Mapping of an NH2-terminal ligand binding site of the insulin receptor by alanine scanning mutagenesis.
Affinity labeling studies and mutational analyses have implicated the involvement of a predicted domain of the insulin receptor (L1, amino acids 1-119) in ligand binding. In order to obtain a higher resolution localization of this ligand binding site, we have performed alanine scanning mutagenesis of this domain. Alanine mutant cDNAs encoding a secreted recombinant insulin receptor extracellular domain were expressed transiently in adenovirus transformed human embryonic kidney cells and the affinity of the expressed receptor for insulin was determined. Mutation of 14 amino acids located in four discontinuous peptide segments to alanine was disruptive of insulin binding: Segment 1, amino acids 12-15; Segment 2, amino acids 34-44; Segment 3, amino acids 64-67; and Segment 4, amino acids 89-91. The quantitative contribution of the four segments to the free energy of insulin binding was 1 > 3 > 2 > 4. Of the 14 amino acids whose mutation compromised insulin binding, 3 are charged, 3 hydrophobic, 5 aromatic, and 3 are amides.
Department of Medicine, State University of New York, Stony Brook 11794.
P F
PF
Williams
D C
DC
Mynarcik
G Q
GQ
Yu
J
J
Whittaker
D000256:Adenoviridae; D000409:Alanine; D000595:Amino Acid Sequence; D001665:Binding Sites; D002461:Cell Line, Transformed; D006801:Humans; D007328:Insulin / Q000378:metabolism*; D007457:Iodine Radioisotopes; D007668:Kidney; D008024:Ligands; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D017354:Point Mutation; D011869:Radioligand Assay; D011972:Receptor, Insulin / Q000096:biosynthesis / Q000737:chemistry* / Q000378:metabolism*; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000378:metabolism
D007328:Insulin; D007457:Iodine Radioisotopes; D008024:Ligands; D011994:Recombinant Proteins; D011972:Receptor, Insulin; D000409:Alanine