Coupling of the Insulin-like Growth Factor-I Receptor Tyrosine Kinase to Gi2 in Human Intestinal Smooth Muscle
2001; Elsevier BV; Volume: 276; Issue: 10 Linguagem: Inglês
10.1074/jbc.m011145200
ISSN1083-351X
AutoresJohn F. Kuemmerle, Karnam S. Murthy,
Tópico(s)PI3K/AKT/mTOR signaling in cancer
ResumoEndogenous insulin-like growth factor-1 (IGF-I) stimulates growth of cultured human intestinal smooth muscle by activating distinct mitogen-activated protein (MAP) kinase-dependent and phosphatidylinositol 3-kinase-dependent signaling pathways. In Rat1 and Balb/c3T3 fibroblasts and in neurons the IGF-I receptor is coupled to an inhibitory G protein, Gi, which mediates Gβγ-dependent MAP kinase activation. The present study determined whether in normal human intestinal smooth muscle cells the IGF-I receptor activates a heterotrimeric G protein and the role of G protein activation in mediating IGF-I-induced growth. IGF-I elicited IGF-I receptor tyrosine phosphorylation, resulting in the specific activation of Gi2. Gβγ subunits selectively mediated IGF-I-dependent MAP kinase activation; Gαi2 subunits selectively mediated IGF-I-dependent inhibition of adenylyl cyclase actvity. IGF-I-stimulated MAP kinase activation and growth were inhibited by pertussis toxin, an inhibitor of Gi/Goactivation. Cyclic AMP inhibits growth of human intestinal muscle cells. IGF-I inhibited both basal and forskolin-stimulated cAMP levels. This inhibition was attenuated in the presence of pertussis toxin. IGF-I stimulated phosphatidylinositol 3-kinase activation, in contrast to MAP kinase activation, occurred independently of Gi2 activation. These data suggest that IGF-I specifically activates Gi2, resulting in concurrent Gβγ-dependent stimulation of MAP kinase activity and growth, and Gαi2-dependent inhibition of cAMP levels resulting in disinhibition of cAMP-mediated growth suppression. Endogenous insulin-like growth factor-1 (IGF-I) stimulates growth of cultured human intestinal smooth muscle by activating distinct mitogen-activated protein (MAP) kinase-dependent and phosphatidylinositol 3-kinase-dependent signaling pathways. In Rat1 and Balb/c3T3 fibroblasts and in neurons the IGF-I receptor is coupled to an inhibitory G protein, Gi, which mediates Gβγ-dependent MAP kinase activation. The present study determined whether in normal human intestinal smooth muscle cells the IGF-I receptor activates a heterotrimeric G protein and the role of G protein activation in mediating IGF-I-induced growth. IGF-I elicited IGF-I receptor tyrosine phosphorylation, resulting in the specific activation of Gi2. Gβγ subunits selectively mediated IGF-I-dependent MAP kinase activation; Gαi2 subunits selectively mediated IGF-I-dependent inhibition of adenylyl cyclase actvity. IGF-I-stimulated MAP kinase activation and growth were inhibited by pertussis toxin, an inhibitor of Gi/Goactivation. Cyclic AMP inhibits growth of human intestinal muscle cells. IGF-I inhibited both basal and forskolin-stimulated cAMP levels. This inhibition was attenuated in the presence of pertussis toxin. IGF-I stimulated phosphatidylinositol 3-kinase activation, in contrast to MAP kinase activation, occurred independently of Gi2 activation. These data suggest that IGF-I specifically activates Gi2, resulting in concurrent Gβγ-dependent stimulation of MAP kinase activity and growth, and Gαi2-dependent inhibition of cAMP levels resulting in disinhibition of cAMP-mediated growth suppression. insulin-like growth factor insulin receptor substrate Src-homology/collagen phosphatidylinositol 3-kinase pertussis toxin Dulbecco's modified Eagle's medium 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid guanosine 5′-3-O-(thio)triphosphate insulin-like growth factor I receptor β subunit insulin-like growth factor-binding protein mitogen-activated protein Insulin-like growth factor (IGF)1-I mediates three distinct regulatory effects on cell growth by activation of the IGF-I receptor. IGF-I stimulates proliferation of cells and may be required for optimal growth of these cells (1Baserga R. Sell C. Porcu P. Rubini M. Cell Prolif. 1994; 27: 62-71Crossref Scopus (134) Google Scholar, 2Cambrey A.D. Kwon O.J. Gray A.J. Harrison N.K. Yacoub M. Barnes P.J. Laurent G.J. Chung K.F. Clin. Sci. 1995; 89: 611-617Crossref PubMed Scopus (54) Google Scholar, 3Reape T.J. Kanczler J.M. Ward J.P.T. Thomas C.R. Am. J. Physiol. 1996; 270: H1141-H1148PubMed Google Scholar, 4Yamamoto M. Yamamoto K. Exp. Cell Res. 1994; 212: 62-68Crossref PubMed Scopus (39) Google Scholar). Transformation and maintenance of the transformed state also require IGF-I receptor activation in some cells (4Yamamoto M. Yamamoto K. Exp. Cell Res. 1994; 212: 62-68Crossref PubMed Scopus (39) Google Scholar, 5Sell C. Rubini M. Rubin R. Liu J.-P. Efstatriadis A. Baserga R. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 11217-11221Crossref PubMed Scopus (530) Google Scholar). IGF-I can also protect cells from apoptosis (6Parrizas M. Saltiel A.R. LeRoith D. J. Biol. Chem. 1997; 272: 154-161Abstract Full Text Full Text PDF PubMed Scopus (598) Google Scholar, 7Wu Y. Tewari M. Cui S Rubin R. J. Cell. Physiol. 1996; 168: 499-509Crossref PubMed Scopus (98) Google Scholar). Three proteins have been identified which are rapidly recruited to the membrane after IGF-I receptor tyrosine phosphorylation: insulin receptor substrate (IRS)-1/2, Src-homology/collagen (Shc), and CT-10-regulated kinase (8Hernandez-Sanchez C. Blakesley V. Kalebic T. Helman L. LeRoith D. J. Biol. Chem. 1995; 270: 29176-29181Abstract Full Text Full Text PDF PubMed Scopus (76) Google Scholar, 9Sasaoka T. Rose D.W. Jhun B.H. Saltiel A.R. Draznin B. Olefsky J.M. J. Biol. Chem. 1994; 269: 13689-13694Abstract Full Text PDF PubMed Google Scholar, 10Beitner-Johnson D. LeRoith D. J. Biol. Chem. 1995; 270: 5187-5190Abstract Full Text Full Text PDF PubMed Scopus (107) Google Scholar). Through these substrates IGF-I mediates activation of two main signaling cascades, the MAP kinase and PI 3-kinase pathways, which can act either in conjunction, in opposition, or individually to mediate the response to IGF-I whether proliferative, transforming, or anti-apoptotic (6Parrizas M. Saltiel A.R. LeRoith D. J. Biol. Chem. 1997; 272: 154-161Abstract Full Text Full Text PDF PubMed Scopus (598) Google Scholar, 11Coolican S.A. Samuel D.S. Ewton D.Z. McWade F.J. Florini J.R. J. Biol. Chem. 1997; 272: 6653-6662Abstract Full Text Full Text PDF PubMed Scopus (538) Google Scholar, 12Foncea R. Andersson M. Ketterman A. Blakesley V. Sapag-Hagart M. Sugden P.H. LeRoith D. Lavender S. J. Biol. Chem. 1997; 272: 19115-19124Abstract Full Text Full Text PDF PubMed Scopus (172) Google Scholar). In Rat1 fibroblasts IGF-I activates a pertussis toxin (PTx)-sensitive heterotrimeric G protein leading to Gβγ-mediated, Ras-dependent MAP kinase stimulation (13Luttrell L.M. van Beisen T. Hawes B.E. Koch W.J. Touhara K. Lefkowitz R.J. J. Biol. Chem. 1995; 270: 16495-16498Abstract Full Text Full Text PDF PubMed Scopus (185) Google Scholar). The signal is transmitted by Gβγ subunits dissociated from an IGF-I-activated inhibitory (PTx-sensitive) G protein. This mechanism of ras-dependent MAP kinase activation is shared by both the IGF-I receptor tyrosine kinases and G protein-coupled receptors, such as the lysophosphatidic acid receptors (13Luttrell L.M. van Beisen T. Hawes B.E. Koch W.J. Touhara K. Lefkowitz R.J. J. Biol. Chem. 1995; 270: 16495-16498Abstract Full Text Full Text PDF PubMed Scopus (185) Google Scholar, 14van Beisen T. Hawes B.E. Luttrell D.K. Krueger K.M. Touhara K. Porfiri E. Sakaue M. Luttrell L.M. Lefkowitz R.J. Nature. 1995; 376: 781-784Crossref PubMed Scopus (520) Google Scholar, 15Selbie L.A. Hill S.J. Trends Pharmacol. Sci. 1998; 19: 87-93Abstract Full Text Full Text PDF PubMed Scopus (254) Google Scholar, 16Della, Rocca G.J. Maudsley S. Daaka Y. Lefkowitz R.J. Luttrell L.J. J. Biol. Chem. 1999; 274: 13978-13984Abstract Full Text Full Text PDF PubMed Scopus (222) Google Scholar). Activation of either receptor tyrosine kinases or the G protein-coupled receptors induces rapid tyrosine phosphorylation of docking proteins, e.g. Shc and Grb2, which function as membrane scaffolds for the recruitment of Ras guanine nucleotide exchange factors, e.g. mSOS, that regulate Ras activity. The regulation of Ras activity by this pathway has further been shown to involve the participation of either Src family nonreceptor tyrosine kinases or focal adhesion kinases depending on the ligand and the cell type examined (15Selbie L.A. Hill S.J. Trends Pharmacol. Sci. 1998; 19: 87-93Abstract Full Text Full Text PDF PubMed Scopus (254) Google Scholar, 16Della, Rocca G.J. Maudsley S. Daaka Y. Lefkowitz R.J. Luttrell L.J. J. Biol. Chem. 1999; 274: 13978-13984Abstract Full Text Full Text PDF PubMed Scopus (222) Google Scholar). IGF-II acting through its cognate IGF-II/mannose 6-phosphate receptor stimulates growth and metabolic effects. Coupling of this receptor to the inhibitory heterotrimeric G protein, Gi2, has also been described in both membranes derived from mouse Balb/3T3 fibroblasts and COS cells transfected with IGF-II/Man-6-P receptor cDNA (17Nishimoto I. Muruyama Y. Katada T. Ui M. Ogata E. J. Biol. Chem. 1989; 264: 14029-14038Abstract Full Text PDF PubMed Google Scholar, 18Okamoto T. Nishimoto I. Muruyama Y. Ohkuni Y. Ogata E. Biochem. Biophys. Res. Commun. 1990; 168: 1201-1210Crossref PubMed Scopus (28) Google Scholar, 19Ikezu T. Okamoto T. Giambarella U. Yokota T. Nishimoto I. J. Biol. Chem. 1995; 270: 29224-29228Abstract Full Text Full Text PDF PubMed Scopus (64) Google Scholar). Upon stimulation by IGF-II, but not by Man-6-P, the activated IGF-II/Man-6-P receptor interacts with Gi2 through the Arg2410-Lys2423 sequence in its C-terminal intracellular domain (19Ikezu T. Okamoto T. Giambarella U. Yokota T. Nishimoto I. J. Biol. Chem. 1995; 270: 29224-29228Abstract Full Text Full Text PDF PubMed Scopus (64) Google Scholar). In contrast to IGF-I-induced IGF-I receptor activation where MAP kinase stimulation is Gβγ-dependent, IGF-II-induced activation of the IGF-II/Man-6-P receptor results in Gαi2-dependent inhibition of adenylate cyclase activity, an effect only potentiated by the Gβγ subunits derived from Gi2activation (19Ikezu T. Okamoto T. Giambarella U. Yokota T. Nishimoto I. J. Biol. Chem. 1995; 270: 29224-29228Abstract Full Text Full Text PDF PubMed Scopus (64) Google Scholar). Human intestinal smooth muscle cells produce IGF-I, which plays a autocrine role in the regulation of growth in culture (20Kuemmerle J.F. Gastroenterology. 1997; 113: 817-824Abstract Full Text PDF PubMed Scopus (52) Google Scholar). In these cells IGF-I-stimulated growth is mediated by activation of distinct MAP kinase-dependent and PI 3-kinase-dependent signaling cascades (21Kuemmerle J.F. Bushman T.L. Am. J. Physiol. 1998; 175: G151-G158Google Scholar). Whether IGF-I-stimulated MAP kinase activation and growth in these cells involve the activation of a heterotrimeric G protein, and the roles of the G protein subunits in mediating this effect are not known. The specific G protein activated by IGF-I and the roles of the α and βγ subunits derived from G protein activation on growth have not been examined. In the present study we show that IGF-I specifically activates the PTx-sensitive inhibitory G protein, Gi2. Gi2activation results in concurrent Gβγ-dependent stimulation of MAP kinase activity and growth, and Gαi2-dependent inhibition of adenylyl cyclase activation, cAMP production and results in disinhibition of cAMP-mediated growth suppression. Muscle cells were isolated from the circular muscle layer of human jejunum as described previously (20Kuemmerle J.F. Gastroenterology. 1997; 113: 817-824Abstract Full Text PDF PubMed Scopus (52) Google Scholar, 21Kuemmerle J.F. Bushman T.L. Am. J. Physiol. 1998; 175: G151-G158Google Scholar). Segments of normal jejunum were obtained from patients undergoing surgery according to a protocol approved by the Institutional Committee on the Conduct of Human Research. Briefly, muscle cells were isolated by enzymatic digestion for 60 min at 31 °C in a medium containing 0.2% collagenase (CLS type II) and 0.1% soybean trypsin inhibitor. The medium consisted of (in mm): 120 NaCl, 4 KCl, 2.6 KH2PO4, 2 CaCl2, 0.6 MgCl2, 25 HEPES, 14 glucose, and 2.1% Eagle's essential amino acid mixture. Primary cultures of human intestinal muscle cells were initiated and maintained in Dulbecco's modified Eagle's medium containing 10% fetal bovine serum (DMEM-10) and containing 200 units/ml penicillin, 200 μg/ml streptomycin, 100 μg/ml gentamycin, and 2 μg/ml amphotericin B. The cells were plated at a concentration of 5 × 105 cells/ml and incubated in a 10% CO2 environment at 37 °C. All subsequent studies were performed in first passage cultured cells after 7 days, at which time the cells were confluent. Proliferation of smooth muscle cells in culture was measured by the incorporation of [3H]thymidine as described previously (20Kuemmerle J.F. Gastroenterology. 1997; 113: 817-824Abstract Full Text PDF PubMed Scopus (52) Google Scholar, 21Kuemmerle J.F. Bushman T.L. Am. J. Physiol. 1998; 175: G151-G158Google Scholar). Briefly, the cells were washed free of serum and incubated for 24 h in DMEM-0. After a 24-h incubation in the absence of serum, the cells were incubated for an additional 24 h with a maximally effective concentration of IGF-I (100 nm) in the presence and absence of various test agents. During the final 4 h of this incubation period, 1 μCi/ml [3H]thymidine was added to the medium. [3H]Thymidine incorporation into the perchloric acid-extractrable pool was used as a measure of DNA synthesis. DNA content was measured fluorometrically using Hoescht 33528 with excitation at 356 nm and emission at 492 nm. Calf thymus DNA was used as a standard. [3H]Thymidine incorporation was expressed as cpm/ng DNA. G proteins selectively activated by IGF-I were identified by the method of Okamotoet al. (22Okamoto T. Ikezu T. Muruyama Y. Ogata E. Nishimoto I. FEBS Lett. 1992; 305: 125-128Crossref PubMed Scopus (38) Google Scholar) as described previously by us (23Murthy K.S. Coy D.H. Makhlouf G.M. J. Biol. Chem. 1996; 271: 23458-23463Abstract Full Text Full Text PDF PubMed Scopus (123) Google Scholar, 24Murthy K.S. Makhlouf G.M. Mol. Pharmacol. 1996; 50: 870-877PubMed Google Scholar, 25Murthy K.S. Makhlouf G.M. J. Biol. Chem. 1997; 272: 21317-21324Abstract Full Text Full Text PDF PubMed Scopus (74) Google Scholar). Confluent human intestinal muscle cells growing in 100-mm dishes were scraped off the plate and homogenized in 20 mm HEPES medium (pH 7.4) containing 2 mm MgCl2, 1 mm EDTA, and 2 mm dithiothreitol. After centrifugation at 27,000 × g for 15 min, the crude membranes were solubilized for 60 min at 4 °C in 20 mmHEPES medium (pH 7.4) containing 2 mm EDTA, 240 mm NaCl, and 1% CHAPS. The membranes were incubated for 20 min with 60 nm [35S]GTPγS in a solution containing 10 mm HEPES (pH 7.4), 100 μm EDTA, and 10 mm MgCl2. The reaction was stopped with 10 volumes of 100 mm Tris-HCl medium (pH 8.0) containing 10 mm MgCl2, 100 mm NaCl, and 20 μm GTP, and the mixture was placed in wells precoated with specific G protein antibodies. After incubation for 2 h on ice, the wells were washed three times with phosphate-buffered saline containing 0.05% Tween 20, and radioactivity from each well was counted. Coating with G protein antibodies (1:1,000) was done after the wells were coated with anti-rabbit IgG (1:1,000) for 2 h on ice. The selective IGF-I receptor tyrosine kinase inhibitor, tyrphostin AG 1024 (100 μm) (26Parrizas M. Gazit A. Levitzki A. Wertheimer E. LeRoith D. Endocrinology. 1997; 138: 1427-1433Crossref PubMed Scopus (0) Google Scholar), was used to identify to role of IGF-I receptor tyrosine kinase phosphorylation in G protein activation. Binding of IGF-I to the IGF-I receptor results in tyrosine phosphorylation of the IGF-I receptor β subunit (IGF-IRβ). Tyrosine phosphorylation of the IGF-IRβ was measured by immunoprecipitation of the IGF-IRβ and subsequent Western blotting of tyrosine-phosphorylated proteins. Confluent muscle cells growing in 100-mm plates were incubated in serum-free DMEM for 72 h. The cells were stimulated for 2 min with increasing concentrations (1–100 nm) of IGF-I, IGF-II, and del(1–6)IGF-II, a synthetic IGF-II agonist that does not interact with IGF-binding proteins (33Francis G.L. Aplin S.E. Milner S.J. McNeil K.A. Ballard F.J. Wallace J.C. Biochem. J. 1993; 293: 719Crossref Scopus (72) Google Scholar). The reaction was terminated by washing with ice-cold phosphate-buffered saline. The cells were lysed in immunoprecipitation buffer consisting of phosphate-buffered saline with added 1% (v/v) Nonidet P-40, 0.5% (w/v) sodium deoxycholate, 0.1% (w/v) SDS, 10 mm phenylmethylsulfonyl fluoride, 1% (w/v) aprotinin, and 1 mm sodium orthovanadate. Cell lysates containing equal amounts of protein were incubated with 2 μg/ml antibody to the IGF-IRβ for 2 h at 4 °C. 20 μl of protein A/G-agarose was added, and the incubation was continued overnight. The resulting immune complexes were collected by centrifugation and washed four times in immunoprecipitation buffer. The final pellet was resuspended in 40 μl of electrophoresis sample buffer and boiled for 5 min. Proteins were separated by SDS-polyacrylamide gel electrophoresis on 10% gels under reducing conditions and then transferred to nitrocellulose membranes. The membranes were incubated overnight with a 1:1,000 dilution of anti-phosphotyrosine antibody (4G10) and then for 90 min with a 1:2,000 dilution of goat anti-mouse IgG-horseradish peroxidase. The bands were visualized with enhanced chemiluminescence and quantitated with densitometry. MAP kinase activity was measured as described previously (21Kuemmerle J.F. Bushman T.L. Am. J. Physiol. 1998; 175: G151-G158Google Scholar). Briefly, confluent muscle cells growing in six-well plates were incubated in serum-free DMEM for 72 h. The cells were stimulated with 100 nm IGF-I for various time periods, 0–240 min, in the presence and absence of test agents. The cells were lysed in a buffer containing (in mm): 10 Tris (pH 7.4), 150 NaCl, 2 EGTA, 2 dithiothreitol, 1 orthovanadate, 1 phenylmethylsulfonyl fluoride, with added 10 μg/ml leupeptin and 10 μg/ml aprotinin. Cellular debris in the lysates was precipitated by centrifugation at 12,000 × g for 20 min at 4 °C. MAP kinase activity was measured in duplicate in aliquots of cell lysate containing equal amounts of protein by the incorporation of phosphate from [γ-32P]ATP (1 μCi/30 μl of reaction volume) into a synthetic MAP kinase substrate (Amersham Pharmacia Biotech) for a 30-min incubation at 30 °C. The reaction was terminated, and phosphorylated peptide substrate was separated using phosphocellulose microfuge spin tubes (Pierce). The results are expressed in pmol of phosphate incorporated/min/mg of protein. Confluent muscle cells growing in 100-mm dishes were permeabilized by modification of techniques described previously (23Murthy K.S. Coy D.H. Makhlouf G.M. J. Biol. Chem. 1996; 271: 23458-23463Abstract Full Text Full Text PDF PubMed Scopus (123) Google Scholar, 24Murthy K.S. Makhlouf G.M. Mol. Pharmacol. 1996; 50: 870-877PubMed Google Scholar, 25Murthy K.S. Makhlouf G.M. J. Biol. Chem. 1997; 272: 21317-21324Abstract Full Text Full Text PDF PubMed Scopus (74) Google Scholar, 27Kuemmerle J.F. Makhlouf G.M. J. Biol. Chem. 1995; 270: 25488-25494Abstract Full Text Full Text PDF PubMed Scopus (133) Google Scholar). The effects of specific G protein subunits were investigated by immunoneutralization using selective antibodies to specific G protein subunits as described previously. This technique has been validated previously and used to identify the signaling mechanisms and functional effects mediated via specific G protein subunits activated by somatostatin, opioid, and muscarinic receptors on intestinal smooth muscle cells (23Murthy K.S. Coy D.H. Makhlouf G.M. J. Biol. Chem. 1996; 271: 23458-23463Abstract Full Text Full Text PDF PubMed Scopus (123) Google Scholar, 24Murthy K.S. Makhlouf G.M. Mol. Pharmacol. 1996; 50: 870-877PubMed Google Scholar, 25Murthy K.S. Makhlouf G.M. J. Biol. Chem. 1997; 272: 21317-21324Abstract Full Text Full Text PDF PubMed Scopus (74) Google Scholar). Briefly, muscle cells were released from the culture plate by treatment with 0.5% (w/v) trypsin containing 0.53 mm EDTA. The cells were washed free of enzymes by centrifugation at 150 ×g and resuspended in a cytosol-like buffer containing (in mm): 20 NaCl, 100 KCl, 1 MgSO4, 25 NaHCO3, 1 EGTA, 0.18 Ca2+, and 1% IGF-I-free bovine serum albumin. Cells were permeabilized by incubation with 35 μg/ml saponin at 31 °C for 10 min. The cells were washed free of saponin by centrifugation at 150 × g and resuspended in the same medium with 1.5 mm ATP and ATP-regenerating system (5 mm creatine phosphate and 10 units/ml creatine phosphokinase). The cells were incubated for 1 h with neutralizing antibody to Gαi2 (10 μg/ml) or Gβ (10 μg/ml). The reaction was initiated by addition of 100 nm IGF-I and terminated after 10 min by placing the cells on ice. The cells were rapidly centrifuged at 150 ×g and 4 °C and the supernatant removed. The cells were resuspended in MAP kinase lysis buffer (see above), and MAP kinase activity was measured by in vitro kinase assay as described above. PI 3-kinase activity was measured by a modification of the method of Higaki et al. (28Higaki M. Sakaue H. Ogawa W. Kasuga M. Shimokodo K. J. Biol. Chem. 1996; 271: 29342-29346Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar) as described previously (21Kuemmerle J.F. Bushman T.L. Am. J. Physiol. 1998; 175: G151-G158Google Scholar). Briefly, muscle cells grown to confluence in 100-mm dishes were incubated in serum-free DMEM for 72 h. Cells were stimulated for 10 min with 100 nm IGF-I in the presence and absence of various test agents. The cells were lysed in a buffer consisting of (in mm): 50 Tris-HCl (pH 7.4), 150 NaCl, 1 Na2VO3, 2 EDTA, 1 MgCl2, 1 CaCl2, and 30 nm leupeptin, with added 1% (w/v) trasylol and 1% (v/v) Nonidet P-40. Aliquots of cell lysate containing equal amounts of protein were incubated with 25 μl of anti-phosphotyrosine antibody (PY20) coupled to agarose beads with gentle mixing for 2 h at 4 °C. The beads were collected by centrifugation at 12,000 × g for 5 min at 4 °C and washed three times with lysis buffer and two times with kinase assay buffer. Kinase assay buffer consisted of (in mm): 50 Tris-HCl (pH 7.8), 50 NaCl, 2 MgCl2, and 0.5 EDTA. After the final washing the beads were resuspended in 30 μl of kinase assay buffer to which 10 μl of sonicated 1 mg/ml phosphatidylinositol was added. The reaction was initiated by the addition of 5 μl of 50 mm ATP containing 0.5 μCi of [γ-32P]ATP and continued for 10 min at 30 °C. The reaction was terminated by the addition of 0.5 ml of 1 n HCl and 2 ml of chloroform-methanol (2:1, v/v). Phospholipids were recovered from the lower organic phase and dried under N2 gas. The dried phospholipids were dissolved in chloroform and spotted on Silica H Gel TLC plates impregnated with 1% potassium oxalate. Chromatograms were developed in chloroform, methanol, 28% NH3, water (70:100:15:25, v/v). The plates were air dried and phospholipids visualized with autoradiography. The spots corresponding to authentic phosphatidylinositol 3,4,5-trisphosphate were scraped off the plates and incorporated 32P quantified by β-scintillation counting. Results are expressed as the increase in 32P incorporation into PI-3-P in cpm above basal values. Adenylyl cyclase activity was measured by the method of Salomon et al. (29Salomon Y. Londos S. Rodbell M. Anal. Chem. 1974; 58: 541-548Google Scholar). Briefly, a 0.1-mg sample of membrane protein was incubated for 15 min at 37 °C in 50 mm Tris-HCl (pH 7.4), 1 nmATP, 2 mm cAMP, 0.1 mm GTP, 0.1 mmisobutylmethylxanthine, 5 mm MgCl2, 100 mm NaCl, 5 mm creatine phosphate, 50 units/ml creatine phosphokinase, and 4 × 106 cpm of [3H]ATP. The reaction was terminated by the addition of 2% SDS, 45 mm ATP, and 1.5 mm cAMP. [3H]cAMP was separated from [3H]ATP by sequential chromatography on Dowex AG50W-4X and alumina columns. The results were expressed as pmol of cAMP/mg of protein/min. cAMP production was measured by modification of methods described previously (23Murthy K.S. Coy D.H. Makhlouf G.M. J. Biol. Chem. 1996; 271: 23458-23463Abstract Full Text Full Text PDF PubMed Scopus (123) Google Scholar, 24Murthy K.S. Makhlouf G.M. Mol. Pharmacol. 1996; 50: 870-877PubMed Google Scholar, 25Murthy K.S. Makhlouf G.M. J. Biol. Chem. 1997; 272: 21317-21324Abstract Full Text Full Text PDF PubMed Scopus (74) Google Scholar). Briefly, confluent muscle cells growing in six-well plates were incubated in serum-free DMEM for 72 h. Cells were incubated for 10 min in 1 μm isobutylmethylxanthine. cAMP in the cells was stimulated by activation with 10 μm forskolin, and 100 nm IGF-I was added for an additional 5 min. The reaction was terminated using ice-cold 10% trichloroacetic acid in which the cells were incubated for 15 min at 4 °C. The supernatants were extracted three times with water-saturated diethyl ether. The resulting aqueous phase was frozen and lyophilized. The samples were reconstituted for radioimmunoassay in 500 μl of 50 mmsodium acetate (pH 6.2) and acetylated with triethylamine/acetic anhydride (3:1 v/v) for 30 min. cAMP was measured in duplicate using 100-μl aliquots and expressed as pmol/mg of protein. Values given represent the mean ± S.E. of n experiments where n represents the number of experiments on cells derived from separate primary cultures. Statistical significance was tested by Student's t test for either paired or unpaired data as was appropriate. Recombinant human IGF-I and IGF-II were from Austral Biologicals (San Ramon, CA). Collagenase and soybean trypsin inhibitor were from Worthington. HEPES was from Research Organics (Cleveland, OH). DMEM was from Mediatech Inc. (Herndon, VA). Fetal bovine serum was from Summit Biotechnologies, Inc. (Fort Collins, CO). The MAP kinase assay kit, [γ-32P]ATP (specific activity 3,000 Ci/mmol), [3H]thymidine (specific activity 6 Ci/mmol), [3H]ATP (specific activity 26.5 Ci/mmol), and [125I]cAMP (specific activity 2,000 Ci/mmol) were from Amersham Pharmacia Biotech. [35S]GTPγS (specific activity 1,250 Ci/mmol) was from PerkinElmer Life Sciences. Western blotting materials and the protein assay kit were from Bio-Rad. Anti-phosphotyrosine PY20- agarose beads were from Transduction Laboratories (Lexington, KY). Phosphocellulose spin columns were from Pierce. Thin layer chromatography plates were from Analtech (Newark, DE). Plastic cultureware was from Corning (Corning, NY). Antibodies to Gαi1, Gαi3, Gαq/11 and Gβ were from Santa Cruz Biotechnology (Santa Cruz, CA). Antibody to Gαi2 was from Chemicon (Temecula, CA). Anti-phosphotyrosine antibody 4G10 was from Upstate Biotechnology (Lake Placid, NY). Tyrphostin AG 1024, forskolin, and PTx were from Calbiochem. Phosphatidylinositol and all other chemicals were obtained from Sigma. IGF-I increased [3H]thymidine incorporation by 289 ± 9% above basal (basal, 34 ± 2 cpm/ng DNA) (Fig.1). The growth elicited by IGF-I was partially inhibited, 35 ± 5%, in the presence of 200 ng/ml PTx. We have shown previously that IGF-I-induced growth is mediated jointly by activation of distinct MAP kinase-dependent, PI 3-kinase-independent and a MAP kinase-independent, PI 3-kinase-dependent pathways (21Kuemmerle J.F. Bushman T.L. Am. J. Physiol. 1998; 175: G151-G158Google Scholar). To determine whether PTx-sensitive growth elicited by IGF-I was mediated by activation of the MAP kinase-dependent or the PI 3-kinase-dependent pathways, cells were incubated with the MAP kinase kinase inhibitor PD98059 (10 μm) or the PI 3-kinase inhibitor LY294002 (10 μm). We have shown previously that at the concentrations used these inhibitors selectively block activation of MAP kinase and PI 3-kinase, respectively, in these cells (21Kuemmerle J.F. Bushman T.L. Am. J. Physiol. 1998; 175: G151-G158Google Scholar). Thymidine incorporation in response to 100 nmIGF-I was partly inhibited (42 ± 4%) by the MAP kinase kinase inhibitor, partly inhibited (52 ± 3%) by the PI 3-kinase inhibitor, and partly inhibited (35 ± 5%) by PTx (Fig. 1) (21Kuemmerle J.F. Bushman T.L. Am. J. Physiol. 1998; 175: G151-G158Google Scholar,30Dudley D.T. Pang L. Decker S.J. Bridges A.J. Saltiel A.J. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 7686-7689Crossref PubMed Scopus (2557) Google Scholar, 31Vlahos C.J. Matter W.F. Hui K.Y. Brown R.F. J. Biol. Chem. 1994; 269: 5241-5248Abstract Full Text PDF PubMed Google Scholar). The MAP kinase kinase inhibitor had only a minor additive effect to that of PTx (MAP kinase kinase inhibitor + PTx: 42 ± 10% inhibition versus PTx alone: 35 ± 5% inhibition,p = not significant) on growth stimulated by IGF-I (Fig. 1). In contrast, the addition of the PI 3-kinase inhibitor and PTx was fully additive, strongly inhibiting growth stimulated by IGF-I (PI 3-K inhibitor + PTx: 90 ± 2% versus PTx alone: 35 ± 5%, p < 0.01) (Fig. 1). These results suggest that the portion of IGF-I-induced growth mediated by activation of the MAP kinase pathway was sensitive to PTx, whereas the portion mediated by activation of the PI 3-kinase pathway was insensitive to PTx. IGF-II and insulin were also examined for their ability to stimulate growth of these cells. 100 nm IGF-II increased [3H]thymidine incorporation by 40 ± 25% above basal, and 100 nm insulin increased [3H]thymidine incorporation by 35 ± 15% above basal values. The increase in [3H]thymidine incorporation elicited by IGF-II or insulin, however, was not inhibited in the presence of PTx (38 ± 8 and 36 ± 20% above basal, respectively). The rank order of potency of these three peptides to stimulate growth, IGF-I > IGF-II > insulin, implied that the growth stimulated by IGF-I reflected predominantly activation of its cognate IGF-I receptor. The ability of IGF-I and IGF-II to stimulate tyrosine phosphorylation of the IGF-IRβ was examined in anti-phosphotyrosine Western blots of IGF-IRβ immunoprec
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