CHARACTERIZATION OF LIGAND-BINDING OF A SOLUBLE HUMAN INSULIN-LIKE GROWTH-FACTOR-I RECEPTOR VARIANT SUGGESTS A LIGAND-INDUCED CONFORMATIONAL CHANGE

Details of the signal transduction mechanisms of the tyrosine kinase f amily of growth factor receptors remain elusive. In this work, we desc ribe an extensive study of kinetic and thermodynamic aspects of growth factor binding to a soluble extracellular human insulin-like growth f actor-I receptor (sIGF-I-R) variant. The extracellular receptor domain s were produced fused to an IgG-binding protein domain (Z) in transfec ted human 293 cells as a correctly processed secreted alpha-beta'-Z di mer. The receptor was purified using IgG affinity chromatography, rend ering 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, insul in-like growth factor-II, and insulin ligand binding rate constants to the immobilized IGF-I-R-Z. The association equilibrium constant, K-a for the IGF-I interaction is determined to 2.8 x 10(8) M(-1) (25 degre es C). Microcalorimetric titrations on IGF-I/IGF-I-R-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, equ ilibrium constants for the 1:1 (IGF-I:(alpha-beta'-Z)(2)) receptor com plex 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 (Delta S) at 25 degrees C is large and negative. Far- and near-UV circular dichroism m easurements display significant changes over the entire wavelength ran ge upon binding of IGF-I to IGF-I-R-Z. These data are all consistent w ith a significant change in structure of the system upon IGF-I binding .