Characterization of Ligand Binding of a Soluble Human Insulin-like Growth Factor I Receptor Variant Suggests a Ligand-induced Conformational Change (1997)

Details of the signal transduction mechanisms of the tyrosine kinase family of growth factor receptors remain elusive. In this work, we describe an extensive study of kinetic and thermodynamic aspects of growth factor binding to a soluble extracellular human insulin-like growth factor-I receptor (sIGF-IR) variant. The extracellular receptor domains were produced fused to an IgG-binding protein domain (Z) in transfected human 293 cells as a correctly processed secreted α-β′-Z dimer. The receptor was purified using IgG affinity chromatography, rendering a pure and homogenous protein in yields from 1 to 5 mg/liter of conditioned cell media. Biosensor technology (BIAcore) was applied to measure the insulin-like growth factor-I (IGF-I), des(1-3)IGF-I, insulin-like growth factor-II, and insulin ligand binding rate constants to the immobilized IGF-IR-Z. The association equilibrium constant, Ka, for the IGF-I interaction is determined to 2.8 × 108 M−1 (25 °C). Microcalorimetric titrations on IGF-I/IGF-IR-Z were performed at three different temperatures (15, 25, and 37 °C) and in two different buffer systems at 25 °C. From these measurements, equilibrium constants for the 1:1 (IGF-I:(α-β′-Z)2) receptor complex in solution are deduced to 0.96 × 108 M−1 (25 °C). The determined heat capacity change for the process is large and negative, −(1)0.51 kcal (K mol)−1. Further, the entropy change (βS) at 25 °C is large and negative. Far- and near-UV circular dichroism measurements display significant changes over the entire wavelength range upon binding of IGF-I to IGF-IR-Z. These data are all consistent with a significant change in structure of the system upon IGF-I binding. Details of the signal transduction mechanisms of the tyrosine kinase family of growth factor receptors remain elusive. In this work, we describe an extensive study of kinetic and thermodynamic aspects of growth factor binding to a soluble extracellular human insulin-like growth factor-I receptor (sIGF-IR) variant. The extracellular receptor domains were produced fused to an IgG-binding protein domain (Z) in transfected human 293 cells as a correctly processed secreted α-β′-Z dimer. The receptor was purified using IgG affinity chromatography, rendering a pure and homogenous protein in yields from 1 to 5 mg/liter of conditioned cell media. Biosensor technology (BIAcore) was applied to measure the insulin-like growth factor-I (IGF-I), des(1-3)IGF-I, insulin-like growth factor-II, and insulin ligand binding rate constants to the immobilized IGF-IR-Z. The association equilibrium constant, Ka, for the IGF-I interaction is determined to 2.8 × 108 M−1 (25 °C). Microcalorimetric titrations on IGF-I/IGF-IR-Z were performed at three different temperatures (15, 25, and 37 °C) and in two different buffer systems at 25 °C. From these measurements, equilibrium constants for the 1:1 (IGF-I:(α-β′-Z)2) receptor complex in solution are deduced to 0.96 × 108 M−1 (25 °C). The determined heat capacity change for the process is large and negative, −(1)0.51 kcal (K mol)−1. Further, the entropy change (βS) at 25 °C is large and negative. Far- and near-UV circular dichroism measurements display significant changes over the entire wavelength range upon binding of IGF-I to IGF-IR-Z. These data are all consistent with a significant change in structure of the system upon IGF-I binding. INTRODUCTIONCellular receptor structure and function relationships have become the focus of an increasing amount of research, relevant for biological understanding as well as for possible pharmaceutical applications. The insulin-like growth factor-I receptor (IGF-IR) 1The abbreviations used are: IGF-IRIGF-I receptorHBSHepes-buffered salineIGF-Iinsulin-like growth factor-IIGF-IIinsulin-like growth factor-IIZsynthetic IgG binding domain from Staphylococcal protein AsIGF-IR-Zsoluble variant of IGF-IR-Z fusioninsRinsulin receptorPBSphosphate-buffered saline. is a transmembrane glycoprotein and belongs to the receptor tyrosine kinase family that includes, among others, the insulin (insR), epidermal growth factor, and platelet-derived growth factor receptors (1Heldin C.H. Cell. 1995; 80: 213-223Abstract Full Text PDF PubMed Scopus (1427) Google Scholar). Insulin-like growth factor-I (IGF-I) is a major regulator of both cellular growth and metabolism (2Siddle K. Prog. Growth Factor Res. 1992; 4: 301-320Abstract Full Text PDF PubMed Scopus (30) Google Scholar). The components of the IGF hormone signaling system are tightly regulated, both in tissue-specific and developmentally specific manners. The IGF molecular system contains three known peptide ligands (IGF-I, IGF-II, and insulin), three types of cellular receptors (IGF-IR, insR, and IGF-II/mannose 6-phosphate), and six known subtypes of circulating IGF-binding proteins (IGFBPs) (IGFBP-1 to IGFBP-6). The molecular components of the IGF signaling system have been the subject of several recent review articles (3Rechler M.M. Vit. Horm. 1993; 47: 1-114Crossref PubMed Scopus (444) Google Scholar, 4Jones J.I. Clemmons D.R. Endocr. Rev. 1995; 16: 3-34Crossref PubMed Google Scholar). The mechanism by which insulin and IGF molecules interact with their respective receptor to mediate signaling is still not understood in great detail at the molecular level. The ligand-induced receptor activation has been suggested to involve a conformational switch in the quaternary structure upon ligand binding, with movements of the extracellular α parts and a congregation of the cytoplasmic tyrosine kinase regions to enable activation (5McDonald N.Q. Murray-Rust J. Blundell T.J. Structure. 1995; 3: 1-6Abstract Full Text Full Text PDF PubMed Scopus (16) Google Scholar, 6Hubbard S.R. Wei L. Ellis L. Hendrickson W.A. Nature. 1994; 372: 746-754Crossref PubMed Scopus (954) Google Scholar). However, the complexity of IGF-I signaling remains rather elusive almost 10 years after the molecular cloning of the human IGF-I receptor gene (7Ullrich A. EMBO J. 1986; 5: 2503-2512Crossref PubMed Scopus (1494) Google Scholar).IGF-IR is active as a preformed heterotetrameric receptor containing two extracellular α-domains with ligand binding sites and two transmembrane-spanning β-domains, also harboring the cytoplasmic tyrosine kinase activity (Fig. 1A). The chains are disulfide-linked in a β-α-α-β arrangement with possibly three disulfide linkages between the α chains and one disulfide bond between each α-β chain pair (8Ward C.W. Hoyne P.A. Flegg R.H. Proteins. 1995; 22: 141-153Crossref PubMed Scopus (86) Google Scholar). The gene is transcribed and translated as a single α-β polypeptide chain and post-translationally processed and assembled into a mature receptor. It has been shown for the homologous insulin receptor that receptor maturation is achieved through proteolytic processing, oligosaccharide attachment, and disulfidelinked homodimerization during the transport from endoplasmic reticulum to Golgi and further to the cell surface (9Yamada K. Goncalves E. Carpentier J.L. Kahn C.R. Shoelson S.E. Biochemistry. 1995; 34: 946-954Crossref PubMed Scopus (10) Google Scholar).In this paper, the ligand binding properties of a purified extracellular portion of the IGF-IR have been characterized using BIAcore™ biosensor analysis. From the BIAcore data, molecular association and disassociation rates as well as binding equilibrium constants were calculated. Binding properties of the soluble IGF-IR receptor variant were analyzed for the ligands IGF-I, des(1-3)-IGF-I, IGF-II, and insulin, respectively. Near- and far-UV circular dichroism measurements were performed to establish an estimation of possible secondary structure changes upon ligand association.Microcalorimetric titration binding experiments of IGF-I to the heterotetrameric IGF-IR-Z were performed at three different temperatures, from which enthalpies, βH0, Gibbs free energies, βG0, entropies, βS0, and heat capacity, βCp0, of the binding process could be calculated. In this work, we have used the model proposed by Freire et al. (10Freire E. Haynie D.T. Xie D. Proteins. 1993; 17: 111-123Crossref PubMed Scopus (39) Google Scholar, 11Freire E. Murphy K.P. Sanchez R.J. Galisteo M.L. Privalov P.L. Biochemistry. 1992; 31: 250-256Crossref PubMed Scopus (103) Google Scholar, 12Murphy K.P. Freire E. Adv. Protein Chem. 1992; 43: 313-361Crossref PubMed Google Scholar, 13Xie D. Freire E. Proteins. 1994; 19: 291-301Crossref PubMed Scopus (127) Google Scholar) to analyze microcalorimetric titration data of the binding of IGF-I to the heterotetrameric IGF-IR-Z. From this analysis, the change in hydration-accessible polar and nonpolar surface areas were calculated, as well as the entropic contribution to changes in conformational degree of upon is to the using both BIAcore and titration of ligand binding, of a growth factor receptor from the insulin-like growth factor family of tyrosine kinase extracellular portion of the receptor was produced as a soluble secreted with an affinity fused to the The of the affinity has been in the of a large of proteins in systems L. PubMed Scopus Google Scholar) and is in this used in a receptor was and in the and the was to be in with the (7Ullrich A. EMBO J. 1986; 5: 2503-2512Crossref PubMed Scopus (1494) Google 293 cells were transfected for of the IGF-I receptor. In 293 the receptor were for of cell The 293 cell and with a of proteolytic cell growth used in the to a from 1 to 5 of of conditioned media. The of the receptor was determined using (Fig. of the affinity using (Fig. a single in the in to soluble receptor. In the a to the of the is and containing the were The determined to is from not of two different to a correctly processed after signal peptide and a of of IGF-IR-Z. of IGF-IR-Z. in are by to the and association were analyzed by analysis. is on the that the are to P.A. J. Chem. Full Text PDF PubMed Google Scholar). in the of α-β′-Z in to the receptor. The in the of one in the to the of receptor. The two of and to and respectively. The the of two to receptor and a of molecular which to the α-β′-Z receptor. The molecular determined from to changes in from and different of in the as well as possible from the of the receptor a of by The of is as and The was to be molecular on the molecular from is for the receptor. to the molecular of the receptor using not using of both free and receptor an from The molecular of free receptor at 25 °C was and the of the complex was The determined to from the in this analysis. From the data the receptor was to be a homogenous receptor The in between free and receptor is the of the ligand and different from the in the of molecular was used to possible changes in secondary structure upon ligand binding. The far-UV of free and receptor are shown in The of the free IGF-I ligand is from the receptor of the secondary structure components using a structure for the free receptor of and of IGF-IR-Z with an of ligand (IGF-I) of the ligand over the wavelength range that these are a of binding IGF-I to receptor. is a in for the complex over the entire The of receptor is and The of secondary structure to contribution to from of the far-UV of free and receptor. The signal of free IGF-I is from the to the signal of receptor near-UV of free receptor signal contribution and of receptor as in The of IGF-I and in is shown for near-UV of the free of the receptor with ligand signal and of the signal of free receptor and are shown in In to the far-UV is an in upon ligand binding in the near-UV The at is by and at is by The near-UV is to chain and Binding binding of IGF-I to purified using IGF-I as a is shown in The from the binding is binding of binding to using IGF-I as a The is to kinetic measurements were performed for a of known ligands to the IGF-I, IGF-II, des(1-3)-IGF-I, and IGF-IR-Z was and for the different ligand are shown in of the rate and equilibrium constants is in IGF-I affinity was determined in three using of each and affinity for the ligands was determined in one also using The in the determined association constants from all was to be as the of the of the of the in analysis, and data kinetic that in both association rates and disassociation rates the in association equilibrium constants for the The variant has an association rate that is that of IGF-I, the association rate for is IGF-I and have disassociation that of is that of The insulin association was to be using BIAcore technology at the association was analyzed for insulin at to used for The in disassociation rate between the ligands are insulin the association The data for insulin disassociation is to a model in to the of the two disassociation one is that of IGF-I, and the is 10 The association equilibrium constants for IGF-IR-Z are for for IGF-I, the for is for both by association and from the of ligand binding to immobilized IGF-IR-Z. The the in after in are for the IGF-I and insulin The ligands are by in the rate constants of IGF-I receptor ligand binding from BIAcore measurements as in M−1 not determined using this different rates not determined using this not determined using this different rates in a for IGF-I Binding to of binding were at all temperatures The heat was at the at each titration and from to (Fig. and 25 the heat in the buffer were the of of equilibrium However, to the at 37 °C in and at 25 °C in data could be to a 1:1 binding model with (Fig. thermodynamic binding a complex with of were also to establish the thermodynamic model for the binding These could be on analysis. and 25 °C in the of binding were from The from the titration experiments are in The from the of the thermodynamic data are in The thermodynamic properties are by the large heat is for in which the of nonpolar is to the large heat capacity the and the entropy are the large heat capacity change be by of large surface The large and also a of the affinity constant, which be by the in at 25 and 37 °C. The of in the was determined to be at to the entropy change upon binding from the from the of in the binding process and to a in the conformational degree of in the IGF-I and IGF-IR-Z contribution was to be in at 25 °C. the is at is not possible to for specific to of to the of the to the different thermodynamic However, the major to and heat capacity from changes in of the two the of the to the thermodynamic properties be to be the for 25 of IGF-I, each of into the microcalorimetric containing of IGF-IR-Z in at and 25 °C. The were with a and the data shown are the as K.P. Freire E. Adv. Protein Chem. 1992; 43: 313-361Crossref PubMed Google of processed microcalorimetric data in for of of IGF-I have been The are data, and the to the by is the to the amount of IGF-I at each from titration measurements of IGF-I binding to (K (K in a entropy and hydration-accessible surface and upon binding of IGF-I to IGF-IR-Z at 25 in nonpolar in polar from change in nonpolar from change in polar from change in to conformational entropic in a to the of the thermodynamic is a significant in the of for the proteins upon binding, which is by the large and from the binding surface areas of IGF-I and the large change in surface have in changes in of the proteins to conformational The surface for free IGF-I is on the structure Biochemistry. PubMed Scopus Google Scholar) and the and J. PubMed Scopus Google Scholar) in the this paper, we have the ligand binding properties of The of the IGF-IR in of ligand binding kinetic secondary structure analysis, and thermodynamic with the of specific and receptor conformational as well as the 1:1 are to the understanding of the IGF-I signaling mechanism and titration thermodynamic and in the large entropy upon ligand binding, are consistent with a conformational by ligand binding. of this could be by a conformational The titration data that of IGF-I to be through the of binding on both the ligand and the receptor from changes to a of surface in of the this that of IGF-I upon binding. is an large portion of the the that a conformational change in the receptor. The of the IGF-I receptor that the secondary structure of the receptor changes upon ligand binding. The far-UV of free and IGF-IR-Z changes in (Fig. and the near-UV changes are in both the changes are and The in the between the complex and the of the of free receptor and free ligand that changes in of chains in the receptor upon ligand These changes could be to secondary quaternary both PubMed Scopus Google structure of the insulin receptor cytoplasmic tyrosine kinase domain has been determined using protein S.R. Wei L. Ellis L. Hendrickson W.A. Nature. 1994; 372: 746-754Crossref PubMed Scopus (954) Google Scholar). These data that the activation of the tyrosine kinase possible by of the domains and in the protein kinase active possible that signal transduction the is the of a ligand-induced quaternary The of this change for involve a of the receptor a of the a of these Binding of ligand to both IGF-I and insulin receptors is proposed to involve 1994; PubMed Scopus Google Scholar). The mechanism has been suggested to involve the of both and affinity binding sites on each α ligand to a affinity on one conformational changes the ligand to the two by also binding to the affinity on the on IGF-IR-Z a of 1:1 (IGF-I:(α-β′-Z)2) with which could two binding of binding to the produced IGF-I receptor for the different ligands is in with performed using ligand D. in Insulin-like Growth Factor 1994; Scholar). The a association rate IGF-I and a has 5 affinity IGF-I to both on rates and The in affinity between the BIAcore and the titration are in with and be to degree of conformational for the IGF-IR-Z in solution is immobilized on a surface in conformational of upon surface the affinity for the The receptor binding properties of a of IGF-I have been using both BIAcore technology and and in that the of binding between used a of molecules have the of the be also for the interaction of human growth hormone to the human growth hormone receptor J. 1993; PubMed Scopus Google Scholar) and for binding of and their binding to IgG L. J. 1995; PubMed Scopus Google Scholar) using and BIAcore In the of association equilibrium constants determined by BIAcore kinetic has been in by J. J. A. 1995; PubMed Scopus Google Scholar) and et al. J. 1994; PubMed Scopus Google in both the of biosensor and titration was used to and J. J. A. 1995; PubMed Scopus Google Scholar) that measurements were in the biosensor In the by et al. J. 1994; PubMed Scopus Google was between kinetic and biosensor measurements as well as titration the kinetic data to be on the The association is between a ligand and a large immobilized the of using proteins transport that a large receptor with a immobilized The of used to the association rate was in the used The disassociation rate not change with in the and the of free protein in the disassociation not that not The rate the to the of the ligands and the of in IGF-IR The association rate of insulin binding to IGF-IR-Z is to be determined using the is to the disassociation rate from the in to the a model the for insulin The rates of disassociation are 10 and for the two insulin the rate for for this is that IGF-I to the IGF-I receptor and a conformational change by the and affinity sites 1994; PubMed Scopus Google Scholar). on the to the one to each α with affinity is to conformational changes and is from these two of ligand binding in the of different the IGF-I receptor A. K. J. Chem. 1992; Full Text PDF PubMed Google Scholar) that signal transduction be of ligand binding. be the the the ligand binding and a conformational further signal In the study by et al. A. K. J. Chem. 1992; Full Text PDF PubMed Google Scholar) is a further that IGF-I ligand binding also the affinity of insulin for the IGF-I receptor to These are consistent with the that insulin on is to between the regions of the IGF-I receptor. The could possibly switch the receptor to an by the in a of an insulin affinity receptor has been shown by et al. E. 1995; Google using conformational that the IGF-I receptor an conformational change in cytoplasmic which is from ligand-induced conformational In this we have data a conformational change in the extracellular domains of a IGF-I receptor. of the structure of the homologous insulin receptor have been K. J. J. J. A. PubMed Scopus Google Scholar, A. Ellis L. J. Chem. 1992; Full Text PDF PubMed Google Scholar). and K. J. J. J. A. PubMed Scopus Google Scholar) the structure of receptor from both in the and of two different of the one an and a in The of insulin not a single the could not be that changes ligand binding and receptor et al. A. Ellis L. J. Chem. 1992; Full Text PDF PubMed Google Scholar) the insulin receptor extracellular using a to the extracellular IGF-IR in this and of to by and These the of large of the receptor α The that the in these the of receptor with ligand is Further, et al. A. Ellis L. J. Chem. 1992; Full Text PDF PubMed Google Scholar) applied to the insulin receptor and that changes in secondary structure insulin was of ligand binding of extracellular receptors a to of the mature have the of an affinity protein system for the of large of a soluble of using the affinity system has and an pure receptor using a (Fig. The protein affinity from the domain of Staphylococcal protein has been used in a large of for the and of proteins L. PubMed Scopus Google Scholar). Growth have not been as a of the of this protein in the 293 cell The produced receptor variant proteolytic after cell data in this the that extracellular IGF-I receptor domains a conformational change upon ligand binding that changes in both the amount of surface and the amount of secondary structure The of in the binding process from of between receptor and It has been shown by that the IGF-I domain are for affinity IGF-I receptor binding J. Chem. Full Text PDF PubMed Google Scholar). The could also be of the conformational change in the receptor These two different be this we have shown that the produced soluble IGF-IR-Z one IGF-I with affinity and is of possibly in the activation of signal The of transmembrane and cytoplasmic regions not affinity ligand binding INTRODUCTIONCellular receptor structure and function relationships have become the focus of an increasing amount of research, relevant for biological understanding as well as for possible pharmaceutical applications. The insulin-like growth factor-I receptor (IGF-IR) 1The abbreviations used are: IGF-IRIGF-I receptorHBSHepes-buffered salineIGF-Iinsulin-like growth factor-IIGF-IIinsulin-like growth factor-IIZsynthetic IgG binding domain from Staphylococcal protein AsIGF-IR-Zsoluble variant of IGF-IR-Z fusioninsRinsulin receptorPBSphosphate-buffered saline. is a transmembrane glycoprotein and belongs to the receptor tyrosine kinase family that includes, among others, the insulin (insR), epidermal growth factor, and platelet-derived growth factor receptors (1Heldin C.H. Cell. 1995; 80: 213-223Abstract Full Text PDF PubMed Scopus (1427) Google Scholar). Insulin-like growth factor-I (IGF-I) is a major regulator of both cellular growth and metabolism (2Siddle K. Prog. Growth Factor Res. 1992; 4: 301-320Abstract Full Text PDF PubMed Scopus (30) Google Scholar). The components of the IGF hormone signaling system are tightly regulated, both in tissue-specific and developmentally specific manners. The IGF molecular system contains three known peptide ligands (IGF-I, IGF-II, and insulin), three types of cellular receptors (IGF-IR, insR, and IGF-II/mannose 6-phosphate), and six known subtypes of circulating IGF-binding proteins (IGFBPs) (IGFBP-1 to IGFBP-6). The molecular components of the IGF signaling system have been the subject of several recent review articles (3Rechler M.M. Vit. Horm. 1993; 47: 1-114Crossref PubMed Scopus (444) Google Scholar, 4Jones J.I. Clemmons D.R. Endocr. Rev. 1995; 16: 3-34Crossref PubMed Google Scholar). The mechanism by which insulin and IGF molecules interact with their respective receptor to mediate signaling is still not understood in great detail at the molecular level. The ligand-induced receptor activation has been suggested to involve a conformational switch in the quaternary structure upon ligand binding, with movements of the extracellular α parts and a congregation of the cytoplasmic tyrosine kinase regions to enable activation (5McDonald N.Q. Murray-Rust J. Blundell T.J. Structure. 1995; 3: 1-6Abstract Full Text Full Text PDF PubMed Scopus (16) Google Scholar, 6Hubbard S.R. Wei L. Ellis L. Hendrickson W.A. Nature. 1994; 372: 746-754Crossref PubMed Scopus (954) Google Scholar). However, the complexity of IGF-I signaling remains rather elusive almost 10 years after the molecular cloning of the human IGF-I receptor gene (7Ullrich A. EMBO J. 1986; 5: 2503-2512Crossref PubMed Scopus (1494) Google Scholar).IGF-IR is active as a preformed heterotetrameric receptor containing two extracellular α-domains with ligand binding sites and two transmembrane-spanning β-domains, also harboring the cytoplasmic tyrosine kinase activity (Fig. 1A). The chains are disulfide-linked in a β-α-α-β arrangement with possibly three disulfide linkages between the α chains and one disulfide bond between each α-β chain pair (8Ward C.W. Hoyne P.A. Flegg R.H. Proteins. 1995; 22: 141-153Crossref PubMed Scopus (86) Google Scholar). The gene is transcribed and translated as a single α-β polypeptide chain and post-translationally processed and assembled into a mature receptor. It has been shown for the homologous insulin receptor that receptor maturation is achieved through proteolytic processing, oligosaccharide attachment, and disulfidelinked homodimerization during the transport from endoplasmic reticulum to Golgi and further to the cell surface (9Yamada K. Goncalves E. Carpentier J.L. Kahn C.R. Shoelson S.E. Biochemistry. 1995; 34: 946-954Crossref PubMed Scopus (10) Google Scholar).In this paper, the ligand binding properties of a purified extracellular portion of the IGF-IR have been characterized using BIAcore™ biosensor analysis. From the BIAcore data, molecular association and disassociation rates as well as binding equilibrium constants were calculated. Binding properties of the soluble IGF-IR receptor variant were analyzed for the ligands IGF-I, des(1-3)-IGF-I, IGF-II, and insulin, respectively. Near- and far-UV circular dichroism measurements were performed to establish an estimation of possible secondary structure changes upon ligand association.Microcalorimetric titration binding experiments of IGF-I to the heterotetrameric IGF-IR-Z were performed at three different temperatures, from which enthalpies, βH0, Gibbs free energies, βG0, entropies, βS0, and heat capacity, βCp0, of the binding process could be calculated. In this work, we have used the model proposed by Freire et al. (10Freire E. Haynie D.T. Xie D. Proteins. 1993; 17: 111-123Crossref PubMed Scopus (39) Google Scholar, 11Freire E. Murphy K.P. Sanchez R.J. Galisteo M.L. Privalov P.L. Biochemistry. 1992; 31: 250-256Crossref PubMed Scopus (103) Google Scholar, 12Murphy K.P. Freire E. Adv. Protein Chem. 1992; 43: 313-361Crossref PubMed Google Scholar, 13Xie D. Freire E. Proteins. 1994; 19: 291-301Crossref PubMed Scopus (127) Google Scholar) to analyze microcalorimetric titration data of the binding of IGF-I to the heterotetrameric IGF-IR-Z. From this analysis, the change in hydration-accessible polar and nonpolar surface areas were calculated, as well as the entropic contribution to changes in conformational degree of upon is to the using both BIAcore and titration of ligand binding, of a growth factor receptor from the insulin-like growth factor family of tyrosine kinase