The structural basis for insulin-like growth factor I receptor high affinity binding.
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.
Tollefsen
S E
SE
Edward Mallinckrodt Department of Pediatrics, Washington University School of Medicine, St. Louis, Missouri 63110.
Thompson
K
K
D001667:Binding, Competitive; D002462:Cell Membrane / Q000378:metabolism; D004229:Dithiothreitol / Q000494:pharmacology; D005260:Female; D006801:Humans; D007334:Insulin-Like Growth Factor I / Q000378:metabolism* / Q000494:pharmacology; D007700:Kinetics; D046911:Macromolecular Substances; D008970:Molecular Weight; D010766:Phosphorylation; D010920:Placenta / Q000378:metabolism; D011247:Pregnancy; D011972:Receptor, Insulin / Q000187:drug effects / Q000302:isolation & purification / Q000378:metabolism*; D017451:Receptors, Somatomedin; D013002:Somatomedins / Q000378:metabolism*
D046911:Macromolecular Substances; D017451:Receptors, Somatomedin; D013002:Somatomedins; D007334:Insulin-Like Growth Factor I; D011972:Receptor, Insulin; D004229:Dithiothreitol