The structural basis for insulin-like growth factor I receptor high affinity binding

• Published in: The Journal of biological chemistry Vol. 263; no. 31; pp. 16267 - 16273
• Main Authors: Tollefsen, S E, Thompson, K
• Format: Journal Article
• Language: English
• Published: Bethesda, MD Elsevier Inc 05.11.1988; American Society for Biochemistry and Molecular Biology
• Subjects: Binding, Competitive; Biological and medical sciences; Cell Membrane - metabolism; Cell physiology; Dithiothreitol - pharmacology; Female; Fundamental and applied biological sciences. Psychology; Hormonal regulation; Humans; IGF-I; Insulin-Like Growth Factor I - metabolism; Insulin-Like Growth Factor I - pharmacology; Kinetics; Macromolecular Substances; Molecular and cellular biology; Molecular Weight; Phosphorylation; Placenta - metabolism; Pregnancy; Receptor, Insulin - drug effects; Receptor, Insulin - isolation & purification; Receptor, Insulin - metabolism; Receptors, Somatomedin; Somatomedins - metabolism; Human; Affinity; Molecular interaction; Membrane receptor; Somatomedin C
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1016/S0021-9258(18)37588-4

We have recently identified high and low affinity insulin-like growth factor I (IGF I) binding sites in solubilized human placental membranes and purified the high affinity IGF I receptor by IGF I affinity chromatography (Tollefsen, S. E., Thompson, K., and Petersen, D. J. (1987) J. Biol. Chem. 262, 16461-16469). To define the structural basis for high affinity IGF I binding, we have examined the effect of disulfide bond reduction on the binding parameters of the high affinity IGF I receptor. We find that the disulfide bonds linking the two alpha beta dimers of the IGF I receptor heterotetramer are reduced by incubation at pH 8.75 with 2 mM dithiothreitol (DTT) for 5 min at room temperature. Gel filtration chromatography on a Superose 12 fast protein liquid chromatography column indicates that the alpha beta dimers do not remain associated by noncovalent interactions after reduction. Scatchard plots of IGF I binding to the IGF I receptor incubated at pH 8.75 with or without DTT indicate that the IGF I receptor alpha beta dimers have a 6.1 +/- 1.6 (mean +/- S.D.) times lower affinity than the heterotetramer for IGF I. The total binding capacity of the IGF I receptor treated with DTT is 1.6 +/- 0.3 (mean +/- S.D.) times higher than that of an equal amount of receptor treated without DTT. These results are consistent with a model in which the heterotetramer binds a single IGF I molecule with high affinity, whereas each of the two alpha beta dimers binds an IGF I molecule with lower affinity after dissociation. We conclude that association of two alpha beta dimers is required for formation of an IGF I receptor with high affinity for its ligand.