Identification and characterization of the ligand-binding domain of insulin receptor by use of an anti-peptide antiserum against amino acid sequence 241-251 of the α subunit

The authors previously reported that a 23-kDa receptor proteolytic fragment containing an insulin-binding site was localized within residues 205-316 in the cysteine-rich region of the insulin receptor α subunit and postulated that sequence 241-251 plays a major role in insulin binding. In the present study, they have used an antiserum raised against a synthetic peptide containing sequence 241-251 to test this postulate and to study the role of sequence 241-251 in insulin binding. The antiserum immunoprecipitated the 23-kDa fragment, confirming their sequence assignment of this fragment. It also immunoprecipitated the intact α subunit of the insulin receptor that had been denatured by reduction and alkylation. However, sequence 241-251 in the native receptor was inaccessible to the antiserum since the antiserum did not block [125I]iodoinsulin binding and did not precipitate either photoaffinity-labeled insulin receptors or insulin receptors labeled with 125I. However, using a radioactive photoaffinity probe ([125I]-AZAP-insulin) that allows cleavage and removal of insulin after photolabeling. They found that sequence 241-251 became accessible to the antiserum after removal of insulin. They conclude therefore that sequence 241-251 forms part of the insulin-binding domain of the insulin receptor and that the binding of insulin to the receptor induces a conformational change that allows exposure of this domain after removal of insulin. Such a conformational change may play a role in activation of the receptor and transmembrane signaling