Alanine-scanning mutagenesis of a C-terminal ligand binding domain of the insulin receptor alpha subunit

A recent affinity labeling study has suggested that amino acids 704-717 of the C terminus of the insulin receptor represent a contact site for insulin. To determine whether these amino acids are part of a ligand binding site, we have performed alanine-scanning mutagenesis of this region, Mutant cDNAs encoding recombinant secreted receptors were transiently expressed in 293 EBNA cells, and their insulin binding properties were evaluated, Of the 14 residues in this region only 4 amino acids, Asp-707, Val-712, Pro-716, and Arg-717, could be mutated to alanine without compromising insulin binding, The reduction in affinity resulting from the individual mutation of the remaining amino acids varied from an increase in K-d to 3.69 x 10(-9) M (Asn-711) to greater than 10(-6) M (Thr-704, Phe-705, Glu-706, and His-710); the K-d of native secreted recombinant receptor is 0.56 x 10(-9) M.