Distinct Interactions of GTP, UTP, and CTP with GsProteins
2002; Elsevier BV; Volume: 277; Issue: 37 Linguagem: Inglês
10.1074/jbc.m204259200
ISSN1083-351X
AutoresAndreas Gille, Hui-Yu Liu, Stephen R. Sprang, Roland Seifert,
Tópico(s)Peptidase Inhibition and Analysis
ResumoEarly studies showed that in addition to GTP, the pyrimidine nucleotides UTP and CTP support activation of the adenylyl cyclase (AC)-stimulating Gs protein. The aim of this study was to elucidate the mechanism by which UTP and CTP support Gs activation. As models, we used S49 wild-type lymphoma cells, representing a physiologically relevant system in which the β2-adrenoreceptor (β2AR) couples to Gs, and Sf9 insect cell membranes expressing β2AR-Gαs fusion proteins. Fusion proteins provide a higher sensitivity for the analysis of β2AR-Gs coupling than native systems. Nucleoside 5′-triphosphates (NTPs) supported agonist-stimulated AC activity in the two systems and basal AC activity in membranes from cholera toxin-treated S49 cells in the order of efficacy GTP ≥ UTP > CTP > ATP (ineffective). NTPs disrupted high affinity agonist binding in β2AR-Gαs in the order of efficacy GTP > UTP > CTP > ATP (ineffective). In contrast, the order of efficacy of NTPs as substrates for nucleoside diphosphokinase, catalyzing the formation of GTP from GDP and NTP was ATP ≥ UTP ≥ CTP ≥ GTP. NTPs inhibited β2AR-Gαs-catalyzed [γ-32P]GTP hydrolysis in the order of potency GTP > UTP > CTP. Molecular dynamics simulations revealed that UTP is accommodated more easily within the binding pocket of Gαsthan CTP. Collectively, our data indicate that GTP, UTP, and CTP interact differentially with Gs proteins and that transphosphorylation of GDP to GTP is not involved in this G protein activation. In certain cell systems, intracellular UTP and CTP concentrations reach ∼10 nmol/mg of protein and are higher than intracellular GTP concentrations, indicating that G protein activation by UTP and CTP can occur physiologically. G protein activation by UTP and CTP could be of particular importance in pathological conditions such as cholera and Lesch-Nyhan syndrome. Early studies showed that in addition to GTP, the pyrimidine nucleotides UTP and CTP support activation of the adenylyl cyclase (AC)-stimulating Gs protein. The aim of this study was to elucidate the mechanism by which UTP and CTP support Gs activation. As models, we used S49 wild-type lymphoma cells, representing a physiologically relevant system in which the β2-adrenoreceptor (β2AR) couples to Gs, and Sf9 insect cell membranes expressing β2AR-Gαs fusion proteins. Fusion proteins provide a higher sensitivity for the analysis of β2AR-Gs coupling than native systems. Nucleoside 5′-triphosphates (NTPs) supported agonist-stimulated AC activity in the two systems and basal AC activity in membranes from cholera toxin-treated S49 cells in the order of efficacy GTP ≥ UTP > CTP > ATP (ineffective). NTPs disrupted high affinity agonist binding in β2AR-Gαs in the order of efficacy GTP > UTP > CTP > ATP (ineffective). In contrast, the order of efficacy of NTPs as substrates for nucleoside diphosphokinase, catalyzing the formation of GTP from GDP and NTP was ATP ≥ UTP ≥ CTP ≥ GTP. NTPs inhibited β2AR-Gαs-catalyzed [γ-32P]GTP hydrolysis in the order of potency GTP > UTP > CTP. Molecular dynamics simulations revealed that UTP is accommodated more easily within the binding pocket of Gαsthan CTP. Collectively, our data indicate that GTP, UTP, and CTP interact differentially with Gs proteins and that transphosphorylation of GDP to GTP is not involved in this G protein activation. In certain cell systems, intracellular UTP and CTP concentrations reach ∼10 nmol/mg of protein and are higher than intracellular GTP concentrations, indicating that G protein activation by UTP and CTP can occur physiologically. G protein activation by UTP and CTP could be of particular importance in pathological conditions such as cholera and Lesch-Nyhan syndrome. G protein-coupled receptor(s) adenylyl cyclase β2-adrenoceptor fusion protein containing the β2AR and Gαolf fusion protein containing the β2AR and the long splice variant of Gαs fusion protein containing the β2AR and the short splice variant of Gαs confidence interval cholera toxin dihydroalprenolol unspecified G protein α subunit guanosine 5′-O-(3-thiotriphosphate) (−)-isoproterenol nucleoside diphosphokinase nucleoside 5′-triphosphate salbutamol S49 wild-type lymphoma cells G proteins consist of an α subunit and a βγ complex and serve as signal transducers between agonist-activated GPCRs1 and effector systems (1Gilman A.G. Annu. Rev. Biochem. 1987; 56: 615-649Crossref PubMed Scopus (4669) Google Scholar, 2Birnbaumer L. Abramowitz J. Brown A.M. Biochim. Biophys. Acta. 1990; 1031: 163-224Crossref PubMed Scopus (958) Google Scholar, 3Sprang S.R. Annu. Rev. Biochem. 1997; 66: 639-678Crossref PubMed Scopus (875) Google Scholar, 4Iiri T. Farfel Z. Bourne H.R. Nature. 1998; 394: 35-38Crossref PubMed Scopus (162) Google Scholar). Upon binding of an agonist, GPCRs undergo a conformational change causing GDP dissociation from Gα. GDP dissociation is the rate-limiting step of the G protein cycle. Agonist-occupied GPCRs then form a ternary complex with the nucleotide-free G protein. The ternary complex possesses high agonist affinity. Subsequently, GPCRs promote binding of GTP to Gα. The binding of GTP to Gα induces the active conformation of the G protein, leading to the dissociation of the heterotrimer into Gα-GTP and the βγ complex. Both Gα-GTP and βγ can regulate the activity of effector systems. Gα possesses GTPase activity. The GTPase cleaves GTP into GDP and Pi and thereby deactivates the G protein. Gα-GDP and βγ reassociate, completing the G protein cycle. Intriguingly, not only the purine nucleotide GTP but also pyrimidine nucleotides exhibit effects on G proteins. Particularly, various natural and synthetic uracil nucleotides disrupt the complex between the photoexcited light receptor rhodopsin and the retinal G protein transducin, but the uracil nucleotides are less effective in this regard than the corresponding guanine nucleotides (5Klinker J.F. Seifert R. Biochem. Biophys. Res. Commun. 1999; 262: 341-345Crossref PubMed Scopus (5) Google Scholar). [γ-32P]GTP hydrolysis and [35S]GTPγS binding competition studies showed that pyrimidine nucleotides bind to G proteins with low affinity (5Klinker J.F. Seifert R. Biochem. Biophys. Res. Commun. 1999; 262: 341-345Crossref PubMed Scopus (5) Google Scholar, 6Northup J.K. Smigel M.D. Gilman A.G. J. Biol. Chem. 1982; 257: 11416-11423Abstract Full Text PDF PubMed Google Scholar, 7Milligan G. Klee W.A. J. Biol. Chem. 1985; 260: 2057-2063Abstract Full Text PDF PubMed Google Scholar, 8Chidiac P. Wells J.W. Biochemistry. 1992; 31: 10908-10921Crossref PubMed Scopus (39) Google Scholar). Moreover, early studies revealed that UTP and CTP support GPCR-mediated AC activation in membranes (9Rodbell M. Birnbaumer L. Pohl S.L. Krans H.M. J. Biol. Chem. 1971; 246: 1877-1882Abstract Full Text PDF PubMed Google Scholar, 10Wolff J. Cook G.H. J. Biol. Chem. 1973; 248: 350-355Abstract Full Text PDF PubMed Google Scholar, 11Bilezikian J.P. Aurbach G.D. J. Biol. Chem. 1974; 249: 157-161Abstract Full Text PDF PubMed Google Scholar). However, it remained unclear whether the effects of UTP and CTP on AC were mediated via NDPK, catalyzing the formation of GTP from GDP and NTP (12Otero A.D. Biochem. Pharmacol. 1990; 39: 1399-1404Crossref PubMed Scopus (102) Google Scholar, 13Piacentini L. Niroomand F. Mol. Cell. Biochem. 1996; 157: 59-63Crossref PubMed Scopus (24) Google Scholar), or via direct interaction of UTP and CTP with Gαs (6Northup J.K. Smigel M.D. Gilman A.G. J. Biol. Chem. 1982; 257: 11416-11423Abstract Full Text PDF PubMed Google Scholar). The aim of the present study was to elucidate the mechanism by which UTP and CTP support Gs activation. To achieve our aim we have studied AC regulation in S49 membranes. S49 cells are a widely used and physiologically relevant model system for the analysis of β2AR/Gs/AC interactions (14Kaslow H.R. Farfel Z. Johnson G.L. Bourne H.R. Mol. Pharmacol. 1979; 15: 472-483PubMed Google Scholar, 15Ransna¨s L.A. Insel P.A. J. Biol. Chem. 1988; 263: 9482-9485Abstract Full Text PDF PubMed Google Scholar, 16Alousi A.A. Jasper J.R. Insel P.A. Motulsky H.J. FASEB J. 1991; 5: 2300-2303Crossref PubMed Scopus (119) Google Scholar, 17Bertin B. Freissmuth M. Jockers R. Strosberg A.D. Marullo S. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 8827-8831Crossref PubMed Scopus (108) Google Scholar). Additionally, we have studied fusion proteins of the β2AR with individual Gαs isoforms, i.e.β2AR-GαsS, β2AR-GαsL, and β2AR-Gαolf, expressed in Sf9 insect cells. Fusion proteins provide close proximity of the coupling partners and ensure efficient GPCR-G protein-effector coupling (18Seifert R. Wenzel-Seifert K. Kobilka B.K. Trends Pharmacol. Sci. 1999; 20: 383-389Abstract Full Text Full Text PDF PubMed Scopus (129) Google Scholar, 19Milligan G. Trends Pharmacol. Sci. 2000; 21: 24-28Abstract Full Text Full Text PDF PubMed Scopus (108) Google Scholar). In addition, fusion proteins allow for the analysis of the coupling of a given GPCR to various Gα isoforms under defined experimental conditions (20Seifert 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, 21Wenzel-Seifert K. Seifert R. Mol. Pharmacol. 2000; 58: 954-966Crossref PubMed Scopus (142) Google Scholar, 22Seifert R. J. Pharmacol. Exp. Ther. 2001; 298: 840-847PubMed Google Scholar, 23Liu H.-Y. Wenzel-Seifert K. Seifert R. J. Neurochem. 2001; 78: 325-338Crossref PubMed Scopus (41) Google Scholar). Here, we report on distinct interactions of GTP, UTP, and CTP with Gs proteins. The generation of baculoviruses encoding for β2AR-GαsS, β2AR-GαsL, and β2AR-Gαolf was described elsewhere (20Seifert 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, 23Liu H.-Y. Wenzel-Seifert K. Seifert R. J. Neurochem. 2001; 78: 325-338Crossref PubMed Scopus (41) Google Scholar,24Seifert R. Lee T.W. Lam V.T. Kobilka B.K. Eur. J. Biochem. 1998; 255: 369-382Crossref PubMed Scopus (102) Google Scholar). [γ-32P]GTP (6,000 Ci/mmol), [α-32P]ATP (3,000 Ci/mmol), [32P]Pi (8,500–9,000 Ci/mmol), [3H]DHA (85–90 Ci/mmol), and [3H]GDP (30–39 Ci/mmol) were from PerkinElmer Life Sciences. Unlabeled ATP (special quality, < 0.01% GTP as assessed by high performance liquid chromatography), GTP, UTP, and CTP were of the highest quality available and were obtained from Roche Molecular Biochemicals. [γ-32P]UTP and [γ-32P]CTP (∼6,000 Ci/mmol each) were synthesized as described (5Klinker J.F. Seifert R. Biochem. Biophys. Res. Commun. 1999; 262: 341-345Crossref PubMed Scopus (5) Google Scholar, 25Walseth T.F. Johnson R.A. Biochim. Biophys. Acta. 1979; 562: 11-31Crossref PubMed Scopus (426) Google Scholar). S49 cells were obtained from the Cell Culture Facility of the University of California at San Francisco. ISO, SAL, (±)-alprenolol and CTX were obtained from Sigma. Sf9 cells were cultured and infected with recombinant baculoviruses as described (20Seifert 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,24Seifert R. Lee T.W. Lam V.T. Kobilka B.K. Eur. J. Biochem. 1998; 255: 369-382Crossref PubMed Scopus (102) Google Scholar, 26Seifert R. Gether U. Wenzel-Seifert K. Kobilka B.K. Mol. Pharmacol. 1999; 56: 348-358Crossref PubMed Scopus (108) Google Scholar). S49 cells were grown at 37 °C in suspension in Dulbecco’s modified Eagle’s medium supplemented with 4.5 g/literd-glucose, 2 mml-glutamine, 1,000 units/ml penicillin, 100 μg/ml streptomycin, and 10% (v/v) heat-inactivated horse serum in a humidified atmosphere containing 7% (v/v) CO2. S49 cells were maintained at a density of 0.2–2.0 × 106 cells/ml. To inactivate the GTPase of Gαs, S49 cells were treated with 1 μg/ml CTX for 24 h before membrane preparation (27Moss J. Vaughan M. Adv. Enzymol. Relat. Areas Mol. Biol. 1988; 61: 303-379PubMed Google Scholar). Dulbecco’s modified Eagle’s medium was from Cellgro Mediatech (Herndon, VA). All other constituents for the culture of S49 cells were obtained from BioWhittaker (Walkersville, MD). S49 and Sf9 membranes were prepared according to the protocol described previously (24Seifert R. Lee T.W. Lam V.T. Kobilka B.K. Eur. J. Biochem. 1998; 255: 369-382Crossref PubMed Scopus (102) Google Scholar). Membranes were suspended in binding buffer (12.5 mmMgCl2, 1 mm EDTA, and 75 mmTris-HCl, pH 7.4) at a concentration of ∼1–2 mg of protein/ml and stored at –80 °C until use. Immediately prior to [3H]DHA binding, AC, NTPase, and NDPK experiments, membrane aliquots were thawed, suspended in binding buffer, and centrifuged for 15 min at 4 °C and 15,000 × g to remove, as far as possible, any remaining endogenous nucleotides (23Liu H.-Y. Wenzel-Seifert K. Seifert R. J. Neurochem. 2001; 78: 325-338Crossref PubMed Scopus (41) Google Scholar). The expression levels of β2AR-GαsS, β2AR-GαsL, and β2AR-Gαolf were determined by saturation binding using the β2AR antagonist [3H]DHA (20Seifert 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, 24Seifert R. Lee T.W. Lam V.T. Kobilka B.K. Eur. J. Biochem. 1998; 255: 369-382Crossref PubMed Scopus (102) Google Scholar). 500-μl tubes contained Sf9 membranes (10–20 μg of protein/tube) expressing fusion proteins, 10 nm[3H]DHA, and binding buffer. Nonspecific binding was determined in the presence of 10 μm (±)-alprenolol. Incubations were performed for 90 min at 25 °C and shaking at 250 rpm. Bound [3H]DHA was separated from free [3H]DHA by filtration through GF/C filters (Schleicher & Schuell). For generation of concentration/response curves for the inhibitory effects of NTPs on high affinity agonist binding, reaction mixtures contained Sf9 membranes (20 μg of protein/tube) expressing β2AR-Gαs fusion proteins, 1 μm SAL, 1 nm [3H]DHA as radioligand, and NTPs at increasing concentrations (26Seifert R. Gether U. Wenzel-Seifert K. Kobilka B.K. Mol. Pharmacol. 1999; 56: 348-358Crossref PubMed Scopus (108) Google Scholar). For determination of the extent of ternary complex formation and its sensitivity to disruption by NTPs, reaction mixtures contained Sf9 membranes (20 μg of protein/tube) expressing β2AR-GαsL, 1 nm[3H]DHA as radioligand, and ISO at increasing concentrations in the absence and presence of NTPs at a fixed concentration (1 mm each) (23Liu H.-Y. Wenzel-Seifert K. Seifert R. J. Neurochem. 2001; 78: 325-338Crossref PubMed Scopus (41) Google Scholar, 24Seifert R. Lee T.W. Lam V.T. Kobilka B.K. Eur. J. Biochem. 1998; 255: 369-382Crossref PubMed Scopus (102) Google Scholar). The determination of AC activity in S49 membranes and Sf9 membranes expressing β2AR-Gαs fusion proteins was performed under identical experimental conditions and followed the protocol published previously (23Liu H.-Y. Wenzel-Seifert K. Seifert R. J. Neurochem. 2001; 78: 325-338Crossref PubMed Scopus (41) Google Scholar). Briefly, 30-μl tubes contained membranes (20–50 μg of protein/tube), 5 mm MgCl2, 0.4 mm EDTA, 30 mm Tris-HCl, pH 7.4, and NTPs at various concentrations in the absence or presence of ISO. Tubes were incubated for 3 min at 37 °C before the addition of 20 μl of reaction mixture containing (final) [α-32P]ATP (1.0–1.5 μCi/tube) plus 40 μm ATP, 2.7 mmmono(cyclohexyl)ammonium phosphoenolpyruvate, 0.125 IU of pyruvate kinase, 1 IU of myokinase, and 0.1 mm cAMP. Reactions were conducted for 20 min at 37 °C. Stopping of reactions and separation of [α-32P]ATP from [32P]cAMP were performed as described previously (23Liu H.-Y. Wenzel-Seifert K. Seifert R. J. Neurochem. 2001; 78: 325-338Crossref PubMed Scopus (41) Google Scholar). High affinity GTPase activity in Sf9 membranes expressing β2AR-Gsα fusion proteins was determined as described (23Liu H.-Y. Wenzel-Seifert K. Seifert R. J. Neurochem. 2001; 78: 325-338Crossref PubMed Scopus (41) Google Scholar). Briefly, tubes (80 μl) contained membranes (10 μg of protein/tube), 1.0 mmMgCl2, 0.1 mm EDTA, 0.1 mm ATP, 1 mm adenylyl imidodiphosphate, 5 mm creatine phosphate, 40 μg of creatine kinase, 30 nm unlabeled GTP, 10 μm ISO, and 0.05% (mass/volume) bovine serum albumin in 50 mm Tris-HCl, pH 7.4. Reaction mixtures were incubated for 3 min at 25 °C before the addition of 20 μl of [γ-32P]GTP (0.2 μCi/tube). Nonenzymatic [γ-32P]GTP hydrolysis was determined in the presence of a large excess of unlabeled GTP (1 mm). Reactions were conducted for 20 min at 25 °C. Stopping of reactions and recovery of32Pi were performed as described (23Liu H.-Y. Wenzel-Seifert K. Seifert R. J. Neurochem. 2001; 78: 325-338Crossref PubMed Scopus (41) Google Scholar). UTPase and CTPase activities in Sf9 membranes were determined essentially as GTPase activity except that reaction mixtures contained [γ-32P]UTP or [γ-32P]CTP (up to 2.5 μCi/tube) instead of [γ-32P]GTP. In addition, reaction mixtures contained unlabeled UTP or CTP (0.1–100 μm) instead of unlabeled GTP. Nonenzymatic UTP and CTP hydrolysis was determined in the presence of 1 mm unlabeled UTP and CTP, respectively. NDPK activity in S49 wild-type lymphoma membranes and Sf9 membranes was determined as described for soluble transducin preparations with modifications (28Klinker J.F. Seifert R. Eur. J. Biochem. 1999; 261: 72-80Crossref PubMed Scopus (21) Google Scholar). Briefly, reaction mixtures contained S49 membranes (5.0–10.0 μg of protein/tube) or Sf9 membranes (0.5–1.0 μg of protein/tube), NTPs at various concentrations, 0.1% (mass/volume) bovine serum albumin, 1 mm MgCl2, and 0.1 mmEDTA in 50 mm Tris-HCl, pH 7.4. Reaction mixtures were preincubated for 3 min at 37 °C before the addition of 0.5 μm carrier-free [3H]GDP. The total reaction volume was 50 μl. Reactions were conducted for 10 min. To obtain blank values, tubes containing all components described above except for membranes were processed in parallel. Stopping of reactions, separation of nucleotides on poly(ethyleneimine)-cellulose TLC plates (Schleicher & Schuell), elution of nucleotides, and counting of radioactivity were performed exactly as described (28Klinker J.F. Seifert R. Eur. J. Biochem. 1999; 261: 72-80Crossref PubMed Scopus (21) Google Scholar). Potential energy minimization and molecular dynamics simulations were carried out using the AMBER 6.0 program package (29Perlman D.A. Case D.A. Caldwell J.W. Ross W.S. Cheatham T.E.I. DeBolt S. Ferguson D. Seibel G.L. Kollman P.A. Comp. Phys. Commun. 1995; 91: 1-41Crossref Scopus (2605) Google Scholar), using the force field of Cornell et al. (30Cornell W.D. Cieplak P. Bayly C.I. Gould I.R. Merz K.M.J. Ferguson D.M. Spellmeyer D.C. Fox T. Caldwell J.W. Kollman P.A. J. Am. Chem. Soc. 1995; 117: 5179-5197Crossref Scopus (11375) Google Scholar). Initial models for Gαs·Mg2+·NTP complexes were based on the coordinates of Gαs·Mg2+·GTPγS in the complex with the catalytic domains of AC (PDB 1AZS) (31Tesmer J.J. Sunahara R.K. Gilman A.G. Sprang S.R. Science. 1997; 278: 1907-1916Crossref PubMed Scopus (663) Google Scholar). We replaced the γS substituent of GTPγS by an sp2-hybridized oxygen atom. Models of complexes with UTP and CTP were generated by replacing the guanine base with uracil and cytosine, respectively, maintaining the glycosyl torsion angle (O4′-C1′-N9-C8) of GTPγS in the starting model. Partial charges assigned to phosphate groups were obtained from Dr. N. Duclert-Savatier (Institut Pasteur, Paris, France). 2N. Duclert-Savatier, personal communication. Protein-bound water molecules observed in the crystal structure were included in the model. The nonbonded cutoff distance was set at 10 Å. The stereochemistry of the Mg2+ ligand field was restrained as an octahedral complex with the six coordinating oxygen atoms of the β- and γ-phosphate oxygens, the hydroxyl groups of Ser-54 (GαsL and GαsS) and Thr-204 (GαsL) and two water molecules, with oxygen-Mg2+ distances of 2.1 Å. This geometry was maintained by pseudo-van der Waals potentials among all pairs of atoms within the octahedral complex. In constructing models of the Gαs·Mg2+·UTP and Gαs·Mg2+·CTP complexes, we placed a water molecule at the site occupied by the guanine exocyclic C (2Birnbaumer L. Abramowitz J. Brown A.M. Biochim. Biophys. Acta. 1990; 1031: 163-224Crossref PubMed Scopus (958) Google Scholar) amine in the GTP complex. The included water molecule bridges the uracil exocyclic C (2Birnbaumer L. Abramowitz J. Brown A.M. Biochim. Biophys. Acta. 1990; 1031: 163-224Crossref PubMed Scopus (958) Google Scholar) keto with the Ο1γ carboxylate oxygen of Asp-295 (GαsL) and Asp-280 (GαsS). The water molecule placed at this site did not move after energy minimization of the model complexes. Energy relaxations were carried out using the SANDER module of AMBER, using steepest descent minimization for the first 250 cycles, followed by 250 cycles of conjugate gradient minimization. In all cases the total computed potential energy typically declined smoothly from values of −14,500 to −16,750 kcal·mol−1, attaining a constant value after 400–450 cycles of minimization. For molecular dynamics simulations, a box of TIP3P water molecules was used to solvate the protein, leaving a 10 Å border between the edge of the box and the closest atoms of the protein. The system was heated to 300 K using the temperature scaling scheme of Berendsen et al.(32Berendsen H.J.C. Postma J.P.M. van Gunsteren W.D. Dinola A. Haak J.R. J. Chem. Phys. 1984; 81: 3684-3690Crossref Scopus (22550) Google Scholar) and periodic boundary conditions. Simulations were carried out for 10 ps in steps of 1 fs. Protein was determined using the Bio-Rad DC protein assay kit. Data shown in Figs. Figure 1, Figure 2, Figure 3, Figure 4, Figure 5 and Table I were analyzed by nonlinear regression, using the Prism III program (GraphPad, Prism, San Diego).Figure 2Effects of UTP, CTP, GTP, and ATP on ternary complex formation in Sf9 membranes expressing β2AR-GαsS, β2AR-GαsL, or β2AR-Gαolf.Concentration/response curves for NTPs are shown. [3H]DHA binding in Sf9 membranes was performed as described under “Experimental Procedures.” Reaction mixtures contained Sf9 membranes (20 μg of protein/tube) expressing β2AR-GαsS, β2AR-GαsL, or β2AR-Gαolf, 1 nm[3H]DHA, 1 μm SAL, and NTPs (•, GTP; ▪, UTP; ▴, CTP; ♦, ATP) at the concentration indicated on theabscissa. 10−10 designates the absence of added NTP. A, membranes expressing β2AR-GαsS at 2.6–4.4 pmol/mg;B, membranes expressing β2AR-GαsL at 3.0–4.8 pmol/mg;C, membranes expressing β2AR-Gαolf at 3.3–4.2 pmol/mg. Data were analyzed by nonlinear regression and were best fitted to sigmoidal concentration/response curves. Data shown are the means ± S.D. of three to five independent experiments performed in triplicate.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Figure 4Competition of [γ-32P]GTP hydrolysis in Sf9 membranes expressing β2AR-Gαolf, β2AR-GαsS, or β2AR-GαsLby GTP, UTP, and CTP. 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, 30 nm[γ-32P]GTP, 10 μm ISO, and unlabeled GTP (•), UTP (▪), or CTP (▴) at increasing concentrations.10−8 designates the absence of unlabeled UTP, CTP, or GTP. GTPase activities in the absence of competitor (control) were as follows. β2AR-Gαolf (expressed at 13.7 pmol/mg), 8.5 ± 0.4 pmol/mg/min; β2AR-GαsS (expressed at 4.0 pmol/mg/min), 3.3 ± 0.3 pmol/mg/min; β2AR-GαsL(expressed at 6.0 pmol/mg), 5.2 ± 0.6 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 were referred to those calibration points. Data were analyzed by nonlinear regression and were best fitted to monophasic competition curves. Data shown are the means ± S.D. of three independent experiments performed in duplicate.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Figure 5Effects of UTP, CTP, and GTP on AC activity in Sf9 membranes expressing β2AR-GαsS, β2AR-GαsL, or β2AR-Gαolf.AC activity in Sf9 membranes was determined as described under “Experimental Procedures.” Reaction mixtures contained Sf9 membranes (20 μg of protein/tube) expressing β2AR-GαsS, β2AR-GαsL, or β2AR-Gαolf, and NTPs at the concentrations indicated on the abscissa with solvent (basal) (▪) or with 10 μm ISO (▪). 10−9 designates the absence of added UTP, CTP, or GTP. A–C, membranes expressing β2AR-GαsS at 2.3–2.6 pmol/mg; D–F, membranes expressing β2AR-GαsL at 4.8–5.4 pmol/mg;G–I, membranes expressing β2AR-Gαolf at 3.6–4.1 pmol/mg. Data were analyzed by nonlinear regression and were best fitted to sigmoidal concentration/response curves. Data shown are the means ± S.D. of three to five independent experiments performed in duplicate. Note that the scale of the ordinate in A–F is different from the scale in G–I. The different scales were chosen to facilitate comparison of the relative effects of NTPs at the various fusion proteins. Differences in the absolute efficacies of β2AR-Gαs fusion proteins at activating AC were reported before (20Seifert 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, 23Liu H.-Y. Wenzel-Seifert K. Seifert R. J. Neurochem. 2001; 78: 325-338Crossref PubMed Scopus (41) Google Scholar).View Large Image Figure ViewerDownload Hi-res image Download (PPT)Table INonlinear regression analysis of the effects of GTP, UTP, and CTP on ternary complex formation in Sf9 membranes expressing β2AR-GαsLAdditionKhKlRhnmnm%Solvent (control)0.7 (0.3–1.3)91 (70–120)34.3 (29.4–39.2)GTP (1 mm)180 (150–200)UTP (1 mm)0.01 (0.005–1.8)100 (80–120)10.7 (3.4–18.0)CTP (1 mm)2.8 (1.0–8.1)130 (70–250)39.5 (24.6–54.4)[3H]DHA binding in Sf9 membranes was performed as described under “Experimental Procedures.” Reaction mixtures contained Sf9 membranes (20 μg of protein/tube) expressing β2AR-GαsL at 4.5–7.4 pmol/mg. Reaction mixtures additionally contained ISO at the concentrations indicated on the abscissa of Fig. 3 in the presence of solvent (control) or various NTPs at a concentration of 1 mm each. The data shown in Fig. 3were analyzed for best fit to monophasic or biphasic competition isotherms (F test). Data shown are the means of five independent experiments performed in triplicate. Numbers in parentheses represent the 95% confidence intervals. Kh andKl designate the dissociation constants for the high and low affinity state of β2AR-GαsL, respectively.Rh indicates the percentage of high affinity binding sites. Open table in a new tab [3H]DHA binding in Sf9 membranes was performed as described under “Experimental Procedures.” Reaction mixtures contained Sf9 membranes (20 μg of protein/tube) expressing β2AR-GαsL at 4.5–7.4 pmol/mg. Reaction mixtures additionally contained ISO at the concentrations indicated on the abscissa of Fig. 3 in the presence of solvent (control) or various NTPs at a concentration of 1 mm each. The data shown in Fig. 3were analyzed for best fit to monophasic or biphasic competition isotherms (F test). Data shown are the means of five independent experiments performed in triplicate. Numbers in parentheses represent the 95% confidence intervals. Kh andKl designate the dissociation constants for the high and low affinity state of β2AR-GαsL, respectively.Rh indicates the percentage of high affinity binding sites. S49 cells express the β2AR, the Gαs splice variants GαsS and GαsL, and AC (17Bertin B. Freissmuth M. Jockers R. Strosberg A.D. Marullo S. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 8827-8831Crossref PubMed Scopus (108) Google Scholar, 33Jones D.T. Masters S.B. Bourne H.R. Reed R.R. J. Biol. Chem. 1990; 265: 2671-2676Abstract Full Text PDF PubMed Google Scholar, 34O'Donnell J. Sweet R.W. Stadel J.M. Mol. Pharmacol. 1991; 39: 702-710PubMed Google Scholar). In the absence of added UTP, CTP, or GTP, the full βAR agonist ISO at a maximally stimulatory concentration (10 μm) had no stimulatory effect on AC activity in S49 membranes (Fig. 1). However, these experimental conditions do not imply the complete absence of NTP because the AC assay contained 40 μm ATP as AC substrate (see “Experimental Procedures”). UTP, CTP, and GTP had little effects on basal AC activity in the absence of ISO. In agreement with the literature (1Gilman A.G. Annu. Rev. Biochem. 1987; 56: 615-649Crossref PubMed Scopus (4669) Google Scholar, 2Birnbaumer L. Abramowitz J. Brown A.M. Biochim. Biophys. Acta. 1990; 1031: 163-224Crossref PubMed Scopus (958) Google Scholar, 3Sprang S.R. Annu. Rev. Biochem. 1997; 66: 639-678Crossref PubMed Scopus (875) Google Scholar, 4Iiri T. Farfel Z. Bourne H.R. Nature. 1998; 394: 35-38Crossref PubMed Scopus (162) Google Scholar), GTP was potent (EC50, 230 nm; 95% c.i., 150–340 nm) and effective at supporting AC activation by ISO (Fig. 1C). UTP was much less potent (EC50, 82 μm, 95% c.i., 43–160 μm) in this regard than GTP, but it was only moderately less efficient (∼80% efficacy) than GTP (Fig. 1A). CTP only poorly supported AC activation by ISO, both in terms of potency and efficacy (Fig. 1B). CTX-ca
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