Insulin/Insulin-like Growth Factor I Hybrid Receptors Have Different Biological Characteristics Depending on the Insulin Receptor Isoform Involved
2002; Elsevier BV; Volume: 277; Issue: 42 Linguagem: Inglês
10.1074/jbc.m202766200
ISSN1083-351X
AutoresGiuseppe Pandini, Francesco Frasca, Rossana Mineo, Laura Sciacca, Riccardo Vigneri, Antonino Belfiore,
Tópico(s)Pancreatic function and diabetes
ResumoThe insulin receptor (IR) and the insulin-like growth factor I receptor (IGF-IR) have a highly homologous structure, but different biological effects. Insulin and IGF-I half-receptors can heterodimerize, leading to the formation of insulin/IGF-I hybrid receptors (Hybrid-Rs) that bind IGF-I with high affinity. As the IR exists in two isoforms (IR-A and IR-B), we evaluated whether the assembly of the IGF-IR with either IR-A or IR-B moieties may differently affect Hybrid-R signaling and biological role. Three different models were studied: (a) 3T3-like mouse fibroblasts with a disrupted IGF-IR gene (R− cells) cotransfected with the human IGF-IR and with either the IR-A or IR-B cDNA; (b) a panel of human cell lines variably expressing the two IR isoforms; and (c) HepG2 human hepatoblastoma cells predominantly expressing either IR-A or IR-B, depending on their differentiation state. We found that Hybrid-Rs containing IR-A (Hybrid-RsA) bound to and were activated by IGF-I, IGF-II, and insulin. By binding to Hybrid-RsA, insulin activated the IGF-I half-receptor β-subunit and the IGF-IR-specific substrate CrkII. In contrast, Hybrid-RsBbound to and were activated with high affinity by IGF-I, with low affinity by IGF-II, and insignificantly by insulin. As a consequence, cell proliferation and migration in response to both insulin and IGFs were more effectively stimulated in Hybrid-RA-containing cells than in Hybrid-RB-containing cells. The relative abundance of IR isoforms therefore affects IGF system activation through Hybrid-Rs, with important consequences for tissue-specific responses to both insulin and IGFs. The insulin receptor (IR) and the insulin-like growth factor I receptor (IGF-IR) have a highly homologous structure, but different biological effects. Insulin and IGF-I half-receptors can heterodimerize, leading to the formation of insulin/IGF-I hybrid receptors (Hybrid-Rs) that bind IGF-I with high affinity. As the IR exists in two isoforms (IR-A and IR-B), we evaluated whether the assembly of the IGF-IR with either IR-A or IR-B moieties may differently affect Hybrid-R signaling and biological role. Three different models were studied: (a) 3T3-like mouse fibroblasts with a disrupted IGF-IR gene (R− cells) cotransfected with the human IGF-IR and with either the IR-A or IR-B cDNA; (b) a panel of human cell lines variably expressing the two IR isoforms; and (c) HepG2 human hepatoblastoma cells predominantly expressing either IR-A or IR-B, depending on their differentiation state. We found that Hybrid-Rs containing IR-A (Hybrid-RsA) bound to and were activated by IGF-I, IGF-II, and insulin. By binding to Hybrid-RsA, insulin activated the IGF-I half-receptor β-subunit and the IGF-IR-specific substrate CrkII. In contrast, Hybrid-RsBbound to and were activated with high affinity by IGF-I, with low affinity by IGF-II, and insignificantly by insulin. As a consequence, cell proliferation and migration in response to both insulin and IGFs were more effectively stimulated in Hybrid-RA-containing cells than in Hybrid-RB-containing cells. The relative abundance of IR isoforms therefore affects IGF system activation through Hybrid-Rs, with important consequences for tissue-specific responses to both insulin and IGFs. insulin receptor insulin-like growth factor insulin-like growth factor I receptor extracellular signal-regulated kinase insulin/insulin-like growth factor I hybrid receptor insulin/insulin-like growth factor I hybrid receptor containing the insulin receptor A isoform insulin/insulin-like growth factor I hybrid receptor containing the insulin receptor B isoform bovine serum albumin phenylmethylsulfonyl fluoride bromodeoxyuridine enzyme-linked immunosorbent assay green fluorescent protein phosphate-buffered saline reverse transcription 5-bromo-4-chloro-3-indolyl-β-d-galactopyranoside Src homology The insulin receptor (IR)1 and the insulin-like growth factor (IGF) I receptor (IGF-IR) are tetrameric glycoproteins composed of two extracellular α- and two transmembrane β-subunits linked by disulfide bonds. Each α-subunit, containing the ligand-binding site, is ∼130 kDa, whereas each β-subunit, containing the tyrosine kinase domain, is ∼95–97 kDa. These receptors share >50% overall amino acid sequence homology and 84% homology in the tyrosine kinase domains. After ligand binding, activated receptors recruit and phosphorylate docking proteins, including the insulin receptor substrate-1 family proteins Gab1 and Shc (1Avruch J. Mol. Cell. Biochem. 1998; 182: 31-48Crossref PubMed Scopus (319) Google Scholar, 2Roth R.A. Steele-Perkins G. Hari J. Stover C. Pierce S. Turner J. Edman J.C. Rutter W.J. Cold Spring Harbor Symp. Quant. Biol. 1988; 53: 537-543Crossref PubMed Google Scholar, 3White M.F. Mol. Cell. Biochem. 1998; 182: 3-11Crossref PubMed Scopus (622) Google Scholar, 4Laviola L. Giorgino F. Chow J.C. Baquero J.A. Hansen H. Ooi J. Zhu J. Riedel H. Smith R.J. J. Clin. Invest. 1997; 99: 830-837Crossref PubMed Scopus (87) Google Scholar, 5Cheatham B. Kahn C.R. Endocr. Rev. 1995; 16: 117-142Crossref PubMed Google Scholar), leading to the activation of many intracellular mediators, including phosphatidylinositol 3-kinase, Akt, and ERK1/2, involved in the regulation of cell metabolism, proliferation, and survival. Although both the IR and IGF-IR similarly activate major signaling pathways, subtle differences exist in the recruitment of certain docking proteins and intracellular mediators between the two receptors (6Sasaoka T. Ishiki M. Sawa T. Ishihara H. Takata Y. Imamura T. Usui I. Olefsky J.M. Kobayashi M. Endocrinology. 1996; 137: 4427-4434Crossref PubMed Scopus (87) Google Scholar, 7Nakae J. Kido Y. Accili D. Endocr. Rev. 2001; 22: 818-835Crossref PubMed Scopus (357) Google Scholar, 8Dupont J. LeRoith D. Horm. Res. (Basel). 2001; 55 Suppl. 2: 22-26PubMed Google Scholar, 9Koval A.P. Blakesley V.A. Roberts Jr., C.T. Zick Y. LeRoith D. Biochem. J. 1998; 330: 923-932Crossref PubMed Scopus (33) Google Scholar). These differences are the basis for the predominant metabolic effect elicited by IR activation and the predominant mitogenic, transforming, and anti-apoptotic effect elicited by IGF-IR activation (10Baserga R. Cancer Res. 1995; 55: 249-252PubMed Google Scholar, 11Prisco M. Romano G. Peruzzi F. Valentinis B. Baserga R. Horm. Metab. Res. 1999; 31: 80-89Crossref PubMed Scopus (104) Google Scholar, 12Kido Y. Nakae J. Accili D. J. Clin. Endocrinol. Metab. 2001; 86: 972-979PubMed Google Scholar, 13De Meyts P. Urso B. Christoffersen C.T. Shymko R.M. Ann. N. Y. Acad. Sci. 1995; 766: 388-401Crossref PubMed Scopus (66) Google Scholar). According to the classical view, insulin binds with high affinity to the IR (100-fold higher than to the IGF-IR), whereas both insulin-like growth factors (IGF-I and IGF-II) bind to the IGF-IR (with 100-fold higher affinity than to the IR). Given the high degree of homology, the insulin and IGF-I half-receptors (composed of one α- and one β-subunit) can heterodimerize, leading to the formation of insulin/IGF-I hybrid receptors (Hybrid-Rs) (14Soos M.A. Whittaker J. Lammers R. Ullrich A. Siddle K. Biochem. J. 1990; 270: 383-390Crossref PubMed Scopus (166) Google Scholar, 15Kasuya J. Paz I.B. Maddux B.A. Goldfine I.D. Hefta S.A. Fujita-Yamaguchi Y. Biochemistry. 1993; 32: 13531-13536Crossref PubMed Scopus (59) Google Scholar, 16Seely B.L. Reichart D.R. Takata Y. Yip C. Olefsky J.M. Endocrinology. 1995; 136: 1635-1641Crossref PubMed Scopus (0) Google Scholar). In many tissues, Hybrid-Rs are the most represented receptor subtype (17Bailyes E.M. Nave B.T. Soos M.A. Orr S.R. Hayward A.C. Siddle K. Biochem. J. 1997; 327: 209-215Crossref PubMed Scopus (229) Google Scholar). Hybrid-Rs may also be overexpressed in a variety of human malignancies as a result of both IR and IGF-IR overexpression (18Pandini G. Vigneri R. Costantino A. Frasca F. Ippolito A. Fujita-Yamaguchi Y. Siddle K. Goldfine I.D. Belfiore A. Clin. Cancer Res. 1999; 5: 1935-1944PubMed Google Scholar, 19Belfiore A. Pandini G. Vella V. Squatrito S. Vigneri R. Biochimie (Paris). 1999; 81: 403-407Crossref PubMed Scopus (89) Google Scholar, 20Papa V. Pezzino V. Costantino A. Belfiore A. Giuffrida D. Frittitta L. Vannelli G.B. Brand R. Goldfine I.D. Vigneri R. J. Clin. Invest. 1990; 86: 1503-1510Crossref PubMed Scopus (266) Google Scholar, 21Papa V. Gliozzo B. Clark G.M. McGuire W.L. Moore D. Fujita- Yamaguchi Y. Vigneri R. Goldfine I.D. Pezzino V. Cancer Res. 1993; 53: 3736-3740PubMed Google Scholar). However, the biological role of these Hybrid-Rs is still unclear. Functional studies have indicated that Hybrid-Rs behave more like IGF-IRs than IRs because they bind to and are activated by IGF-I with an affinity similar to that of the typical IGF-IR. In contrast, Hybrid-R activation in response to insulin occurs with much lower affinity (22Soos M.A. Field C.E. Siddle K. Biochem. J. 1993; 290: 419-426Crossref PubMed Scopus (231) Google Scholar, 23Frattali A.L. Pessin J.E. J. Biol. Chem. 1993; 268: 7393-7400Abstract Full Text PDF PubMed Google Scholar). Hybrid-Rs are therefore believed to provide additional binding sites to IGF-I and to increase cell sensitivity to this growth factor (17Bailyes E.M. Nave B.T. Soos M.A. Orr S.R. Hayward A.C. Siddle K. Biochem. J. 1997; 327: 209-215Crossref PubMed Scopus (229) Google Scholar, 18Pandini G. Vigneri R. Costantino A. Frasca F. Ippolito A. Fujita-Yamaguchi Y. Siddle K. Goldfine I.D. Belfiore A. Clin. Cancer Res. 1999; 5: 1935-1944PubMed Google Scholar, 19Belfiore A. Pandini G. Vella V. Squatrito S. Vigneri R. Biochimie (Paris). 1999; 81: 403-407Crossref PubMed Scopus (89) Google Scholar). These studies have not, however, taken into account the different IR isoform contribution to Hybrid-R formation and function. The human IR exists in two isoforms (IR-A and IR-B), generated by alternative splicing of the insulin receptor gene that either excludes or includes 12 amino acid residues encoded by a small exon (exon 11) at the carboxyl terminus of the IR α-subunit (see Table I). The relative abundance of IR isoforms is regulated by tissue-specific and unknown factors (24Moller D.E. Yokota A. Caro J.F. Flier J.S. Mol. Endocrinol. 1989; 3: 1263-1269Crossref PubMed Scopus (257) Google Scholar, 25Mosthaf L. Grako K. Dull T.J. Coussens L. Ullrich A. McClain D.A. EMBO J. 1990; 9: 2409-2413Crossref PubMed Scopus (282) Google Scholar). Recently, we found that IR-A (but not IR-B) binds IGF-II with high affinity and behaves as a second physiological receptor for IGF-II in fetal and dedifferentiated (malignant) cells (26Frasca F. Pandini G. Scalia P. Sciacca L. Mineo R. Costantino A. Goldfine I.D. Belfiore A. Vigneri R. Mol. Cell. Biol. 1999; 19: 3278-3288Crossref PubMed Scopus (702) Google Scholar, 27Sciacca L. Costantino A. Pandini G. Mineo R. Frasca F. Scalia P. Sbraccia P. Goldfine I.D. Vigneri R. Belfiore A. Oncogene. 1999; 18: 2471-2479Crossref PubMed Scopus (236) Google Scholar, 28Vella V. Sciacca L. Pandini G. Mineo R. Squatrito S. Vigneri R. Belfiore A. Mol. Pathol. 2001; 54: 121-124Crossref PubMed Scopus (148) Google Scholar). We therefore hypothesized that the relative abundance of the two isoforms may affect the functional properties of Hybrid-Rs and modulate, in this way, the activation of the IGF system.Table IDescription of receptors and tranfected cells studiedDescriptionReceptorsIR-AIR isoform lacking 12 amino acid residues encoded by exon 11IR-BIR isoform containing 12 amino acid residues encoded by exon 11Hybrid-RAReceptor composed of one α- and one β-subunit of the IGF-IR and one α- and one β-subunit of IR-AHybrid-RBReceptor composed of one α- and one β-subunit of the IGF-IR and one α- and one β-subunit of IR-BCellsR−3T3-like fetal fibroblasts derived from IGF-IR knockout miceR−IR-AR− cells transfected with a construct encoding IR-AR−IR-BR− cells transfected with a construct encoding IR-BR+R− cells transfected with the human IGF-IR geneR+AR+ cells transfected with a construct encoding IR-A to obtain cells expressing the Hybrid-RAR+BR+ cells transfected with a construct encoding for IR-B to obtain cells expressing the Hybrid-RB Open table in a new tab To investigate these issues, we used three different cellular models. First, we used R− fibroblasts, which are 3T3-like cells derived from IGF-IR knockout mice. These cells also have low levels of endogenous IR. We cotransfected these cells with both the human IGF-IR gene and a construct encoding either IR-A or IR-B to obtain cells expressing either Hybrid-RsA or Hybrid-RsB, respectively (see Table I). Second, we employed a panel of human cell lines that express the two IR isoforms in variable amounts. Third, we used HepG2 hepatoblastoma cells that express predominantly either IR-A or IR-B depending on the culture conditions (29Kosaki A. Webster N.J. J. Biol. Chem. 1993; 268: 21990-21996Abstract Full Text PDF PubMed Google Scholar). We found that each of the IR isoforms is equally able to form hybrids with the IGF-IR. Hybrid-RsA and Hybrid-RsB, however, have different functional characteristics. Hybrid-RsB have a high affinity only for IGF-I. Hybrid-RsA have an even higher affinity for IGF-I and bind also IGF-II and insulin. Insulin binding to Hybrid-RsAphosphorylates the IGF-IR β-subunit and activates CrkII, an IGF-IR-specific substrate. Accordingly, cell transfection with IR-A cDNA (but not with IR-B cDNA) markedly increases cell motility in response not only to IGF-I, but also to insulin and IGF-II. These data therefore suggest that the relative abundance of IR isoforms modulates the activation of the IGF system by regulating both binding and signaling characteristics of Hybrid-Rs. They also provide clues to the mechanism by which insulin may activate the IGF-IR phosphorylation cascade and biological effects in a tissue-specific manner. These findings may have important implications for cell biological responses to insulin, IGF-I, and IGF-II. The pNTK2 expression vectors containing the cDNAs for the A (Ex11−) and B (Ex11+) isoforms of the human IR were kindly provided by Dr. Axel Ullrich (Max Planck Institute of Biochemistry, Martinsried, Germany). The pECE expression vector containing the cDNA encoding the human IGF-IR was a gift of Dr. R. Roth (Department of Molecular Pharmacology, Stanford University, Stanford, CA). The pCH110 expression vector for β-galactosidase was kindly provided by Dr. F. Tatò (Universitá di Roma “La Sapienza,” Rome, Italy). The expression vector for pBOS-H2B-GFP was kindly provided by Dr. J. Y. Wang (University of California at San Diego, San Diego, CA). The following materials were purchased from the indicated manufacturers: fetal calf serum, glutamine, LipofectAMINE, and DNase I from Invitrogen (Paisley, UK); RPMI 1640 medium, Dulbecco's modified Eagle's medium, minimum essential medium, Ham's nutrient mixture F-12, bovine serum albumin (BSA; radioimmunoassay grade), bacitracin, phenylmethylsulfonyl fluoride (PMSF), puromycin, bromodeoxyuridine (BrdUrd), and porcine insulin from Sigma; protein G-Sepharose from Amersham Biosciences (Uppsala, Sweden); and125I-labeled IGF-I (specific activity of 11.1 MBq/μg) from PerkinElmer Life Sciences (Zaventem, Belgium). IGF-I and IGF-II were obtained from Calbiochem, and FuGENE 6 transfection reagent was obtained from Roche Molecular Biochemicals (Mannheim, Germany). The following anti-IR antibodies were employed: monoclonal antibodies MA-10 and MA-20 (which recognize the IR α-subunit, but only poorly recognize the Hybrid-R) (Dr. I. D. Goldfine, University of California at San Francisco, San Francisco, CA) (30Forsayeth J.R. Montemurro A. Maddux B.A. DePirro R. Goldfine I.D. J. Biol. Chem. 1987; 262: 4134-4140Abstract Full Text PDF PubMed Google Scholar, 31Roth R.A. Cassell D.J. Wong K.Y. Maddux B.A. Goldfine I.D. Proc. Natl. Acad. Sci. U. S. A. 1982; 79: 7312-7316Crossref PubMed Scopus (75) Google Scholar); monoclonal antibody CT-1 (which recognizes the IR β-subunit) and monoclonal antibody 83-7 (which recognizes the α-subunits of both the IR and Hybrid-R) (Dr. K. Siddle, University of Cambridge, Cambridge, UK) (32Soos M.A. Siddle K. Baron M.D. Heward J.M. Luzio J.P. Bellatin J. Lennox E.S. Biochem. J. 1986; 235: 199-208Crossref PubMed Scopus (135) Google Scholar, 33Ganderton R.H. Stanley K.K. Field C.E. Coghlan M.P. Soos M.A. Siddle K. Biochem. J. 1992; 288: 195-205Crossref PubMed Scopus (45) Google Scholar); a rabbit polyclonal antibody that recognizes the IR β-subunit (Transduction Laboratories, Lexington, KY); and polyclonal antibody 29B4 (which recognizes the IR β-subunit) (Santa Cruz Biotechnology Inc., Santa Cruz, CA). The following anti-IGF-IR antibodies were employed: monoclonal antibody αIR-3 (which recognizes the IGF-IR α-subunit and only poorly recognizes the Hybrid-R) (Oncogene Research, Cambridge, MA) (34Kull Jr., F.C. Jacobs S., Su, Y.F. Svoboda M.E. Van Wyk J.J. Cuatrecasas P. J. Biol. Chem. 1983; 258: 6561-6566Abstract Full Text PDF PubMed Google Scholar); monoclonal antibody 17-69 (which recognizes the α-subunits of both the IGF-IR and Hybrid-R) (Dr. K. Siddle) (35Soos M.A. Field C.E. Lammers R. Ullrich A. Zhang B. Roth R.A. Andersen A.S. Kjeldsen T. Siddle K. J. Biol. Chem. 1992; 267: 12955-12963Abstract Full Text PDF PubMed Google Scholar); and a chicken polyclonal antibody that recognizes the IGF-IR α-subunit (Upstate Biotechnology, Inc., Lake Placid, NY). Anti-phospho-ERK1/2 and anti-phospho-Akt antibodies were purchased from New England Biolabs (Beverly, MA); anti-phosphotyrosine monoclonal antibody 4G10 was from Upstate Biotechnology, Inc.; and anti-BrdUrd antibody was from BD PharMingen (Erembodegem, Belgium). ARO cells were kindly provided by Dr. A. Pontecorvi (Regina Elena Cancer Institute, Rome, Italy). A549, IM-9, HepG2, MDA-MB157, and PC-3 cells were obtained from American Type Culture Collection. R− mouse fibroblasts (3T3-like mouse cells derived from animals with a targeted disruption of the IGF-IR gene, expressing ∼5 × 103insulin receptors/cell) were kindly provided by Dr. R. Baserga (Kimmel Cancer Center, Jefferson University, Philadelphia, PA) (Table I). HepG2 and MDA-MB157 cells were routinely grown in minimum essential medium supplemented with 10% fetal bovine serum. A549, PC-3, IM-9 and ARO cells were routinely grown in RPMI 1640 medium supplemented with 10% fetal bovine serum. The R− mouse fibroblasts were routinely grown in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum. R− cells were grown in 35-mm plates until 60–70% confluent. They were first transfected with 2 μg of pECE expression vector containing the cDNA encoding the IGF-IR (36Steele-Perkins G. Turner J. Edman J.C. Hari J. Pierce S.B. Stover C. Rutter W.J. Roth R.A. J. Biol. Chem. 1988; 263: 11486-11492Abstract Full Text PDF PubMed Google Scholar) and cotransfected with 0.2 μg of pSV2 plasmid encoding the hygromycin resistance gene by the LipofectAMINE method according to the manufacturer's protocol. Cells were then subjected to antibiotic selection in medium supplemented with 400 μg/ml hygromycin for 3 weeks. Stably transfected clones were tested for receptor content by ELISA. Cell clones were further transfected with the pNTK2 expression vector containing the cDNA for either the A (Ex11−) or B (Ex11+) isoform of the human IR (37Ullrich A. Gray A. Tam A.W. Yang-Feng T. Tsubokawa M. Collins C. Henzel W., Le Bon T. Kathuria S. Chen E. Jacobs S. Francke U. Ramachandran R. Fujita-Yamagughi Y. EMBO J. 1986; 5: 2503-2512Crossref PubMed Scopus (1489) Google Scholar) and cotransfected with the pPDV6+ plasmid encoding the puromycin resistance gene. Cells were subsequently subjected to antibiotic selection in medium supplemented with 400 μg/ml hygromycin and 2.4 μg/ml puromycin for 3 weeks. Receptor content was evaluated in selected clones by ELISA. Cell clones expressing similar amounts of either IR-A or IR-B, IGF-IR, and Hybrid-R (either the Hybrid-RA or Hybrid-RB) were selected for subsequent studies. For migration studies, HepG2 cells were transiently transfected by the FuGENE 6 method according to the manufacturer's protocol. Briefly, 4 × 105 cells were seeded in six-well plates and grown for 24 h in complete medium (minimum essential medium with 10% fetal bovine serum). Thereafter, a transfection mixture containing 2 μg of pNTK2-IR-A/IR-B + 0.2 μg of β-galactosidase or histone H2B-GFP + 12 μl of FuGENE 6 in 100 μl of minimal essential medium without serum or antibiotics was added to each well. Cells were grown in complete medium; and after 48 h, they were assayed for β-galactosidase activity or scored under a fluorescence microscope for GFP expression. Cells were grown until 80% confluent and serum-starved 24 h before stimulation with the various ligands. For receptor and ERK/Akt activation, cells were stimulated with 10 nminsulin, IGF-I, or IGF-II for 10 min. For in vitroCrk phosphorylation, cells were stimulated with 50 nminsulin, IGF-I, or IGF-II for 5 min. After three washes with ice-cold PBS, cells were lysed in cold radioimmune precipitation assay buffer containing 50 mm Tris (pH 7.4), 150 mmNaCl, 0.5% Nonidet P-40, 0.5% Triton X-100, 0.25% sodium deoxycholate, 10 mm sodium pyrophosphate, 1 mmNaF, 1 mm sodium orthovanadate, 2 mm PMSF, 10 μg/ml aprotinin, 10 μg/ml pepstatin, and 10 μg/ml leupeptin. After being scraped, samples were rotated for 15 min at 4 °C. Insoluble material was separated from the soluble extract by microcentrifugation at 10,000 × g for 10 min at 4 °C. Protein concentration was determined by the Bradford assay. Either the Hybrid-RA or Hybrid-RB was captured by incubating cell lysates for 22 h in Maxisorp Break-Apart immunoplates (Nunc, Roskilde, Denmark) precoated with 2 μg/ml antibody 83-7. After washing, the immunocaptured receptors were incubated with 125I-labeled IGF-I (10 pm in 50 mm HEPES-buffered saline (pH 7.6) containing 0.05% Tween 20, 1% BSA, 2 mm sodium orthovanadate, 1 mg/ml bacitracin, and 1 mm PMSF) in the presence or absence of increasing concentrations of various unlabeled ligands (insulin, IGF-I, and IGF-II). After 2 h at room temperature, the plates were washed, and the radioactivity in each well was counted in a γ-counter. Cell lysates were prepared as described above and used for receptor measurement both by ELISA and Western blot analysis. The characteristics and specificity of these ELISAs have been previously described (18Pandini G. Vigneri R. Costantino A. Frasca F. Ippolito A. Fujita-Yamaguchi Y. Siddle K. Goldfine I.D. Belfiore A. Clin. Cancer Res. 1999; 5: 1935-1944PubMed Google Scholar). Receptors were captured by incubating lysates (0.5–60 μg/well) in Maxisorp immunoplates precoated with the specific monoclonal antibody (2 μg/ml) indicated below. After washing, the immunocaptured receptors were incubated with the specific biotinylated monoclonal antibody indicated below (0.3 μg/ml in 50 mm HEPES-buffered saline (pH 7.6) containing 0.05% Tween 20, 1% BSA, 2 mm sodium orthovanadate, 1 mg/ml bacitracin, and 1 mm PMSF) and then with peroxidase-conjugated streptavidin. The peroxidase activity was determined colorimetrically by adding 100 μl of 3,3′,5,5′-tetramethylbenzidine (0.4 mg/ml in 0.1 mcitrate/phosphate buffer (pH 5.0) with 0.4 μl/ml 30% H2O2). The reaction was stopped by the addition of 1.0 m H3PO4, and the absorbance was measured at 450 nm. IRs were captured with anti-IR antibody MA-20 and detected with biotinylated anti-IR antibody CT-1 (30Forsayeth J.R. Montemurro A. Maddux B.A. DePirro R. Goldfine I.D. J. Biol. Chem. 1987; 262: 4134-4140Abstract Full Text PDF PubMed Google Scholar, 33Ganderton R.H. Stanley K.K. Field C.E. Coghlan M.P. Soos M.A. Siddle K. Biochem. J. 1992; 288: 195-205Crossref PubMed Scopus (45) Google Scholar). IGF-IRs were captured with anti-IGF-IR antibody αIR-3 and detected with biotinylated antibody 17-69 (34Kull Jr., F.C. Jacobs S., Su, Y.F. Svoboda M.E. Van Wyk J.J. Cuatrecasas P. J. Biol. Chem. 1983; 258: 6561-6566Abstract Full Text PDF PubMed Google Scholar, 35Soos M.A. Field C.E. Lammers R. Ullrich A. Zhang B. Roth R.A. Andersen A.S. Kjeldsen T. Siddle K. J. Biol. Chem. 1992; 267: 12955-12963Abstract Full Text PDF PubMed Google Scholar). Hybrid-Rs were captured with anti-IR antibody 83-7, which recognizes both the Hybrid-R and IR, and detected with biotinylated anti-IGF-IR antibody 17-69 (32Soos M.A. Siddle K. Baron M.D. Heward J.M. Luzio J.P. Bellatin J. Lennox E.S. Biochem. J. 1986; 235: 199-208Crossref PubMed Scopus (135) Google Scholar, 35Soos M.A. Field C.E. Lammers R. Ullrich A. Zhang B. Roth R.A. Andersen A.S. Kjeldsen T. Siddle K. J. Biol. Chem. 1992; 267: 12955-12963Abstract Full Text PDF PubMed Google Scholar). The receptor content was evaluated by comparing each sample with a standard curve, as previously described (18Pandini G. Vigneri R. Costantino A. Frasca F. Ippolito A. Fujita-Yamaguchi Y. Siddle K. Goldfine I.D. Belfiore A. Clin. Cancer Res. 1999; 5: 1935-1944PubMed Google Scholar). The minimal detectable amount of hybrids was 0.125 ng/well (1.25 ng/ml). The assay was linear from 0.125 to 1.0 ng/well. There was no interference from either 1 ng/well purified IR (from human IR cDNA-transfected NIH/3T3 cells) or 1 ng/well purified IGF-IR (from human IGF-IR cDNA-transfected Chinese hamster ovary cells). Multiple dilutions of cells and tissues containing either Hybrid-RsA or Hybrid-RsB produced dose-response curves parallel to those obtained with the purified IR/IGF-IR hybrid standard (Ref. 18Pandini G. Vigneri R. Costantino A. Frasca F. Ippolito A. Fujita-Yamaguchi Y. Siddle K. Goldfine I.D. Belfiore A. Clin. Cancer Res. 1999; 5: 1935-1944PubMed Google Scholar and data not shown). Intra-assay coefficients of variation were <7% at 0.5 ng/tube and <8% at 1.0 ng/tube. Inter-assay coefficients of variation were <8 and <10%, respectively (18Pandini G. Vigneri R. Costantino A. Frasca F. Ippolito A. Fujita-Yamaguchi Y. Siddle K. Goldfine I.D. Belfiore A. Clin. Cancer Res. 1999; 5: 1935-1944PubMed Google Scholar). The ELISAs for the IR and IGF-IR had similar characteristics of sensitivity and specificity, as previously described (18Pandini G. Vigneri R. Costantino A. Frasca F. Ippolito A. Fujita-Yamaguchi Y. Siddle K. Goldfine I.D. Belfiore A. Clin. Cancer Res. 1999; 5: 1935-1944PubMed Google Scholar). Purified IGF-IR or Hybrid-R (up 1 ng/well) did not interfere in the IR assay, and purified IR or Hybrid-R did not interfere in the IGF-IR assay. The minimal detectable amounts were 0.05 ng/tube for the IR and 0.0625 ng/tube for the IGF-IR. Intra-assay coefficients of variation were <8%, and inter-assay coefficients of variation were <10% for both assays (18Pandini G. Vigneri R. Costantino A. Frasca F. Ippolito A. Fujita-Yamaguchi Y. Siddle K. Goldfine I.D. Belfiore A. Clin. Cancer Res. 1999; 5: 1935-1944PubMed Google Scholar). To confirm data obtained by ELISA, aliquots of the same lysates were subjected to Western blot analysis. Cell lysates were incubated at 4 °C under constant rotation for 2 h with 4 μg of the specific anti-receptor antibody and then for 2 h with protein G-Sepharose. Immunoprecipitates were eluted and subjected to SDS-PAGE and then immunoblotted (1 μg/ml) as described below. IRs were immunoprecipitated with anti-IR antibody MA-20 and blotted with the rabbit anti-IR polyclonal antibody. IGF-IRs were immunoprecipitated with anti-IGF-IR antibody αIR-3 and blotted with the chicken anti-IGF-IR polyclonal antibody. Hybrid-Rs were immunoprecipitated with anti-IR antibody 83-7 and blotted with the chicken anti-IGF-IR polyclonal antibody. Western blot specificity was evaluated by examining the interference of 200 ng of purified receptor of each subtype added to a cell lysate containing ∼200 ng of IR, IGF-IR, or Hybrid-R. Cell lysates were incubated at 4 °C under constant rotation for 1 h with protein G-Sepharose to eliminate antibody MA-10 bound to the IR. After centrifugation, the supernatant was incubated at 4 °C under constant rotation for 2 h with 4 μg of anti-Hybrid-R antibody 83-7 coated with protein G-Sepharose. Immunoprecipitates were eluted and subjected to SDS-PAGE. The resolved proteins were transferred to nitrocellulose membranes, immunoblotted with anti-phosphotyrosine monoclonal antibody 4G10, and revealed by an ECL method. The nitrocellulose membrane was then stripped with Restore stripping buffer (Pierce) for 30 min at room temperature and subsequently reprobed with the chicken anti-IGF-IR polyclonal antibody. As previously described (38Belfiore A. Costantino A. Frasca F. Pandini G. Mineo R. Vigneri P. Maddux B. Goldfine I.D. Vigneri R. Mol. Endocrinol. 1996; 10: 1318-1326PubMed Google Scholar), 100 μl of the cell lysates prepared as described above were immunocaptured in Maxisorp plates coated with antibodies 83-7 (which recognizes both the IR and Hybrid-R) and MA-20 (which recognizes the IR only) at a concentration of 2 μg/ml in 50 mm sodium bicarbonate (pH 9.0) overnight at 4 °C. After washing, the captured phosphorylated proteins were incubated with biotin-conjugated anti-phosphotyrosine antibody 4G10 (0.3 μg/ml in 50 mmHEPES (pH 7.6), 150 mm NaCl, 0.05% Tween 20, 1% BSA, 2 mm sodium orthovanadate, 1 mg/ml bacitracin, and 1 mm PMSF) for 2 h at 22 °C and then with peroxidase-conjugated streptavidin. The peroxidase activity was determined colorimetrically by adding 100 μl of 3,3′,5,5′-tetramethylbenzidine (0.4 mg/ml in 0.1 mcitrate/phosphate buffer (pH 5.0) with 0.4 μl/ml 30% H2O2). The reaction was stopped by the addition of 1.0 m H3PO4, and the absorbance was measured at 450 nm. In vitro receptor tyrosine kinase activity for CrkII was measured as previously described (9Koval A.P. Blakesley V.A. Roberts Jr., C.T. Zick Y. LeRoith D. Biochem. J. 1998; 330: 923-932Crossref PubMed Scopus (33) Google Scholar) with modifications. 500 μg of proteins were immunoprecipitated with either anti-IR monoclonal antib
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