-coupled receptor adenylyl cyclase β2-adrenoceptor fusion protein consisting of the β2-adrenoceptor and Gαolf fusion protein consisting of the β2-adrenoceptor and the long splice variant of Gαs fusion protein consisting of the β2-adrenoceptor and the short splice variant of Gαs N-formyl-l-methionyl-l-leucyl-l-phenylalanine formyl peptide receptor 2,3, fusion protein consisting of the formyl peptide receptor and Gαi1, Gαi2, or Gαi3 non-specified G-protein α-subunit family of three G-protein α-subunits (Gαi1, Gαi2, and Gαi3) that mediates adenylyl cyclase inhibition family of three G-protein α-subunits (GαsL, GαsS, and Gαolf) that mediates adenylyl cyclase activation olfactory Gαs-protein long splice variant of Gαs short splice variant of Gαs guanosine 5′-[β-thio]diphosphate guanosine 5′-[β,γ-imido]-triphosphate guanosine 5′-[γ-thio]triphosphate 2′(3′)-O-(N-methylanthraniloyl)-guanosine 5′-[β,γ-imido]-triphosphate 2′(3′)-O-(N-methylanthraniloyl)-guanosine 5′-[γ-thio]triphosphate S49 wild-type lymphoma cells Gαs-deficient S49 lymphoma cells adenosine 5′-[β,γ-imido]triphosphate adenosine 5′-[α,β-methylene]triphosphate G-proteins are heterotrimeric (αβγ-structure) and serve as signal transducers between agonist-occupied GPCRs1 and effector systems (1Gilman A.G. Annu. Rev. Biochem. 1987; 56: 615-649Crossref PubMed Scopus (5031) Google Scholar, 2Birnbaumer L. Abramowitz J. Brown A.M. Biochim. Biophys. Acta. 1990; 1031: 163-224Crossref PubMed Scopus (1006) Google Scholar). GPCR promotes GDP dissociation from Gα. 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 Gα. GαGTPdissociates from GPCR, thereby disrupting the ternary complex. In addition, GαGTP and βγ dissociate from each other, and both GαGTP and βγ regulate the activity of effector systems. G-proteins are deactivated by the GTPase of Gα that cleaves GTP into GDP and Pi. The GTP hydrolysis-resistant GTP analogs GTPγS and GppNHp (Fig. 1) induce persistent G-protein activation as does AlF4−, the latter mimicking the transition state of GTP hydrolysis as GDP-AlF4− complex (1Gilman A.G. Annu. Rev. Biochem. 1987; 56: 615-649Crossref PubMed Scopus (5031) Google Scholar, 3Eckstein F. Annu. Rev. Biochem. 1985; 54: 367-402Crossref PubMed Google Scholar, 4Sprang S.R. Annu. Rev. Biochem. 1997; 66: 639-678Crossref PubMed Scopus (896) Google Scholar). The hydrolysis-resistant GDP analog GDPβS (Fig. 1) is a partial G-protein activator (5Eckstein F. Cassel D. Levkovitz H. Lowe M. Selinger Z. J. Biol. Chem. 1979; 254: 9829-9834Abstract Full Text PDF PubMed Google Scholar,6Jakobs K.H. Gehring U. Gaugler B. Pfeuffer T. Schultz G. Eur. J. Biochem. 1983; 130: 605-611Crossref PubMed Scopus (48) Google Scholar). 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 (7Jameson D.M. Eccleston J.F. Methods Enzymol. 1997; 278: 363-390Crossref PubMed Scopus (120) Google Scholar). However, only few studies with MANT-nucleotides and G-proteins have been conducted so far, and the data are controversial. MANT-GTPγS 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 GTPγS for purified Gαi1 (8Remmers A.E. Neubig R.R. J. Biol. Chem. 1996; 271: 4791-4797Abstract Full Text Full Text PDF PubMed Scopus (29) Google Scholar, 9Remmers A.E. Anal. Biochem. 1998; 257: 89-94Crossref PubMed Scopus (19) Google Scholar). The maximum fluorescence of Gi/Go-proteins induced by MANT-GTPγS is higher than the maximum fluorescence induced by MANT-GppNHp, suggesting that the two nucleotides stabilize different conformations in G-proteins (8Remmers A.E. Neubig R.R. J. Biol. Chem. 1996; 271: 4791-4797Abstract Full Text Full Text PDF PubMed Scopus (29) Google Scholar, 10Remmers A.E. Posner R. Neubig R.R. J. Biol. Chem. 1994; 269: 13771-13778Abstract Full Text PDF PubMed Google Scholar). Moreover, like GTPγS/GppNHp, MANT-GTPγS/MANT-GppNHp confer protease protection to Gi/Go-proteins (8Remmers A.E. Neubig R.R. J. Biol. Chem. 1996; 271: 4791-4797Abstract Full Text Full Text PDF PubMed Scopus (29) Google Scholar). 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 (11Zera E.M. Molloy D.P. Angleson J.K. Lamture J.B. Wensel T.G. Malinski J.A. J. Biol. Chem. 1996; 271: 12925-12931Abstract Full Text Full Text PDF PubMed Scopus (21) Google Scholar). 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-GTPγS/MANT-GppNHp on Gs- and Gi-protein-mediated signaling. As models we used fusion proteins and co-expression systems of the β2AR with the Gαs-proteins, GαsL, GαsS, or Gαolf (12Seifert R. Lee T.W. Lam V.T. Kobilka B.K. Eur. J. Biochem. 1998; 255: 369-382Crossref PubMed Scopus (102) Google Scholar, 13Seifert R. Wenzel-Seifert K. Lee T.W. Gether U. Sanders-Bush E. Kobilka B.K. J. Biol. Chem. 1998; 273: 5109-5116Abstract Full Text Full Text PDF Scopus (131) Google Scholar, 14Liu H.-Y. Wenzel-Seifert K. Seifert R. J. Neurochem. 2001; 78: 325-338Crossref PubMed Scopus (42) Google Scholar), fusion proteins, and co-expression systems of the FPR with the Gαi-proteins, Gαi1, Gαi2, or Gαi3 (15Wenzel-Seifert K. Hurt C.M. Seifert R. J. Biol. Chem. 1998; 273: 24181-24189Abstract Full Text Full Text PDF PubMed Scopus (96) Google Scholar, 16Wenzel-Seifert K. Arthur J.M. Liu H.Y. Seifert R. J. Biol. Chem. 1999; 274: 33259-33266Abstract Full Text Full Text PDF PubMed Scopus (86) Google Scholar), and individually expressed GαsS. As physiologically relevant systems, we studied S49 wt lymphoma cell membranes, a standard model for the analysis of Gαs-proteins (17Ransnäs L.A. Insel P.A. J. Biol. Chem. 1988; 263: 9482-9485Abstract Full Text PDF PubMed Google Scholar) and S49cyc− cell membranes, a Gαs-deficient S49 mutant cell line serving as model for the analysis of Gi-proteins (6Jakobs K.H. Gehring U. Gaugler B. Pfeuffer T. Schultz G. Eur. J. Biochem. 1983; 130: 605-611Crossref PubMed Scopus (48) Google Scholar, 18Hildebrandt J.D. Hanoune J. Birnbaumer L. J. Biol. Chem. 1982; 257: 14723-14725Abstract Full Text PDF PubMed Google Scholar). Gαsactivates, and Gαi inhibits, the effector AC (19Sunahara R.K. Dessauer C.W. Gilman A.G. Annu. Rev. Pharmacol. Toxicol. 1996; 36: 461-480Crossref PubMed Scopus (748) Google Scholar, 20Tang W.J. Hurley J.H. Mol. Pharmacol. 1998; 54: 231-240Crossref PubMed Scopus (164) Google Scholar). Surprisingly, we found that MANT-GTPγS/MANT-GppNHp constitute a novel class of potent competitive AC inhibitors. Initially, MANT-GTPγS, 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-GTPγS 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, GTPγS, GppNHp, GDPβS, 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 GαsS and Gαi2 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 β1γ2 complex was donated Dr. P. Gierschik (Department of Pharmacology and Toxicology, University of Ulm, Germany). S49 wt and S49cyc− lymphoma cells were obtained from the Cell Culture Facility of the University of California, San Francisco. The construction of baculoviruses encoding β2AR-Gαs- and FPR-Gαi fusion proteins, β2AR, and FPR have been described elsewhere (12Seifert R. Lee T.W. Lam V.T. Kobilka B.K. Eur. J. Biochem. 1998; 255: 369-382Crossref PubMed Scopus (102) Google Scholar, 13Seifert R. Wenzel-Seifert K. Lee T.W. Gether U. Sanders-Bush E. Kobilka B.K. J. Biol. Chem. 1998; 273: 5109-5116Abstract Full Text Full Text PDF Scopus (131) Google Scholar, 15Wenzel-Seifert K. Hurt C.M. Seifert R. J. Biol. Chem. 1998; 273: 24181-24189Abstract Full Text Full Text PDF PubMed Scopus (96) Google Scholar, 16Wenzel-Seifert K. Arthur J.M. Liu H.Y. Seifert R. J. Biol. Chem. 1999; 274: 33259-33266Abstract Full Text Full Text PDF PubMed Scopus (86) Google Scholar, 21Gether U. Lin S. Kobilka B.K. J. Biol. Chem. 1995; 270: 28268-28275Abstract Full Text Full Text PDF PubMed Scopus (258) Google Scholar). [3H]Dihydroalprenolol (85–90 Ci/mmol), [3H]FMLP (56 Ci/mmol), [α-32P]ATP (3,000 Ci/mmol), and [γ-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. Sf9 cells were cultured and infected with 1:100 dilutions of high titer virus stocks as described (22Houston C. Wenzel-Seifert K. Bürckstümmer T. Seifert R. J. Neurochem. 2002; 80: 678-696Crossref PubMed Scopus (57) Google Scholar). Sf9 membranes were prepared as described (12Seifert R. Lee T.W. Lam V.T. Kobilka B.K. Eur. J. Biochem. 1998; 255: 369-382Crossref PubMed Scopus (102) Google Scholar) and stored at −80 °C until use. S49 wt and S49cyc− cells were cultured under the conditions described recently (23Gille A. Liu H.Y. Sprang S.R. Seifert R. J. Biol. Chem. 2002; 277: 34434-34442Abstract Full Text Full Text PDF PubMed Scopus (14) Google Scholar). 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. 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 β2AR-Gαs fusion proteins and β2AR 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-Gαi 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 β2AR-GαsS(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 + Gαi2 + β1γ2 (30–50 μg of protein/tube), 10 nm [3H]FMLP, and guanine nucleotides at increasing concentrations. 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 (14Liu H.-Y. Wenzel-Seifert K. Seifert R. J. Neurochem. 2001; 78: 325-338Crossref PubMed Scopus (42) Google Scholar). Assay tubes contained Sf9 membranes expressing fusion proteins (10 μg of protein/tube), 1.0 mm MgCl2, 0.1 mmEDTA, 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 (β2AR-Gαs fusion proteins) or 10 μm FMLP (FPR-Gαi fusion proteins). Reaction mixtures (80 μl) were incubated for 3 min at 25 °C before the addition of 20 μl of [γ-32P]GTP (0.2–0.5 μCi/tube). All stock and work dilutions of [γ-32P]GTP were prepared in 20 mm Tris/HCl, pH 7.4, because [γ-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 mmNaH2PO4, 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 [γ-32P]GTP degradation was determined in the presence of 1 mm unlabeled GTP and was <1% of the total amount of radioactivity added. 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 (14Liu H.-Y. Wenzel-Seifert K. Seifert R. J. Neurochem. 2001; 78: 325-338Crossref PubMed Scopus (42) Google Scholar). Briefly, tubes contained various membranes (15–50 μg of protein/tube), 5 mmMgCl2, 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 μmAlCl3, 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) [α-32P]ATP (1.0–1.5 μCi/tube) plus 40 μm unlabeled ATP, 2.7 mmmono(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 mmunlabeled ATP/Mn2+ plus 10 mmMnCl2. Reactions were conducted for 20 min at 37 °C. Reactions were terminated by the addition of 20 μl of 2.2n 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 [α-32P]ATP by elution of [32P]cAMP with 4 ml of 0.1 m ammonium acetate, pH 7.0 (24Alvarez R. Daniels D.V. Anal. Biochem. 1990; 187: 98-103Crossref PubMed Scopus (75) Google Scholar). Recovery of [32P]cAMP was ∼80%. Blank values were routinely ∼0.01% of the total amount of [α-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 GαsS in the presence of MANT-GTPγS/MANT-GppNHp) or high isotopic dilution of [α-32P]ATP (such as those in the presence of 1 mm unlabeled ATP). [32P]cAMP was determined by liquid scintillation counting. 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). We determined the affinities of GTPγS and GppNHp and their MANT-derivatives for Gαs- and Gαi-proteins by measuring agonist-stimulated steady-state GTP hydrolysis of β2AR-Gαs and FPR-Gαi fusion proteins expressed in Sf9 insect cell membranes (13Seifert R. Wenzel-Seifert K. Lee T.W. Gether U. Sanders-Bush E. Kobilka B.K. J. Biol. Chem. 1998; 273: 5109-5116Abstract Full Text Full Text PDF Scopus (131) Google Scholar, 14Liu H.-Y. Wenzel-Seifert K. Seifert R. J. Neurochem. 2001; 78: 325-338Crossref PubMed Scopus (42) Google Scholar, 16Wenzel-Seifert K. Arthur J.M. Liu H.Y. Seifert R. J. Biol. Chem. 1999; 274: 33259-33266Abstract Full Text Full Text PDF PubMed Scopus (86) Google Scholar,23Gille A. Liu H.Y. Sprang S.R. Seifert R. J. Biol. Chem. 2002; 277: 34434-34442Abstract Full Text Full Text PDF PubMed Scopus (14) Google Scholar). The affinity profiles of nucleotides were similar for Gαs-proteins (GαsL, GαsS, and Gαolf) (Fig. 2,A–C) and Gαi-proteins (Gαi1, Gαi2, and Gαi3) (Fig. 2, D–F). GTPγS inhibited GTP hydrolysis with Ki values ranging from 3.6 (β2AR-Gαolf) to 8.9 nm (FPR-Gαi3). As expected (1Gilman A.G. Annu. Rev. Biochem. 1987; 56: 615-649Crossref PubMed Scopus (5031) Google Scholar, 2Birnbaumer L. Abramowitz J. Brown A.M. Biochim. Biophys. Acta. 1990; 1031: 163-224Crossref PubMed Scopus (1006) Google Scholar), GppNHp inhibited GTP hydrolysis with considerably lower potencies (∼20–140-fold) than GTPγS. The Ki values for GppNHp ranged from 170 (FPR-Gαi2) to 520 nm(β2AR-Gαolf). The introduction of the MANT group at the 2′(3′)-O-position of the ribosyl group reduced the affinity of GTPγS for Gαs- and Gαi-proteins by ∼30–300-fold, i.e. theKi values for MANT-GTPγS ranged from 250 (FPR-Gαi2) to 1100 nm(β2AR-Gαolf). The affinities of MANT-GppNHp for Gαs- and Gαi-proteins were 4.5–11-fold lower than those of GppNHp, i.e. the Kivalues for MANT-GppNHp ranged from 1.2 (FPR-Gαi2) to 5.8 μm (β2AR-Gαolf). We also analyzed GTPases with GTP at increasing concentrations in the presence of MANT-GTPγS 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 (25Klinker J.F. Seifert R. Biochem. Pharmacol. 1997; 54: 551-562Crossref PubMed Scopus (17) Google Scholar, 26Klinker J.F. Seifert R. Biochem. Biophys. Res. Commun. 1999; 262: 341-345Crossref PubMed Scopus (5) Google Scholar), we expected that the linear regression lines intersect in the y axis, reflecting competitive interaction of GTP with MANT-GTPγS. In fact, MANT-GTPγS exhibited competitive interaction with GTP at Gαs (Fig.3A) and Gαi (Fig. 3B). Collectively, our data show that MANT-GTPγS and MANT-GppNHp bind to Gαs- and Gαi-proteins, although with low affinity. In case of the β2AR/Gαs 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 β2AR and, thereby,increase [3H]dihydroalprenolol binding (13Seifert R. Wenzel-Seifert K. Lee T.W. Gether U. Sanders-Bush E. Kobilka B.K. J. Biol. Chem. 1998; 273: 5109-5116Abstract Full Text Full Text PDF Scopus (131) Google Scholar,14Liu H.-Y. Wenzel-Seifert K. Seifert R. J. Neurochem. 2001; 78: 325-338Crossref PubMed Scopus (42) Google Scholar, 27Seifert R. Gether U. Wenzel-Seifert K. Kobilka B.K. Mol. Pharmacol. 1999; 56: 348-358Crossref PubMed Scopus (111) Google Scholar). As reported before (14Liu H.-Y. Wenzel-Seifert K. Seifert R. J. Neurochem. 2001; 78: 325-338Crossref PubMed Scopus (42) Google Scholar), GTPγS potently (IC50, 0.7 nm; CI (CI, 95% confidence interval), 0.2–2.1 nm) and efficaciously disrupted the ternary complex in membranes expressing β2AR-GαsS (Fig.4A). MANT-GTPγS (1 μm) reduced ternary complex formation in β2AR-GαsS by ∼50%, but at higher concentrations, the effect of MANT-GTPγS was reverted. MANT-GTPγS 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-GTPγS inhibited [3H]dihydroalprenolol binding to the β2AR. We examined the effect of MANT-GTPγS (10 μm) on binding of [3H]dihydroalprenolol in Sf9 membranes expressing the β2AR alone, i.e. a system in which ternary complex formation is not detected (12Seifert R. Lee T.W. Lam V.T. Kobilka B.K. Eur. J. Biochem. 1998; 255: 369-382Crossref PubMed Scopus (102) Google Scholar). In fact, MANT-GTPγS (10 μm) inhibited binding of [3H]dihydroalprenolol (1 nm) by 40%. Thus, because of interference of MANT-GTPγS with ligand binding to the β2AR, we could not answer the question whether GTPγS and MANT-GTPγS exhibit similar efficacies at disrupting the ternary complex in β2AR-GαsS. In case of the FPR/Gαi2 couple, ternary complex disruption is measured directly by guanine nucleotide-induced reduction of high affinity agonist ([3H]FMLP) binding (15Wenzel-Seifert K. Hurt C.M. Seifert R. J. Biol. Chem. 1998; 273: 24181-24189Abstract Full Text Full Text PDF PubMed Scopus (96) Google Scholar, 28Gierschik P. Steisslinger M. Sidiropoulos D. Herrmann E. Jakobs K.H. Eur. J. Biochem. 1989; 183: 97-105Crossref PubMed Scopus (56) Google Scholar). As reported before (15Wenzel-Seifert K. Hurt C.M. Seifert R. J. Biol. Chem. 1998; 273: 24181-24189Abstract Full Text Full Text PDF PubMed Scopus (96) Google Scholar, 28Gierschik P. Steisslinger M. Sidiropoulos D. Herrmann E. Jakobs K.H. Eur. J. Biochem. 1989; 183: 97-105Crossref PubMed Scopus (56) Google Scholar), GTPγS potently (IC50, 26 nm; CI, 15–43 nm) and efficaciously disrupted the ternary complex of the FPR/Gαi2 couple (Fig.4B). Considering the Ki values of MANT-GTPγS for Gαi-proteins (250–500 nm) (Fig. 2, D–F), we would have expected maximal disruption of the ternary complex with MANT-GTPγS at 10 μm. However, MANT-GTPγS (10 μm) decreased [3H]FMLP binding by not more than 25%. Thus, MANT-GTPγS is rather inefficient at stabilizing the conformation in Gαi 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 β2AR and Gαs (23Gille A. Liu H.Y. Sprang S.R. Seifert R. J. Biol. Chem. 2002; 277: 34434-34442Abstract Full Text Full Text PDF PubMed Scopus (14) Google Scholar, 27Seifert R. Gether U. Wenzel-Seifert K. Kobilka B.K. Mol. Pharmacol. 1999; 56: 348-358Crossref PubMed Scopus (111) Google Scholar). We analyzed the effects of GTP analogs on AC activity in Sf9 membranes expressing GαsS. GTPγS 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. GTPγS and GppNHp were similarly efficacious at activating AC. In agreement with previous results (5Eckstein F. Cassel D. Levkovitz H. Lowe M. Selinger Z. J. Biol. Chem. 1979; 254: 9829-9834Abstract Full Text PDF PubMed Google Scholar), GDPβS was less efficacious at activating AC than GTPγS/GppNHp, i.e.GDPβS acted as a partial Gαs activator. MANT-GTPγS and MANT-GppNHp abolished basal AC activity in Sf9 membranes expressing GαsS (reflecting the activity of GαsS bound to GDP (13Seifert R. Wenzel-Seifert K. Lee T.W. Gether U. Sanders-Bush E. Kobilka B.K. J. Biol. Chem. 1998; 273: 5109-5116Abstract Full Text Full Text PDF Scopus (131) Google Scholar, 29Sunahara R.K. Dessauer C.W. Whisnant R.E. Kleuss C. Gilman A.G. J. Biol. Chem. 1997; 272: 22265-22271Abstract Full Text Full Text PDF PubMed Scopus (161) Google Scholar)) with IC50 values of 7.4 (CI, 3.6–15 μm) and 34 μm (CI, 5.9–110 μm), respectively (Fig.5A). There is evidence to support the concept of multiple Gα states (4Sprang S.R. Annu. Rev. Biochem. 1997; 66: 639-678Crossref PubMed Scopus (896) Google Scholar, 5Eckstein F. Cassel D. Levkovitz H. Lowe M. Selinger Z. J. Biol. Chem. 1979; 254: 9829-9834Abstract Full Text PDF PubMed Google Scholar, 8Remmers A.E. Neubig R.R. J. Biol. Chem. 1996; 271: 4791-4797Abstract Full Text Full Text PDF PubMed Scopus (29) Google Scholar, 23Gille A. Liu H.Y. Sprang S.R. Seifert R. J. Biol. Chem. 2002; 277: 34434-34442Abstract Full Text Full Text PDF PubMed Scopus (14) Google Scholar, 26Klinker J.F. Seifert R. Biochem. Biophys. Res. Commun. 1999; 262: 341-345Crossref PubMed Scopus (5) Google Scholar, 27Seifert R. Gether U. Wenzel-Seifert K. Kobilka B.K. Mol. Pharmacol. 1999; 56: 348-358Crossref PubMed Scopus (111) Google Scholar). Based on the literature and our present data, the hypothesis evolved that MANT-GTPγS/MANT-GppNHp could stabilize an inhibitory Gαs conformation. To test this hypothesis, we examined the interactions of MANT-GTPγS/MANT-GppNHp with GTP, GTPγS, GppNHp, GDPβS and AlF4−, i.e.substances that all bind to the nucleotide-binding pocket of Gα and stabilize distinct conformations (4Sprang S.R. Annu. Rev. Biochem. 1997; 66: 639-678Crossref PubMed Scopus (896) Google Scholar, 5Eckstein F. Cassel D. Levkovitz H. Lowe M. Selinger Z. J. Biol. Chem. 1979; 254: 9829-9834Abstract Full Text PDF PubMed Google Scholar, 8Remmers A.E. Neubig R.R. J. Biol. Chem. 1996; 271: 4791-4797Abstract Full Text Full Text PDF PubMed Scopus (29) Google Scholar, 30Coleman D.E. Sprang S.R. J. Biol. Chem. 1999; 274: 16669-16672Abstract Full Text Full Text PDF PubMed Scopus (49) Google Scholar). If MANT-GTPγS/MANT-GppNHp inhibited AC via GαsS, they should competitively block the stimulatory effects of GTPγS, GppNHp, GTP, GDPβS, and AlF4− on AC. As positive control, we studied the interaction of the full GαsS activator, GTPγS, with the partial GαsS activator, GDPβS, on AC (Fig. 5A) (5Eckstein F. Cassel D. Levkovitz H. Lowe M. Selinger Z. J. Biol. Chem. 1979; 254: 9829-9834Abstract Full Text PDF PubMed Google Scholar). Competitive interaction of MANT-GTPγS with GTP was already observed in the GTPase studies (Fig. 3). GDPβS shifted the concentration/response curves for GTPγS to the right without reducing the maximal AC activity obtained with GTPγS (Fig.5B). These data confirm the competitive interaction of guanine nucleotides at the nucleotide-binding site of Gαs(31Svoboda M. Furnelle J. Eckstein F. Christophe J. FEBS Lett. 1980; 109: 275-279Crossref PubMed Scopus (13)

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