Localization of the insulin-binding site to the cysteine-rich region of the insulin receptor α-subunit

Affinity-purified insulin receptor was photoaffinity labeled with a cleavable radioactive insulin photoprobe. Exhaustive digestion of the labeled α-subunit with endoproteinase Glu-C produced a major radioactive fragment of 23 kDa as a part of the putative insulin-binding domain. This fragment could contain either residues 205–316 or 518–633 of the α-subunit. Rat hepatoma cells and Chinese hamster ovary cells were transfected with cDNA encoding a human insulin receptor mutant with a deletion of the cysteine-rich region spanning amino acid residues 124–319. Insulin binding by these cells was not increased in spite of high numbers of the mutant insulin receptors being expressed. A panel of monoclonal antibodies which was specific for the receptor α-subunit and inhibited insulin binding immunoprecipitated the photolabeled 23-kDa receptor fragment but not the receptor mutant. A synthetic peptide containing residues 243–251 was specifically bound by agarose-insulin beads. We therefore suggest that the 23-kDa fragment contains residues 205–316, and that insulin binding occurs, in part, in the cysteine-rich region of the α-subunit.