title: Characterization of ligand binding of a soluble human insulin-like growth factor I receptor variant suggests a ligand-induced conformational change.
abstract: 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 alpha-beta'-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 x 10(8) M-1 (25 degrees C). Microcalorimetric titrations on IGF-I/IGF-IR-Z were performed at three different temperatures (15, 25, and 37 degrees C) and in two different buffer systems at 25 degrees C. From these measurements, equilibrium constants for the 1:1 (IGF-I:(alpha-beta'-Z)2) receptor complex in solution are deduced to 0.96 x 10(8) M-1 (25 degrees C). The determined heat capacity change for the process is large and negative, -0.51 kcal (K mol)-1. Further, the entropy change (DeltaS) at 25 degrees 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.
authors.affiliation: Department of Biochemistry and Biotechnology, Royal Institute of Technology, S-100 44 Stockholm, Sweden.
authors.forename: M
authors.initials: M
authors.lastname: Jansson
authors.forename: D
authors.initials: D
authors.lastname: Hallén
authors.forename: H
authors.initials: H
authors.lastname: Koho
authors.forename: G
authors.initials: G
authors.lastname: Andersson
authors.forename: L
authors.initials: L
authors.lastname: Berghard
authors.forename: J
authors.initials: J
authors.lastname: Heidrich
authors.forename: E
authors.initials: E
authors.lastname: Nyberg
authors.forename: M
authors.initials: M
authors.lastname: Uhlén
authors.forename: J
authors.initials: J
authors.lastname: Kördel
authors.forename: B
authors.initials: B
authors.lastname: Nilsson
mesh_terms: D015374:Biosensing Techniques; D002151:Calorimetry; D002455:Cell Division; D002460:Cell Line; D002942:Circular Dichroism; D006801:Humans; D007328:Insulin / Q000378:metabolism; D007334:Insulin-Like Growth Factor I / Q000378:metabolism; D007335:Insulin-Like Growth Factor II / Q000378:metabolism; D007668:Kidney / Q000166:cytology; D007834:Lasers; D008958:Models, Molecular; D010446:Peptide Fragments / Q000378:metabolism; D011487:Protein Conformation; D017526:Receptor, IGF Type 1 / Q000378:metabolism*; D011994:Recombinant Proteins / Q000378:metabolism; D012542:Scattering, Radiation; D015398:Signal Transduction; D014162:Transfection
chemical_list: D007328:Insulin; D010446:Peptide Fragments; D011994:Recombinant Proteins; C058957:insulin-like growth factor 1, des-(1-3)-; D007334:Insulin-Like Growth Factor I; D007335:Insulin-Like Growth Factor II; D017526:Receptor, IGF Type 1