Analog Binding Properties of Insulin Receptor Mutants (original) (raw)

1997, Journal of Biological Chemistry

Recent studies utilizing alanine scanning mutagenesis have identified a major ligand binding domain of the secreted recombinant insulin receptor composed of two subdomains, one between amino acids 1 and 120 and the other between amino acids 704 and 716. In order to obtain a more detailed characterization of these subdomains, we examined the binding of an insulin superanalog, des-(B25-30)-[His-A8, Asp-B10, Tyr-B25 ␣-carboxamide]insulin, to alanine mutants of the ligand binding determinants of these subdomains. cDNAs encoding mutant secreted recombinant receptors were transiently expressed in 293 EBNA cells, and the binding properties for this analog of the expressed receptors were evaluated. In general des-(B25-30)-[His-A8, Asp-B10, Tyr-B25 ␣-carboxamide]insulin binding correlated with insulin binding, suggesting that both peptides bound to the receptor in a similar manner. Alanine mutations of eight amino acids (Asn 15 , Phe 64 , Phe 705 , Glu 706 , Tyr 708 , Leu 709 , Asn 711 , and Phe 714) of the receptor produced the most profound decreases in affinity for des-(B25-30)-[His-A8, Asp-B10, Tyr-B25 ␣-carboxamide]insulin, suggesting that interactions with these amino acids contributed the major part of the free energy of the ligand-receptor interaction. Mutation of Arg 14 and His 710 to Ala produced receptors with undetectable insulin binding but an affinity for des-(B25-30)-[His-A8, Asp-B10, Tyr-B25 ␣-carboxamide]insulin only 8-23-fold less than for native receptor. Further analog studies were performed to elucidate this paradox. The receptor binding potencies of His-A8 and Asp-B10 insulins for these receptor mutants appeared to parallel their relative potencies for native receptor. In contrast the receptor binding potency of des-(B25-30)-[Tyr-B25 ␣-carboxamide]insulin was disproportionately increased for these mutants when compared with its potency for native receptor.