M. Jansson et al., CHARACTERIZATION OF LIGAND-BINDING OF A SOLUBLE HUMAN INSULIN-LIKE GROWTH-FACTOR-I RECEPTOR VARIANT SUGGESTS A LIGAND-INDUCED CONFORMATIONAL CHANGE, The Journal of biological chemistry, 272(13), 1997, pp. 8189-8197
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
.