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BIBTEX RIS

An Investigation of the Ligand Binding Properties and Negative Cooperativity of Soluble Insulin-like Growth Factor Receptors

2007; Elsevier BV; Volume: 283; Issue: 9 Linguagem: Inglês

10.1074/jbc.m707054200

ISSN

1083-351X

Autores

Kathy H. Surinya, Briony E. Forbes, Filomena Occhiodoro, Grant W. Booker, Geoffrey L. Francis, Kenneth Siddle, John C. Wallace, Leah Cosgrove,

Tópico(s)

Metabolism, Diabetes, and Cancer

Resumo

To investigate the interaction of the insulin-like growth factor (IGF) ligands with the insulin-like growth factor type 1 receptor (IGF-1R), we have generated two soluble variants of the IGF-1R. We have recombinantly expressed the ectodomain of IGF-1R or fused this domain to the constant domain from the Fc fragment of mouse immunoglobulin. The ligand binding properties of these soluble IGF-1Rs for IGF-I and IGF-II were investigated using conventional ligand competition assays and BIAcore biosensor technology. In ligand competition assays, the soluble IGF-1Rs both bound IGF-I with similar affinities and a 5-fold lower affinity than that seen for the wild type receptor. In addition, both soluble receptors bound IGF-II with similar affinities to the wild type receptor. BIAcore analyses showed that both soluble IGF-1Rs exhibited similar ligand-specific association and dissociation rates for IGF-I and for IGF-II. The soluble IGF-1R proteins both exhibited negative cooperativity for IGF-I, IGF-II, and the 24-60 antibody, which binds to the IGF-1R cysteine-rich domain. We conclude that the addition of the self-associating Fc domain to the IGF-1R ectodomain does not affect ligand binding affinity, which is in contrast to the soluble ectodomain of the IR. This study highlights some significant differences in ligand binding modes between the IGF-1R and the insulin receptor, which may ultimately contribute to the different biological activities conferred by the two receptors. To investigate the interaction of the insulin-like growth factor (IGF) ligands with the insulin-like growth factor type 1 receptor (IGF-1R), we have generated two soluble variants of the IGF-1R. We have recombinantly expressed the ectodomain of IGF-1R or fused this domain to the constant domain from the Fc fragment of mouse immunoglobulin. The ligand binding properties of these soluble IGF-1Rs for IGF-I and IGF-II were investigated using conventional ligand competition assays and BIAcore biosensor technology. In ligand competition assays, the soluble IGF-1Rs both bound IGF-I with similar affinities and a 5-fold lower affinity than that seen for the wild type receptor. In addition, both soluble receptors bound IGF-II with similar affinities to the wild type receptor. BIAcore analyses showed that both soluble IGF-1Rs exhibited similar ligand-specific association and dissociation rates for IGF-I and for IGF-II. The soluble IGF-1R proteins both exhibited negative cooperativity for IGF-I, IGF-II, and the 24-60 antibody, which binds to the IGF-1R cysteine-rich domain. We conclude that the addition of the self-associating Fc domain to the IGF-1R ectodomain does not affect ligand binding affinity, which is in contrast to the soluble ectodomain of the IR. This study highlights some significant differences in ligand binding modes between the IGF-1R and the insulin receptor, which may ultimately contribute to the different biological activities conferred by the two receptors. Insulin-like growth factors (IGFs 2The abbreviations used are:IGFinsulin-like growth factorIRinsulin receptorIGF-1Rinsulin-like growth factor type 1 receptorsIGF-1Rsoluble insulin-like growth factor type 1 receptor ectodomainsIGF-1R/Fcsoluble insulin-like growth factor type 1 receptor ectodomain-Fc fusionwtIGF-1Rwild type IGF-1RmAbmonoclonal antibodyEu-IGFeuropium-labeled IGFL1 and L2large homologous domain 1 and 2, respectfullyCRcysteine-richFnfibronectin.; IGF-I and IGF-II) mediate their biological functions primarily through the type 1 IGF receptor (IGF-1R). Both IGF-I and IGF-II are essential for normal growth and development. Targeted gene disruption of IGF-I or IGF-II results in mice with reduced birth weight compared with their wild type littermates (1DeChiara T.M. Efstratiadis A. Robertson E.J. Nature. 1990; 345: 78-80Crossref PubMed Scopus (1410) Google Scholar, 2Liu J.P. Baker J. Perkins A.S. Robertson E.J. Efstratiadis A. Cell. 1993; 75: 59-72Abstract Full Text PDF PubMed Scopus (2595) Google Scholar), whereas disruption of the IGF-1R gene results in death at birth from respiratory failure (2Liu J.P. Baker J. Perkins A.S. Robertson E.J. Efstratiadis A. Cell. 1993; 75: 59-72Abstract Full Text PDF PubMed Scopus (2595) Google Scholar). Conversely, overexpression of the IGF-1R or IGFs in cancer tissues leads to the potentiation of cancer cell growth and survival (3Samani A.A. Yakar S. Leroith D. Brodt P. Endocr. Rev. 2007; 28: 20-47Crossref PubMed Scopus (860) Google Scholar). Also, the IGF-1R is essential for transformation to a malignant phenotype (4Sell C. Dumenil G. Deveaud C. Miura M. Coppola D. DeAngelis T. Rubin R. Efstratiadis A. Baserga R. Mol. Cell. Biol. 1994; 14: 3604-3612Crossref PubMed Scopus (505) Google Scholar). Interestingly, there is increasing evidence to support a role for IGF-II in a variety of cancers, where elevated IGF-II levels are associated with increased cancer risk (5Gicquel C. Bertagna X. Schneid H. Francillard-Leblond M. Luton J.P. Girard F. Le Bouc Y. J. Clin. Endocrinol. Metab. 1994; 78: 1444-1453Crossref PubMed Scopus (198) Google Scholar, 6Quinn K.A. Treston A.M. Unsworth E.J. Miller M.J. Vos M. Grimley C. Battey J. Mulshine J.L. Cuttitta F. J. Biol. Chem. 1996; 271: 11477-11483Abstract Full Text Full Text PDF PubMed Scopus (175) Google Scholar, 7Renehan A.G. Jones J. Potten C.S. Shalet S.M. O'Dwyer S.T. Br. J. Cancer. 2000; 83: 1344-1350Crossref PubMed Scopus (100) Google Scholar, 8Vella V. Pandini G. Sciacca L. Mineo R. Vigneri R. Pezzino V. Belfiore A. J. Clin. Endocrinol. Metab. 2002; 87: 245-254Crossref PubMed Scopus (159) Google Scholar), reviewed in Ref. 3Samani A.A. Yakar S. Leroith D. Brodt P. Endocr. Rev. 2007; 28: 20-47Crossref PubMed Scopus (860) Google Scholar. insulin-like growth factor insulin receptor insulin-like growth factor type 1 receptor soluble insulin-like growth factor type 1 receptor ectodomain soluble insulin-like growth factor type 1 receptor ectodomain-Fc fusion wild type IGF-1R monoclonal antibody europium-labeled IGF large homologous domain 1 and 2, respectfully cysteine-rich fibronectin. Despite their similarity in sequence and structure IGF-I and IGF-II can stimulate both overlapping and distinct biological functions (reviewed in Ref. 9Denley A. Cosgrove L.J. Booker G.W. Wallace J.C. Forbes B.E. Cytokine Growth Factor Rev. 2005; 16: 421-439Crossref PubMed Scopus (328) Google Scholar). This is evident in patients with IGF-I deficiency, which results in severe growth and mental retardation, where IGF-II does not compensate for the loss of IGF-I activity (10Woods K.A. Camacho-Hubner C. Barter D. Clark A.J. Savage M.O. Acta Paediatr. Suppl. 1997; 423: 39-45Crossref PubMed Google Scholar, 11Walenkamp M.J. Karperien M. Pereira A.M. Hilhorst-Hofstee Y. van Doorn J. Chen J.W. Mohan S. Denley A. Forbes B. van Duyvenvoorde H.A. van Thiel S.W. Sluimers C.A. Bax J.J. de Laat J.A. Breuning M.B. Romijn J.A. Wit J.M. J. Clin. Endocrinol. Metab. 2005; 90: 2855-2864Crossref PubMed Scopus (275) Google Scholar, 12Denley A. Wang C.C. McNeil K.A. Walenkamp M.J. van Duyvenvoorde H. Wit J.M. Wallace J.C. Norton R.S. Karperien M. Forbes B.E. Mol. Endocrinol. 2005; 19: 711-721Crossref PubMed Scopus (55) Google Scholar). Therefore, in order to understand how both IGF-I and IGF-II stimulate their respective biological outcomes, we first need to understand the mechanism by which both ligands bind and in turn activate the IGF-1R. The IGF-1R is a member of the tyrosine kinase family of receptors and, together with the insulin receptor (IR) and insulin-related receptor, forms a subfamily with similar structural organization (reviewed in Ref. 9Denley A. Cosgrove L.J. Booker G.W. Wallace J.C. Forbes B.E. Cytokine Growth Factor Rev. 2005; 16: 421-439Crossref PubMed Scopus (328) Google Scholar). The IGF-1R and IR are homodimers composed of two α and two β subunits and are synthesized as a single precursor polypeptide, which is then post-translationally processed by dimerization, proteolytic cleavage, and glycosylation. The amino-terminal regions of the α chain of these receptors are composed of three domains, two structurally homologous subdomains designated large homologous domain 1 (L1) and large homologous domain 2 (L2), which are separated by a cysteine-rich (CR) domain of ∼160 amino acids (3Samani A.A. Yakar S. Leroith D. Brodt P. Endocr. Rev. 2007; 28: 20-47Crossref PubMed Scopus (860) Google Scholar) (see supplemental Fig. S1). The major ligand binding determinants of the structurally related IGF-1R and IR reside within the extracellular α subunits of the receptors (reviewed in Refs. 9Denley A. Cosgrove L.J. Booker G.W. Wallace J.C. Forbes B.E. Cytokine Growth Factor Rev. 2005; 16: 421-439Crossref PubMed Scopus (328) Google Scholar, 13Adams T.E. Epa V.C. Garrett T.P. Ward C.W. Cell Mol. Life Sci. 2000; 57: 1050-1093Crossref PubMed Scopus (486) Google Scholar, and 14De Meyts P. Growth Horm. IGF Res. 2002; 12: 81-83Crossref PubMed Scopus (14) Google Scholar). 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Chem. 1997; 272: 8189-8197Abstract Full Text Full Text PDF PubMed Scopus (39) Google Scholar), suggesting that all of the determinants required for high affinity ligand binding reside within the extracellular domain of the IGF-1R. Chemical cross-linking studies, the generation of chimeric IGF-I/IR, and alanine-scanning mutagenesis have been used to further define the ligand binding sites within the ectodomain (24Fabry M. Schaefer E. Ellis L. Kojro E. Fahrenholz F. Brandenburg D. J. Biol. Chem. 1992; 267: 8950-8956Abstract Full Text PDF PubMed Google Scholar, 25Schumacher R. Soos M.A. Schlessinger J. Brandenburg D. Siddle K. Ullrich A. J. Biol. Chem. 1993; 268: 1087-1094Abstract Full Text PDF PubMed Google Scholar, 26Kurose T. Pashmforoush M. Yoshimasa Y. Carroll R. Schwartz G.P. Burke G.T. Katsoyannis P.G. Steiner D.F. J. Biol. Chem. 1994; 269: 29190-29197Abstract Full Text PDF PubMed Google Scholar, 27Kjeldsen T. Wiberg F.C. Andersen A.S. J. Biol. 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J. 2007; 403: 603-613Crossref PubMed Scopus (162) Google Scholar). Interestingly, a unique binding site for IGF-I exists within the CR domain of the IGF-1R and provides the specificity for that ligand (37Gustafson T.A. Rutter W.J. J. Biol. Chem. 1990; 265: 18663-18667Abstract Full Text PDF PubMed Google Scholar, 38Zhang B. Roth R.A. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 9858-9862Crossref PubMed Scopus (79) Google Scholar, 39Andersen A.S. Kjeldsen T. Wiberg F.C. Vissing H. Schaffer L. Rasmussen J.S. De Meyts P. Moller N.P. J. Biol. Chem. 1992; 267: 13681-13686Abstract Full Text PDF PubMed Google Scholar). Recently, the crystal structure of the IR ectodomain has been determined in the absence of bound ligand (40McKern N.M. Lawrence M.C. Streltsov V.A. Lou M.Z. Adams T.E. Lovrecz G.O. Elleman T.C. Richards K.M. Bentley J.D. Pilling P.A. Hoyne P.A. Cartledge K.A. Pham T.M. Lewis J.L. Sankovich S.E. Stoichevska V. Da Silva E. Robinson C.P. Frenkel M.J. Sparrow L.G. Fernley R.T. Epa V.C. Ward C.W. Nature. 2006; 443: 218-221Crossref PubMed Scopus (240) Google Scholar) and reveals a folded over conformation, which could accommodate two potential ligand binding sites. This arrangement is consistent with the evidence that ligand binding involves interaction with both receptor halves. Kinetic analysis of the ligand-receptor interaction with wild type IR and IGF-1R reveals high and low affinity ligand states that are involved in the characteristic negative cooperativity mode of binding (reviewed in Refs. 41De Meyts P. Diabetologia. 1994; 37: S135-S148Crossref PubMed Scopus (259) Google Scholar, 42De Meyts P. Whittaker J. Nat. Rev. Drug Discov. 2002; 1: 769-783Crossref PubMed Scopus (486) Google Scholar, 43De Meyts P. BioEssays. 2004; 26: 1351-1362Crossref PubMed Scopus (253) Google Scholar). The accelerated dissociation of bound ligand from the IR in the presence of unlabeled insulin suggests the presence of at least two interacting binding sites. In contrast, those soluble IR ectodomain fragments that have low affinity for ligand have a linear Scatchard plot and do not show negative cooperativity (41De Meyts P. Diabetologia. 1994; 37: S135-S148Crossref PubMed Scopus (259) Google Scholar, 42De Meyts P. Whittaker J. Nat. Rev. Drug Discov. 2002; 1: 769-783Crossref PubMed Scopus (486) Google Scholar). Although the wild type full-length IGF-1R exhibits negative cooperativity for IGF-I (44De Meyts P. Wallach B. Christoffersen C.T. Urso B. Gronskov K. Latus L.J. Yakushiji F. Ilondo M.M. Shymko R.M. Horm. Res. 1994; 42: 152-169Crossref PubMed Scopus (199) Google Scholar), it is yet to be determined whether a similar mode of binding exists for the related IGF-II ligand. In addition, truncation of the IGF-1R at the transmembrane domain is assumed to remove the characteristic feature of the wild type IGF-1R, negative cooperativity, but this has not been formally demonstrated. In the present study, we have assembled and expressed two soluble human IGF-1R variants, comprising the ectodomain of the IGF-1R and a soluble chimeric IGF-1R-Fc fusion protein of the IGF-1R ectodomain, fused to the Fc region of the heavy chain of mouse immunoglobulin (IgG1), designated sIGF-1R and sIGF-1R/Fc, respectively. These soluble IGF-1Rs exhibit high affinity IGF-I and IGF-II binding and negative cooperativity determined by immunocapture competition binding assays and BIAcore biosensor analysis. The sIGF-1R/Fc and sIGF-1R both exhibit high affinity IGF-I and IGF-II binding and negative cooperativity. We have also utilized a number of conformation-dependent anti-IGF-1R antibodies (45Soos 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) as additional probes for receptor conformation and have examined their effect on IGF-I and IGF-II binding to either solubilized wild type or sIGF-1Rs. Construction of Soluble IGF-1 Receptor cDNA Plasmids—A schematic representation of the IGF-1R domain structure and the soluble receptors used in this study is provided in supplemental Fig. S1. Two soluble human IGF-1R variants were generated, comprising the ectodomain of the IGF-1R and a soluble chimeric IGF-1R-Fc fusion protein of the IGF-1R ectodomain fused to the Fc region of the heavy chain of mouse immunoglobulin (IgG1), designated sIGF-1R and sIGF-1R/Fc, respectively. The cloning strategy, the generation of stable cell lines expressing the soluble IGF-1 receptors, screening of transfected cell lines, metabolic labeling and immunoprecipitation assays, purification of soluble IGF-1 receptors, and SDS-PAGE and Western blot analysis are all described in the supplemental materials. Ligand Binding Analysis—Wells of microtiter plates (Immulon 4 HBX from Dynex Technologies) were coated with anti-IGF-1R monoclonal antibody 24-31 (10 μg/ml, 100 μl/well in phosphate-buffered saline) (45Soos 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), which does not interfere with receptor binding, 3P. A. Hoyne, unpublished results. and blocked with 0.5% bovine serum albumin (radioimmunoassay grade from Sigma) in phosphate-buffered saline for 2 h at room temperature. Cell culture supernatant from soluble receptor-expressing cell lines or purified soluble receptor was incubated overnight at 4 °C. The wild-type IGF-1R was obtained from the p6 BALB/c3T3 cell line overexpressing the IGF-1R (46Pietrzkowski Z. Lammers R. Carpenter G. Soderquist A.M. Limardo M. Phillips P.D. Ullrich A. Baserga R. Cell Growth Differ. 1992; 3: 199-205PubMed Google Scholar), and cell membranes were solubilized as described by Denley et al. (47Denley A. Bonython E.R. Booker G.W. Cosgrove L.J. Forbes B.E. Ward C.W. Wallace J.C. Mol. Endocrinol. 2004; 18: 2502-2512Crossref PubMed Scopus (166) Google Scholar). Various concentrations of unlabeled receptor grade recombinant human IGF-I or human IGF-II (both prepared in house as previously described (48King R. Wells J.R. Krieg P. Snoswell M. Brazier J. Bagley C.J. Wallace J.C. Ballard F.J. Ross M. Francis G.L. J. Mol. Endocrinol. 1992; 8: 29-41Crossref PubMed Scopus (67) Google Scholar, 49Francis G.L. Aplin S.E. Milner S.J. McNeil K.A. Ballard F.J. Wallace J.C. Biochem. J. 1993; 293: 713-719Crossref PubMed Scopus (75) Google Scholar)) were diluted in binding buffer (100 mm HEPES, 100 mm NaCl, 10 mm MgCl2, 0.05% (w/v) bovine serum albumin, and 0.025% (w/v) Triton X-100), either 25 pm 125I-IGF-I or 125I-IGF-II (GE Healthcare) was added, and plates were then incubated overnight at 4 °C. Receptor was diluted to bind 10–15% of added 125I-IGF-I or 125I-IGF-II tracer in the absence of unlabeled ligand. Plates were washed three times with phosphate-buffered saline, and bound counts were determined using a γ scintillation counter. The background binding was 5% of the total counts added, and this was subtracted to give specific counts bound. The binding data were analyzed using GraphPad Prism 3.03 by curve-fitting with a one-site competition model. Receptor binding affinities were also determined using europium-labeled IGF-I (Eu-IGF-I) and IGF-II (Eu-IGF-II) in microtiter plate assays as described by Denley et al. (47Denley A. Bonython E.R. Booker G.W. Cosgrove L.J. Forbes B.E. Ward C.W. Wallace J.C. Mol. Endocrinol. 2004; 18: 2502-2512Crossref PubMed Scopus (166) Google Scholar). Briefly, IGF-1R was immunocaptured with mAb 24-31 on white Greiner Lumitrac 600 plates and blocked with 0.5% bovine serum albumin/TBST (Tris-buffered saline, 0.1% Tween 20), and then supernatant containing soluble receptor or solubilized p6 cell lysate was added. Bound europium-labeled IGF-I or IGF-II was then incubated on immobilized receptors together with unlabeled IGF-I or IGF-II or 100 nm mAb, as specified for individual experiments. Wells were washed three times in TBST and once in water. 100 μl/well enhancement solution (PerkinElmer Life Sciences) was added and then incubated for 10 min prior to determining the bound europium label by time-resolved fluorescence (47Denley A. Bonython E.R. Booker G.W. Cosgrove L.J. Forbes B.E. Ward C.W. Wallace J.C. Mol. Endocrinol. 2004; 18: 2502-2512Crossref PubMed Scopus (166) Google Scholar). BIAcore Determination of IGF-I and IGF-II Affinities for Soluble IGF-1R—sIGF-1R/Fc and sIGF-1R were coupled to the CM5 biosensor chip using a similar method to that previously described (50Forbes B.E. Hartfield P.J. McNeil K.A. Surinya K.H. Milner S.J. Cosgrove L.J. Wallace J.C. Eur. J. Biochem. 2002; 269: 961-968Crossref PubMed Scopus (46) Google Scholar). Briefly, CM5 sensor chips were activated with 35 μ l of N-ethyl-N′-[(dimethylamino)propyl]carbodiimide and N-hydroxysuccinimide at 5 μl/min. Receptor dialyzed against HBS running buffer (10 mm HEPES, 150 mm NaCl, 3.4 mm EDTA, 0.005% surfactant P20, pH 7.4) was coupled to the CM5 sensor chip by injecting 35 μl of receptor (4 μg) in 10 mm sodium acetate, pH 4.5. Uncoupled surfaces were then deactivated by 1 m ethanolamine, pH 8.5. A blank flow cell was used as a reference on all chips. Binding analyses were performed at 25 °C using a BIAcore 2000 biosensor (BIAcore, Uppsala, Sweden). A sensor surface coupled with 8,000–10,000 response units would routinely result in a response of ∼100 response units with 200 nm IGF-I. Kinetic analyses were performed using a range of IGF-I or IGF-II concentrations (100, 50, 25, 12.5, and 6.25 nm) at 30 μl/min. Surfaces were regenerated using 0.3 m sodium citrate, 0.4 m NaCl, pH 4.5. Data were analyzed using BIAevaluation 3.2 software using a 1:1 Langmuir binding model. This model describes a simple reversible interaction between two molecules in a 1:1 complex. In each of at least two repeat experiments, samples were injected in duplicate, and the response on the reference flow cell was subtracted. Dissociation of Eu-IGF Ligands from IGF-1R—Following the binding of Eu-IGF-I or Eu-IGF-II ligand to immobilized receptors, plates were washed in TBST before the addition of 100 nm unlabeled IGF-I, IGF-II, or 24-60 mAb in 100 μ l of europium-binding buffer at room temperature. At various time points (0–2 h), wells were washed once in water, and then 100 μl/well enhancement solution was added and incubated for 10 min prior to determining the bound europium label by time-resolved fluorescence (47Denley A. Bonython E.R. Booker G.W. Cosgrove L.J. Forbes B.E. Ward C.W. Wallace J.C. Mol. Endocrinol. 2004; 18: 2502-2512Crossref PubMed Scopus (166) Google Scholar). The dose-response experiments for examining the accelerating effect of unlabeled ligand or anti-IGF-1R mAb 24-60 were performed with an increasing concentration of IGF-I or IGF-II (0.01 nm to 5 μm) and 24-60 mAb (0.01 nm-5 μm), respectively. Synthesis, Expression, and Purification of Soluble IGF-1 Receptor Constructs—Two recombinant soluble IGF-I receptor (sIGF-1R) constructs were cloned into the pEEL5.HCMV-GS mammalian expression vector (51Cosgrove L. Lovrecz G.O. Verkuylen A. Cavaleri L. Black L.A. Bentley J.D. Howlett G.J. Gray P.P. Ward C.W. McKern N.M. Protein Expression Purif. 1995; 6: 789-798Crossref PubMed Scopus (43) Google Scholar) driven by the human cytomegaloviral promoter. The first expression plasmid, designated pEEL-sIGF-1R, encoded the ectodomain of the human IGF-1R (sIGF-1R; 1–906 amino acids). In addition, a chimeric human IGF-1R-mouse Fc immunoglobulin fusion protein was encoded by the plasmid pEEL-sIGF-1R/Fc (for more detail, see the supplemental materials). Both of these cDNA expression constructs were transfected into hamster kidney fibroblast BHK-21 cells, and stable cell lines were generated. Cell lines expressing high levels of soluble IGF-1Rs (∼5 mg/liter) were identified by ELISA using conformation-dependent anti-IGF-1R antibodies (45Soos 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). Receptor Binding Assays—We initially established that europium labeling of IGFs resulted in a tracer with identical characteristics in receptor binding assays as the traditional 125I-labeled IGFs. Competition binding assays using captured solubilized IGF-1R with Eu-IGF-I and 125I-IGF-I (Fig. 1A) resulted in binding curves that were superimposable. The same result was seen with Eu-IGF-II and 125I-IGF-II (data not shown). Therefore, europium-labeled tracers were used for all subsequent binding assays. Competition binding using solubilized wtIGF-1R, sIGF-1R/Fc, and sIGF-1R showed that both soluble ectodomain-containing fragments were identical in their binding affinities for IGF-I (Fig. 1B and Table 1). However, they showed a slightly lower binding affinity (IC50 = 0.4 nm) compared with the solubilized wild type IGF-1R (IC50 = 0.23 nm). A similar result was seen with Eu-IGF-II competition binding assays (Fig. 1C), where the ectodomain fragments bound IGF-II with a 1.8-fold lower affinity than solubilized IGF-1R. All receptors had a 2-fold lower affinity for IGF-II compared with IGF-I. BIAcore analyses confirmed the competition binding assays and showed that sIGF-1R/Fc and sIGF-1R have the same relative affinities for IGF-I or IGF-II (Fig. 1D and Table 2).TABLE 1Binding of europium- and 125I-labeled IGFs to wild type and soluble IGF-1RsIC50sIGF-1R/FcsIGF-1RwtIGF-1RnmEu-IGF-I0.42 ± 0.150.41 ± 0.10.25 ± 0.15Eu-IGF-II0.83 ± 0.30.88 ± 0.60.49 ± 0.07125I-IGF-I2.3 ± 2.22.0 ± 1.60.25 ± 0.1 Open table in a new tab TABLE 2BIAcore analysis of IGFs binding to soluble IGF-1Rs coupled to the biosensor surfaceAnalyteSurfaceka × 105kd × 10-3KD × 10-9m-1 s-1s-1mIGF-IsIGF-1R/Fc4.72.55.3sIGF-1R2.92.79.3IGF-IIsIGF-1R/Fc5.65.710sIGF-1R4.28.219 Open table in a new tab Dissociation of Eu-IGF Ligands Bound to sIGF-1Rs—To investigate negative cooperativity of IGF-I and IGF-II binding to the soluble IGF-1Rs, we initially examined the dissociation of bound Eu-IGF-I in the presence or absence of unlabeled IGF-I (Fig. 2). In the absence of unlabeled ligand, the rate of dissociation of bound IGF-I from both soluble receptors (Fig. 2, B and C) was similar and slightly faster than from the solubilized wild type IGF-1R (Fig. 2A). However, the presence of excess unlabeled IGF-I accelerated the dissociation from the sIGF-1R, with ∼80% dissociation seen at 30 min compared with 55% from sIGF-1R/Fc and 50% from the wild type receptor (Fig. 2). Although negative cooperativity of IGF-I binding to the wild type IGF-1R has been previously demonstrated, the effect of IGF-II on negative cooperativity has not been reported. Therefore, we also examined the dissociation of bound Eu-IGF-II in the presence or absence of IGF-II from the wild type and soluble IGF-1Rs (Fig. 2). In buffer alone, dissociation of bound Eu-IGF-II from sIGF-1R/Fc and sIGF-1R (Fig. 2, E and F) was similar and slightly faster than that seen for the solubilized wtIGF-1R (Fig. 2D). The presence of unlabeled IGF-II accelerated the dissociation from the solubilized wtIGF-1R and both sIGF-1Rs (Fig. 2). The solubilized wtIGF-1R exhibited a slower dissociation rate in the presence of unlabeled IGF-II compared with sIGF-1R/Fc (∼30 and ∼50% dissociation at 30 min, respectively), whereas dissociation from sIGF-1R was faster (∼80% dissociation at 30 min Fig. 2). In addition, the dissociation of bound IGF-II with unlabeled ligand from sIGF-1R was similar to that observed with bound IGF-I with unlabeled ligand from the sIGF-1R (∼80% dissociation at 30 min). It was concluded that both of the soluble IGF-1Rs exhibit negative cooperativity of IGF-I and IGF-II binding. Soos and Siddle 4M. A. Soos and K. Siddle, unpublished data. have previously demonstrated that the anti-IGF-1R mAb 24-60 accelerates the dissociation of 125I-IGF-I from solubilized wtIGF-1R. This antibody binds to a similar epitope to that of αIR3 mAb on the IGF-1R (52Keyhanfar M. Booker G.W. Whittaker J. Wallace J.C. Forbes B.E. Biochem. J. 2007; 401: 269-

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