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 alpha subunit.
We 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 alpha subunit and postulated that sequence 241-251 plays a major role in insulin binding [Yip, C. C., Hsu, H., Patel, R. G., Hawley, D. M., Maddux, B. A., & Goldfine, I.D. (1988) Biochem. Biophys. Res. Commun. 157, 321-329]. In the present study, we 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 our sequence assignment of this fragment. It also immunoprecipitated the intact alpha 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, we found that sequence 241-251 became accessible to the antiserum after removal of insulin. We 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.
Yip
C C
CC
Banting and Best Department of Medical Research, University of Toronto, Ontario, Canada.
Grunfeld
C
C
Goldfine
I D
ID
D000345:Affinity Labels; D000595:Amino Acid Sequence; D006801:Humans; D007328:Insulin / Q000378:metabolism*; D007457:Iodine Radioisotopes; D008024:Ligands; D008969:Molecular Sequence Data; D010920:Placenta / Q000378:metabolism*; D011233:Precipitin Tests; D011487:Protein Conformation; D011489:Protein Denaturation; D011972:Receptor, Insulin / Q000737:chemistry* / Q000187:drug effects / Q000378:metabolism
D000345:Affinity Labels; D007328:Insulin; D007457:Iodine Radioisotopes; D008024:Ligands; D011972:Receptor, Insulin