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

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 scanning 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 Phe(701) resulted in a receptor with undetectable IGF-1 binding; alanine substitution of the corresponding amino acid of the insulin receptor, Phe(714), produces a 140-fold reduction in affinity for insulin, Mutation of Asp(8), Asn(11), Phe(58), Phe(692), Glu(693), His(697), and Asn(698) 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 Asp(12) 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 Arg(10), which has normal affinity for IGF-1, and His(697), 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 K-d values for insulin of the corresponding alanine mutants of the insulin receptor, Arg(14) and His(710), are 2-3 orders of magnitude greater than for wild type receptor. However, in contrast, the relative affinity of des(25-30)[Phe(B25) alpha-carboxamide]insulin for these IGF-1 receptor mutants is reduced only 4- and 50-fold, respectively.