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• W2102041216 abstract "An increasing amount of experimental data suggest that cross-talk exists between pathways involving tyrosine kinases and heterotrimeric G proteins. In a previous study, we demonstrated that bradykinin (BK) increases the intracellular accumulation of cAMP in the human epidermoid carcinoma cell line A431 by stimulating adenylate cyclase activity via a stimulatory G protein (Gsα) (Liebmann, C., Graneß, A., Ludwig, B., Adomeit, A., Boehmer, A., Boehmer, F.-D., Nürnberg, B., and Wetzker, R. (1996) Biochem. J. 313, 109-118). Here, we present several lines of evidence indicating the ability of epidermal growth factor (EGF) to suppress BK-induced activation of the cAMP pathway in A431 cells via tyrosine phosphorylation of Gsα. Gsα was specifically immunoprecipitated from A431 cells using the anti-αs antiserum AS 348. Tyrosine phosphorylation of Gsα was detectable in EGF-pretreated cells with monoclonal anti-phosphotyrosine antibodies. Additionally, A431 cells were labeled with [32P]orthophosphate in vivo and treated with EGF, and the resolved immunoprecipitates were subjected to amino acid analysis. The results clearly indicate that EGF induces tyrosine phosphorylation of Gsα in A431 cells. Treatment of A431 cells with EGF decreased BK-induced cAMP accumulation in intact cells as well as the stimulation of adenylate cyclase by BK, NaF, and guanyl nucleotides, but not by forskolin. Also, EGF treatment abolished both the BK- and isoprenaline-induced stimulation of guanosine 5′-O-(3-[35S]thiotriphosphate) binding to Gsα. In contrast, the BK-evoked, Gq-mediated stimulation of inositol phosphate formation in A431 cells was not affected by EGF pretreatment. Thus, EGF-induced tyrosine phosphorylation of Gsα is accompanied by a loss of its susceptibility to G protein-coupled receptors and its ability to stimulate adenylate cyclase via guanyl nucleotide exchange. We propose that Gsα may represent a key regulatory protein in the cross-talk between the signal transduction pathways of BK and EGF in A431 cells. An increasing amount of experimental data suggest that cross-talk exists between pathways involving tyrosine kinases and heterotrimeric G proteins. In a previous study, we demonstrated that bradykinin (BK) increases the intracellular accumulation of cAMP in the human epidermoid carcinoma cell line A431 by stimulating adenylate cyclase activity via a stimulatory G protein (Gsα) (Liebmann, C., Graneß, A., Ludwig, B., Adomeit, A., Boehmer, A., Boehmer, F.-D., Nürnberg, B., and Wetzker, R. (1996) Biochem. J. 313, 109-118). Here, we present several lines of evidence indicating the ability of epidermal growth factor (EGF) to suppress BK-induced activation of the cAMP pathway in A431 cells via tyrosine phosphorylation of Gsα. Gsα was specifically immunoprecipitated from A431 cells using the anti-αs antiserum AS 348. Tyrosine phosphorylation of Gsα was detectable in EGF-pretreated cells with monoclonal anti-phosphotyrosine antibodies. Additionally, A431 cells were labeled with [32P]orthophosphate in vivo and treated with EGF, and the resolved immunoprecipitates were subjected to amino acid analysis. The results clearly indicate that EGF induces tyrosine phosphorylation of Gsα in A431 cells. Treatment of A431 cells with EGF decreased BK-induced cAMP accumulation in intact cells as well as the stimulation of adenylate cyclase by BK, NaF, and guanyl nucleotides, but not by forskolin. Also, EGF treatment abolished both the BK- and isoprenaline-induced stimulation of guanosine 5′-O-(3-[35S]thiotriphosphate) binding to Gsα. In contrast, the BK-evoked, Gq-mediated stimulation of inositol phosphate formation in A431 cells was not affected by EGF pretreatment. Thus, EGF-induced tyrosine phosphorylation of Gsα is accompanied by a loss of its susceptibility to G protein-coupled receptors and its ability to stimulate adenylate cyclase via guanyl nucleotide exchange. We propose that Gsα may represent a key regulatory protein in the cross-talk between the signal transduction pathways of BK and EGF in A431 cells. There is mounting evidence indicative of complex, probably cell-specific interactions between signaling pathways involving heterotrimeric G proteins and tyrosine kinases. For example, the stimulation of G protein-coupled receptors modulates key proteins of the mitogen-activated protein kinase pathway via protein kinase C- or protein kinase A-mediated phosphorylation on serine or threonine residues (1Koich W. Heidecker G. Kochs G. Hummel R. Vahidi H. Mischak H. Finkenzeller G. Marmé D. Rapp U.R. Nature. 1993; 364: 249-252Crossref PubMed Scopus (1157) Google Scholar, 2Blumer K.J. Johnson G.L. Trends Biochem. Sci. 1994; 18: 236-240Abstract Full Text PDF Scopus (421) Google Scholar, 3Cook S.J. McCormick F. Science. 1993; 262: 1069-1072Crossref PubMed Scopus (865) Google Scholar). Furthermore, several isoforms of α subunits of G proteins were shown to be phosphorylated in vitro on tyrosine residues by tyrosine kinase receptors such as the epidermal growth factor (EGF) 1The abbreviations used are: EGFepidermal growth factorBKbradykininGTPγSguanosine 5′-O-(3-thiotriphosphate)DMEMDulbecco's modified Eagle's mediumPAGEpolyacrylamide gel electrophoresisGpp(NH)p5′-guanylylimidodiphosphatePIPESpiperazine-N,N′-bis(2-ethanesulfonic acid). receptor and the insulin receptor or by non-receptor tyrosine kinases of the Src kinase family. EGF was shown to activate cardiac adenylate cyclase via a mechanism requiring both Gsα and the EGF receptor tyrosine kinase (4Nair B.G. Parikh B. Milligan G. Patel T.B. J. Biol. Chem. 1990; 265: 21317-21322Abstract Full Text PDF PubMed Google Scholar, 5Nair B.G. Patel T.B. Biochem. Pharmacol. 1993; 46: 1239-1245Crossref PubMed Scopus (35) Google Scholar). EGF was also found to stimulate phospholipase C in rat hepatocytes (6Liang M. Garrison J.C. J. Biol. Chem. 1991; 266: 13342-13349Abstract Full Text PDF PubMed Google Scholar) or phospholipase A2 in rat kidney (7Teitelbaum I. J. Biol. Chem. 1990; 265: 4218-4222Abstract Full Text PDF PubMed Google Scholar) in a pertussis toxin-sensitive manner, suggesting an involvement of Gi proteins. The molecular mechanism of these receptor tyrosine kinase-mediated effects remained unclear. In reconstituted phospholipid vesicles, the insulin receptor was found to catalyze tyrosine phosphorylation of Giα and Goα, suggesting the possibility of a direct interaction of receptor tyrosine kinases and G proteins (8Krupinsky J. Rajaram R. Lakonishok M. Benovic J.L. Cerione R.A. J. Biol. Chem. 1988; 263: 12333-12341Abstract Full Text PDF PubMed Google Scholar). Further progress in this field came from in vitro studies of Hausdorff et al. (9Hausdorff W.P. Pitcher J.A. Luttrell D.K. Lindner M.E. Kurose H. Parsons S.J. Caron M.G. Lefkowitz R.J. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 5720-5724Crossref PubMed Scopus (87) Google Scholar) on direct interactions between the non-receptor tyrosine kinase pp60c−src and purified G protein α subunits. pp60c−src was shown to phosphorylate recombinant Gsα as well as other Gsα isoforms on tyrosine residues almost stoichiometrically (9Hausdorff W.P. Pitcher J.A. Luttrell D.K. Lindner M.E. Kurose H. Parsons S.J. Caron M.G. Lefkowitz R.J. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 5720-5724Crossref PubMed Scopus (87) Google Scholar). In 1995, the in vitro sites of phosphorylation of Gsα by pp60c−src were identified (10Moyers J.S. Lindner M.E. Shannon J.D. Parsons S.J. Biochem. J. 1995; 305: 411-417Crossref PubMed Scopus (35) Google Scholar). The phosphorylated tyrosine residues are located at N-terminal position 37 and at C-terminal position 377 of Gsα and thus in regions known to participate in nucleotide exchange and receptor interaction (10Moyers J.S. Lindner M.E. Shannon J.D. Parsons S.J. Biochem. J. 1995; 305: 411-417Crossref PubMed Scopus (35) Google Scholar, 11Reus-Domiano S. Hamm H. FASEB J. 1995; 9: 1059-1066Crossref PubMed Scopus (140) Google Scholar). Very recently, using purified EGF receptor and recombinant Gsα, Poppleton et al. (12Poppleton H. Sun H. Fulgham D. Bertics P. Patel T.B. J. Biol. Chem. 1996; 271: 6947-6951Abstract Full Text Full Text PDF PubMed Scopus (75) Google Scholar) demonstrated an activation of Gsα via phosphorylation on tyrosine residues by EGF receptor kinase. However, little is known about tyrosine phosphorylation of Gsα in intact cells and how such phosphorylation might affect the function of the G protein. We have recently shown that bradykinin (BK) activates dual pathways in A431 human epidermoid carcinoma cells, i.e. the phospholipase C-β/protein kinase C pathway and, independently via Gsα, the cyclic AMP/protein kinase A pathway, which represents a negative feedback loop to the BK-induced protein kinase C activation (13Liebmann C. Graneß A. Ludwig B. Adomeit A. Boehmer A. Boehmer F.-D. Nürnberg B. Wetzker R. Biochem. J. 1996; 313: 109-118Crossref PubMed Scopus (55) Google Scholar). At the same time, as observed earlier by Hosoi et al. (14Hosoi K. Kurihara K. Uhea T. J. Cell. Physiol. 1993; 157: 1-12Crossref PubMed Scopus (9) Google Scholar), the BK-induced activation of protein kinase C leads to an increased serine/threonine phosphorylation of EGF receptors and, subsequently, to a reduced binding of EGF. These findings prompted us to investigate whether EGF is able to affect (vice versa) the BK signaling pathways in A431 cells. epidermal growth factor bradykinin guanosine 5′-O-(3-thiotriphosphate) Dulbecco's modified Eagle's medium polyacrylamide gel electrophoresis 5′-guanylylimidodiphosphate piperazine-N,N′-bis(2-ethanesulfonic acid). In this paper, we present experimental evidence that EGF treatment of A431 cells results in both inhibition of BK-induced [35S]GTPγS binding to Gsα and BK-induced stimulation of adenylate cyclase activity in A431 membranes as well as inhibition of BK-induced cAMP accumulation in intact A431 cells. Furthermore, we show that EGF stimulation leads to specific tyrosine phosphorylation, but also to an increase in serine/threonine phosphorylation of Gsα. This is the first report demonstrating possible functional consequences of Gsα tyrosine phosphorylation in intact cells. A431 human epidermoid carcinoma cells were maintained in Dulbecco's modified Eagle's medium (DMEM; Life Technologies, Inc.) supplemented with 4.5 g/liter glucose, 2 mM glutamine, 7.5% fetal calf serum, and antibiotics. Treatment of intact cells with BK and various agents as outlined in the figure legends was performed with nearly confluent cultures. An A431 particulate fraction (referred to as “membranes”) was prepared as described before (13Liebmann C. Graneß A. Ludwig B. Adomeit A. Boehmer A. Boehmer F.-D. Nürnberg B. Wetzker R. Biochem. J. 1996; 313: 109-118Crossref PubMed Scopus (55) Google Scholar). Protein concentration was determined according to Lowry et al. (15Lowry O.H. Rosebrough N.J. Farr A.L. Randall R.J. J. Biol. Chem. 1951; 193: 265-275Abstract Full Text PDF PubMed Google Scholar) or Bradford (16Bradford M.M. Anal. Biochem. 1976; 72: 248-254Crossref PubMed Scopus (215560) Google Scholar) with bovine serum albumin as a standard. The activity of adenylate cyclase in A431 membranes was determined according to Schultz and Jakobs (17Schultz G. Jakobs K.H. Bergmeyer H.H. Bergmeyer J. Graßl M. Methods in Enzymatic Analysis.Vol. IV. Verlag Chemie, Weinheim, Germany1983: 369-388Google Scholar) with slight modifications. Briefly, membranes (80 μg of protein/assay) were incubated for 20 min at 25°C in a standard mixture containing [α-32P]ATP (∼2 × 107 cpm/tube), 5 mM MgCl2, 1 mM 3-isobutyl-1-methylxanthine, 0.1 mM cAMP, 0.1 mM ATP, 5 mM creatine phosphate, 150 units of creatine phosphokinase, 1 mg/ml bovine serum albumin, and 50 mM HEPES, pH 7.2. To inactivate angiotensin-converting enzyme, which degrades BK, the incubation mixture was supplemented with 1 μM captopril. Variable additions such as NaF (as [AlF4]−), forskolin, GTPγS, and BK were added as indicated, giving a total assay volume of 100 μl. The samples were preincubated for 5 min at 25°C, and the reaction was started by adding the membranes. The incubation was terminated by the addition of 400 μl of ZnCl2 solution (125 mM) followed by 500 μl of Na2CO3 solution (125 mM). The samples were centrifuged for 5 min at 10,000 × g, and 800 μl of the supernatant fluid were transferred to alumina oxid 90 (E. Merck, Darmstadt, Germany)-containing columns. After draining of the sample, labeled cAMP was eluted by the subsequent addition of 2 × 2 ml of Tris-HCl buffer (100 mM), pH 7.5, and [32P]Pi was determined by measuring Cerenkov radiation. For each column, cAMP recovery was estimated individually using [3H]cAMP. A431 cells in 24-well plates were treated with serum-free DMEM overnight, and then the medium was changed to HEPES-buffered serum-free DMEM, which had been adjusted to pH 7.2 just prior usage. Then, cells were exposed to the agents tested in 500 μl of serum-free medium supplemented with 100 μM 3-isobutyl-1-methylxanthine and 10 μM captopril. The reaction was stopped by the addition of 1 ml of ice-cold ethanol (96%, v/v), giving a final concentration of 65% (v/v). The cells were scraped off the plates, and the ethanolic extract was centrifuged at 14,000 × g for 6 min at room temperature. The supernatants containing the extracted cAMP were removed, and the pellets were washed with 500 μl of ethanol (65% v/v) and centrifuged as described above. Supernatants were pooled, evaporated to dryness at 60°C, and resolved in 600 μl of 0.05 M acetate buffer. Two samples were taken for estimation of cAMP concentration using the cAMP 125I-labeled scintillation proximity (non-acetylation) assay from Amersham. For each well, the protein content was determined (16Bradford M.M. Anal. Biochem. 1976; 72: 248-254Crossref PubMed Scopus (215560) Google Scholar). Binding of [35S]GTPγS to A431 membranes was determined as described previously (18Liebmann C. Schnittler M. Nawrath S. Jakobs K.H. Eur. J. Pharmacol. Mol. Pharmacol. Sect. 1991; 207: 67-71Crossref PubMed Scopus (12) Google Scholar). The reaction mixture contained [35S]GTPγS (105 cpm/assay), 1 μM GDP, 5 mM MgCl2, 1 mM EDTA, 1 mM dithiothreitol, 10 μM captopril, 50 mM HEPES, pH 7.2, and 15 μg of membrane protein in a total volume of 200 μl. Further details are provided in the figure legends. The incubation was started by the addition of the membranes and was carried out in quadruplicates for 45 min at 4°C (equilibrium conditions). The reaction was terminated by rapid filtration through Whatman GF/C glass-fiber filters under vacuum. The filters were washed with 3 × 2 ml of 50 mM HEPES, pH 7.2, containing 5 mM MgCl2. The filters were dried and counted for radioactivity. Nonspecific binding was determined in the presence of 10 μM unlabeled GTPγS and represents ∼40% of total binding. BK-induced formation of inositol phosphates in A431 cells was determined as described by Tilly et al. (19Tilly B.C. van Paridou P.A. Verlaan I. Wirtz K.W.A. de Laat S.W. Molenaar W.H. Biochem. J. 1987; 244: 129-135Crossref PubMed Scopus (75) Google Scholar). Briefly, cells were prelabeled with 4 μCi/ml [3H]inositol for 24 h in inositol-free DMEM. 2 h prior to stimulation, the medium was changed to serum-free DMEM containing 20 mM HEPES, pH 7.2. Then, the cells were stimulated for 10 min with bradykinin at the concentrations indicated in the presence of 10 mM LiCl2. The reaction was terminated by replacing the medium with 1 ml of 10% trichloroacetic acid. Inositol phosphate-containing extracts were washed four times with 2 volumes of water-saturated diethyl ether, neutralized by adding Tris base, diluted to 4 ml with distilled water, and placed on AG 1-X8 columns (200-400 mesh, formate form, Bio-Rad). The columns were subsequently eluted with 2 ml of water and 5 ml of 60 mM ammonium formate and 5 mM sodium tetraborate, followed by 2 ml of 1.0 M ammonium formate and 0.1 M formic acid for five times, yielding the inositol phosphate fraction. Radioactivity was measured using a Flow-Scint IV scintillator (Packard Instrument Co.). Antiserum AS 348 was raised against the peptide sequence RMMLRQYELL, corresponding to C-terminal region 385-394 of αs and characterized as described elsewhere (20Spicher K. Kalkbrenner F. Zobel A. Herhammer R. Nürnberg B. Söhling A. Schultz G. Biochem. Biophys. Res. Commun. 1994; 198: 906-914Crossref PubMed Scopus (63) Google Scholar, 21Nürnberg B. Spicher K. Herhammer R. Besserhoff A. Frank R. Hilz H. Schultz G. Biochem J. 1994; 300: 387-394Crossref PubMed Scopus (43) Google Scholar). RC20 monoclonal anti-phosphotyrosine antibodies (Transduction Laboratories) were purchased from Dianova (Hamburg, Germany). Subconfluent A431 cells were preincubated in serum-free DMEM overnight. Then, the medium was changed to fresh serum-free DMEM, and the cells were stimulated with EGF (100 ng/ml) at 37°C in the absence or presence of other additions and for various lengths of time as indicated, followed by the preparation of membranes. Immunoprecipitation of Gs proteins was performed according to Laugwitz et al. (22Laugwitz K.-L. Spicher K. Schultz G. Offermanns S. Methods Enzymol. 1994; 237: 283-294Crossref PubMed Scopus (50) Google Scholar). For several experiments, AS 348 antibodies were covalently coupled to protein A-Sepharose by means of dimethyl pimelimidate. Briefly, 200 μl of AS 348 antiserum (or nonimmune serum as a control) and 200 μl of protein A-Sepharose (12.5 mg of beads) were incubated for 2 h and subsequently washed three times with phosphate-buffered saline, pH 7.4, and twice with 0.2 M sodium borate, pH 9.0. The beads were resuspended in 0.1 M borate buffer. The antibodies were cross-linked to the beads by adding 5.2 mg of dimethyl pimelimidate and mixing for 30 min at room temperature. Thereafter, the beads were washed with 0.2 M ethanolamine, pH 8.0, and the incubation was continued with 0.2 M ethanolamine, pH 8.0, for 2 h at room temperature. Finally, the beads were washed two times with 0.1 M glycine, pH 3.0, and three times with phosphate-buffered saline. The covalently coupled antibodies were stored in phosphate-buffered saline, pH 7.4, with 0.02% sodium azide at 4°C. Membranes were solubilized in 40 μl of 2% (w/v) SDS for 10 min at room temperature. Thereafter, 120 μl of precipitating buffer containing 1% (w/v) Triton X-100, 1% (w/v) deoxycholate, 150 mM NaCl, 1 mM dithiothreitol, 1 mM EDTA, 0.2 mM phenylmethylsulfonyl fluoride, 10 μg/ml aprotinin, 100 μM sodium orthovanadate, and 10 mM Tris-HCl, pH 7.4, were added. To remove insoluble material, the solubilized membranes were centrifuged at 4°C and 12,000 × g for 10 min. Covalently coupled antiserum AS 348 (20 μl) or nonimmune serum as a control was added to the supernatants, and the samples were incubated at 4°C for 4 h under constant rotation. Thereafter, the beads were pelleted (14,000 × g, 10 s) and washed twice with 1 ml of washing buffer A containing 1% (w/v) Nonidet P-40, 0.5% (w/v) SDS, 600 mM NaCl, and 50 mM Tris-HCl, pH 7.4, and twice with 1 ml of washing buffer B containing 300 mM NaCl, 10 mM EDTA, and 100 mM Tris-HCl, pH 7.4. The Sepharose beads were resuspended in 40 μl of SDS sample buffer, heated for 10 min at 100°C, and centrifuged, and the supernatant was subjected to SDS-polyacrylamide gel electrophoresis (PAGE) on 10% (w/v) acrylamide gels and transferred onto nitrocellulose filters. The blots were further processed as described previously (22Laugwitz K.-L. Spicher K. Schultz G. Offermanns S. Methods Enzymol. 1994; 237: 283-294Crossref PubMed Scopus (50) Google Scholar) for AS 348 antibodies and according to the instructions of the manufacturer in the case of RC20 anti-phosphotyrosine antibodies. Subconfluent A431 cells in 6-well plates (Nunc) were depleted of serum for 24 h. Then, the medium was changed to phosphate-free DMEM medium. After the addition of 0.3 mCi of [32P]orthophosphate/ml of medium, cells were incubated for 3 h at 37°C and 7.5% CO2 in a humidified atmosphere. After labeling of cells, they were exposed to EGF (100 ng/ml) for 5 min (or not, as indicated), washed twice with 0.5 ml of phosphate-buffered saline containing 0.1% (w/v) bovine serum albumin, and solubilized at 4°C in 160 μl/well lysis buffer containing 10 mM Tris-HCl, pH 7.4, 150 mM NaCl, 1% (w/v) Triton X-100, 1% (w/v) deoxycholate, 0.5% (w/v) SDS, 1 mM dithiothreitol, 1 mM EDTA, 0.2 mM phenylmethylsulfonyl fluoride, 10 μg/ml aprotinin, 0.1 mM sodium vanadate, and 0.1% (w/v) bovine serum albumin. The lysate was cleared by centrifugation at 12,000 × g and 4°C for 10 min. Metabolically labeled Gsα phosphoprotein was immunoprecipitated by protein A-Sepharose-bound anti-αs antiserum AS 348 as described above. Immunocomplexes were washed twice with both buffers A and B, heated with SDS sample buffer, and subjected to 10% SDS-PAGE. The αs protein bands were localized in the fixed dried gels by exposure to Biomax film (Eastman Kodak Co.) for 4 h at −80°C using an intensifying screen. The section of the dried SDS-polyacrylamide gels corresponding to the position of Gsα (∼100-200 cpm) was extracted; the protein was precipitated and hydrolyzed; and phosphoamino acid analysis was performed by two-dimensional separation on thin-layer cellulose plates as described by Boyle et al. (23Boyle W.J. van der Geer P. Hunter T. Methods Enzymol. 1991; 201: 110-149Crossref PubMed Scopus (1275) Google Scholar). The thin-layer plates were analyzed using a Bio-Rad Model GS250 Molecular Imager and prolonged exposure (3-4 days) on BI imaging screens. [α-32P]ATP (800 Ci/mmol), [35S]GTPγS (1200-1400 Ci/mmol), [32P]Pi (8500-9120 Ci/mmol), and myo-[2-3H]inositol (20.5 Ci/mmol) were purchased from New England Nuclear (Dreieich, Germany). [8-3H]cAMP (26 Ci/mmol), the cAMP 125I-labeled scintillation proximity assay kit, and the ECL Western blotting detection reagents were obtained from Amersham (Braunschweig, Germany). Bradykinin, GTPγS, Gpp(NH)p, 3-isobutyl-1-methylxanthine, HEPES, PIPES, dithiothreitol, bacitracin, captopril, phenylmethylsulfonyl fluoride, cAMP, ATP, creatine phosphate, creatine phosphokinase, bovine serum albumin, ovalbumin, peroxidase-conjugated goat anti-rabbit IgG, forskolin, lauryl sulfate, Nonidet P-40, deoxycholate, sodium orthovanadate, Tween 20 (polyoxyethylenesorbitan monolaurate), dimethyl pimelimidate, and protein A-Sepharose were obtained from Sigma (Deisenhofen, Germany). Leupeptin, pepstatin A, and Pefabloc were from Boehringer (Mannheim, Germany). Ammonium formate, sodium tetraborate, and Triton X-100 were purchased from SERVA (Heidelberg, Germany). Hoe 140 (D-Arg[Hyp3, Thi5,D-Tic7, Oic8]BK, where Thi is ß-Z-thienylamine, Tic is D-1,2,3,4-tetraisoquinoline-3-carboxylic acid, and Oic is [3aS, 7aS]octahydroindol-2-carboxylic acid) was kindly provided by Prof. B. Schölkens (Hoechst AG, Frankfurt, Germany). The EGF receptor-specific blocker AG 1478 was purchased from Calbiochem. In intact A431 cells, BK elicited a concentration-dependent increase in intracellular cAMP up to ∼140% of the basal level (100%) after 20 min of stimulation (Fig. 1). Half-maximal effects were seen at ∼3 nM BK. When the cells were pretreated with EGF (100 ng/ml, 5 min), the ability of BK to enhance cAMP accumulation was significantly inhibited (Fig. 1, inset, bar D versus bar B), whereas EGF pretreatment itself had no significant effect on the basal cAMP level (inset, bar C versus bar A). In contrast, in EGF-treated A431 cells, BK further stimulated inositol phosphate formation, but at a higher level. This was probably due to additional stimulation of the phospholipase C-γ isoform by EGF (Fig. 2). Thus, EGF pretreatment of A431 cells seems to inhibit the stimulatory BK effect on the cAMP system, but not phosphoinositide breakdown, which are activated via the separate G proteins Gs and Gq, respectively (13Liebmann C. Graneß A. Ludwig B. Adomeit A. Boehmer A. Boehmer F.-D. Nürnberg B. Wetzker R. Biochem. J. 1996; 313: 109-118Crossref PubMed Scopus (55) Google Scholar, 19Tilly B.C. van Paridou P.A. Verlaan I. Wirtz K.W.A. de Laat S.W. Molenaar W.H. Biochem. J. 1987; 244: 129-135Crossref PubMed Scopus (75) Google Scholar).Fig. 2BK-induced inositol phosphate formation in EGF-treated and untreated A431 cells. myo-[3H]Inositol-prelabeled A431 cells were incubated with increasing concentrations of BK for 10 min. Incubation and determination of total inositol phosphates were performed as described under “Experimental Procedures.”▪, A431 cells without EGF treatment; •, A431 cells pretreated with 100 ng/ml EGF for 5 min prior to the addition of BK. Each point represents the mean ± S.E. of three independent experiments in triplicate determinations.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Adenylate cyclase activity was measured in A431 membranes prepared from cells after treatment with EGF (100 ng/ml, 5 min). In these membranes, indeed, the stimulation of adenylate cyclase activity by NaF (0.1 mM), GTPγS (10 μM), Gpp(NH)p (10 μM), or BK (1 μM) was reduced compared with the effects in membranes prepared from A431 cells not treated with EGF (Fig. 3). The stimulatory effect of forskolin on adenylate cyclase did not significantly differ under either condition (Fig. 3). These results suggest that EGF interferes with activators of adenylate cyclase at the level of the Gsα protein, which is involved in the activation of adenylate cyclase activity by BK in A431 cells (13Liebmann C. Graneß A. Ludwig B. Adomeit A. Boehmer A. Boehmer F.-D. Nürnberg B. Wetzker R. Biochem. J. 1996; 313: 109-118Crossref PubMed Scopus (55) Google Scholar). As shown in Fig. 4, under our assay conditions, BK caused a concentration-dependent and biphasic increase in [35S]GTPγS binding to A431 membranes. For the first increase, half-maximal and maximal stimulations (up to 155% of control) were observed with ∼0.3 and 1 nM BK, respectively. At higher BK concentrations, [35S]GTPγS binding was continuously reduced, but was followed by a second increase at BK concentrations of ∼30 nM for half-maximal binding and 100 nM for maximal binding (up to 175% of control). The first as well as the second increase in [35S]GTPγS binding induced by BK were completely abolished in the presence of the bradykinin B2 receptor antagonist Hoe 140, indicating that both effects are mediated via the same BK receptor type (Table I). The reasons for the biphasic curve shape in the BK-stimulated [35S]GTPγS binding to A431 membranes are not yet known. It should be noted that the basal binding of [35S]GTPγS to membranes prepared from EGF-treated cells was significantly enhanced (24.0 ± 1.0 fmol/mg of protein; n = 6) compared with that to membranes from untreated cells (18.5 ± 2.2 fmol/mg of protein). To identify that part of the complex pattern of BK-induced [35S]GTPγS binding to A431 membranes that may correspond to the activation of αs subunits, we studied the effect of anti-αs antiserum AS 348 in this assay. AS 348 was raised against the C-terminal region of Gsα, corresponding to amino acids 385-394 (20Spicher K. Kalkbrenner F. Zobel A. Herhammer R. Nürnberg B. Söhling A. Schultz G. Biochem. Biophys. Res. Commun. 1994; 198: 906-914Crossref PubMed Scopus (63) Google Scholar), and has been successfully used in A431 cells both to detect Gsα on Western blots and to prevent the BK-induced stimulation of adenylate cyclase activity (13Liebmann C. Graneß A. Ludwig B. Adomeit A. Boehmer A. Boehmer F.-D. Nürnberg B. Wetzker R. Biochem. J. 1996; 313: 109-118Crossref PubMed Scopus (55) Google Scholar). Pretreatment of A431 membranes with the anti-αs antiserum completely abolished the second phase of [35S]GTPγS binding evoked by BK concentrations higher than 1 nM, whereas the first stimulatory phase employing subnanomolar concentrations of BK was not affected. In the presence of nonimmune serum, both the first and the second BK-induced increases in [35S]GTPγS binding were not significantly altered (Fig. 4A). Compared with the effect of BK in the presence of anti-αs antibodies, a similar curve shape was obtained when the effect of BK on [35S]GTPγS binding was studied in membranes prepared from EGF-pretreated A431 cells (Fig. 4B). In these membranes, an increase in [35S]GTPγS binding at subnanomolar BK concentrations was observed, which was attenuated at concentrations higher than 1 nM BK and completely failed at concentrations higher than ∼3 nM BK. In addition, we studied the effect of the β-adrenergic agonist isoprenaline, which is known to activate Gsα selectively, on [35S]GTPγS binding to A431 membranes. Isoprenaline was found to stimulate [35S]GTPγS binding in a concentration-dependent manner (EC50∼ 20 nM), but completely failed to induce this effect in the presence of the anti-αs antiserum as well as in membranes prepared from EGF-treated A431 cells (Fig. 5).Table I.Effect of the bradykinin B2 receptor antagonist Hoe 140 on the BK-induced biphasic stimulation of [33S]GTPγS binding to A431 membranesAdditionsStimulation of [33S]GTPγS bindingfmol/mg proteinNone (basal)18.5 ± 2.2BK (0.3 nM)27.8 ± 3.2aSignificantly different from BK alone (p < 0.01; Student's t test).+Hoe 14017.7 ± 1.9BK (100 nM)33.5 ± 7.3bSignificantly different from BK alone (p < 0.05; Student's t test).+Hoe 14018.1 ± 1.2a Significantly different from BK alone (p < 0.01; Student's t test).b Significantly different from BK alone (p < 0.05; Student's t test). Open table in a new tab Fig. 5Effects of isoprenaline on [35S]GTPγS binding to A431 membranes. Shown is the binding of [35S]GTPγS to A431 membranes (▴), to A431 membranes in the presence of anti-αs antiserum AS 348 (▵), and to A431 membranes from cells pretreated with EGF (100 ng, 5 min) (▿). Experiments were performed as described for" @default.
• W2102041216 created "2016-06-24" @default.
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• W2102041216 title "Tyrosine Phosphorylation of Gsα and Inhibition of Bradykinin-induced Activation of the Cyclic AMP Pathway in A431 Cells by Epidermal Growth Factor Receptor" @default.
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