title: Alanine-scanning mutagenesis of a C-terminal ligand binding domain of the insulin receptor alpha subunit.
abstract: 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 Kd 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 Kd of native secreted recombinant receptor is 0.56 x 10(-9) M.
authors.affiliation: Department of Medicine, State University of New York, Stony Brook 11794, USA.
authors.forename: D C
authors.initials: DC
authors.lastname: Mynarcik
authors.forename: G Q
authors.initials: GQ
authors.lastname: Yu
authors.forename: J
authors.initials: J
authors.lastname: Whittaker
mesh_terms: D000409:Alanine; D000818:Animals; D018076:DNA, Complementary; D007328:Insulin / Q000378:metabolism*; D016297:Mutagenesis, Site-Directed; D011485:Protein Binding; D011972:Receptor, Insulin / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D013552:Swine
chemical_list: D018076:DNA, Complementary; D007328:Insulin; D011972:Receptor, Insulin; D000409:Alanine