Identification of common ligand binding determinants of the insulin and insulin-like growth factor 1 receptors. Insights into mechanisms of ligand binding

• Published in: The Journal of biological chemistry Vol. 272; no. 30; pp. 18650 - 18655
• Main Authors: Mynarcik, D C, Williams, P F, Schaffer, L, Yu, G Q, Whittaker, J
• Format: Journal Article
• Language: English
• Published: United States 25.07.1997
• Subjects: Binding Sites; Cell Line; DNA, Complementary - metabolism; Humans; Insulin - analogs & derivatives; Insulin - metabolism; Insulin-Like Growth Factor I - metabolism; Kinetics; Ligands; Molecular Weight; Mutagenesis, Site-Directed; Protein Binding; Receptor, IGF Type 1 - genetics; Receptor, IGF Type 1 - metabolism; Receptor, Insulin - genetics; Receptor, Insulin - metabolism
• ISSN: 0021-9258
• DOI: 10.1074/jbc.272.30.18650

Insulin and insulin-like growth factor 1 (IGF-1) are peptides that share nearly 50% sequence homology. However, although their cognate receptors also exhibit significant overall sequence homology, the affinity of each peptide for the non-cognate receptor is 2-3 orders of magnitude lower than for the cognate receptor. The molecular basis for this discrimination is unclear, as are the molecular mechanisms underlying ligand binding. We have recently identified a major ligand binding site of the insulin receptor by alanine scannning mutagenesis. These studies revealed that a number of amino acids critical for insulin binding are conserved in the IGF-1 receptor, suggesting that they may play a role in ligand binding. We therefore performed alanine mutagenesis of these amino acids to determine whether this is the case. cDNAs encoding alanine-substituted secreted recombinant IGF-1 receptors were expressed in 293 EBNA cells, and the ligand binding properties of the expressed proteins were evaluated. Mutation of Phe701 resulted in a receptor with undetectable IGF-1 binding; alanine substitution of the corresponding amino acid of the insulin receptor, Phe714, produces a 140-fold reduction in affinity for insulin. Mutation of Asp8, Asn11, Phe58, Phe692, Glu693, His697, and Asn698 produces a 3.5-6-fold reduction in affinity for IGF-1. In contrast, alanine mutation of the corresponding amino acids of the insulin receptor with the exception of Asp12 produces reductions in affinity that are 50-fold or greater. The affinity of insulin for these mutants relative to wild type receptor was similar to that of their relative affinity for IGF-1 with two exceptions; the IC50 values for insulin binding to the mutants of Arg10, which has normal affinity for IGF-1, and His697, which has a 6-fold reduction in affinity for IGF-1, were both at least 2 orders of magnitude greater than for wild type receptor. The Kd values for insulin of the corresponding alanine mutants of the insulin receptor, Arg14 and His710, are 2-3 orders of magnitude greater than for wild type receptor. However, in contrast, the relative affinity of des(25-30)[PheB25 alpha-carboxamide]insulin for these IGF-1 receptor mutants is reduced only 4- and 50-fold, respectively.