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.
Highlights
Cellular 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
insulin-like growth factor-I receptor (IGF-IR) is active as a preformed heterotetrameric receptor containing two extracellular ␣-domains with ligand binding sites and two transmembrane-spanning -domains, harboring the cytoplasmic tyrosine kinase activity (Fig. 1A)
We have described the ligand binding properties of recombinant soluble extracellular human insulinlike growth factor-I receptor (sIGF-IR)
Summary
Ligand Proteins—Native IGF-I, des(1–3)IGF-I, and IGF-II were produced in Escherichia coli and purified as described previously (14, 15). The receptor was analyzed at a concentration of 1 mg/ml in 10 mM phosphate buffer, pH 7.4, at seven different temperatures in the range 4 –25 °C. Following a 15-min incubation, free thiols of the partially reduced receptor were alkylated by adding iodoacetic acid to a final concentration of 100 mM. Immobilized IGF-IR-Z was regenerated after each cycle by injection of 12 l of acidic regeneration solution containing 0.3 M sodium citrate, pH 5, and 0.4 M NaCl. Kinetic measurements were performed by injection of each analyte for 300 s followed by disassociation in buffer flow for 400 s. To correct for dilution enthalpies, additional dilution experiments where IGF-I was added in the buffer solution alone were performed. Adding up the contributions (i–v) we arrive at the following expression for the total change in entropy upon binding
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