The cysteine-rich domains of the insulin and insulin-like growth factor I receptors are primary determinants of hormone binding specificity. Evidence from receptor chimeras.
To delineate the structural determinants of the insulin receptor (IR) and insulin-like growth factor I receptor (IGFIR) which affect hormone binding specificity we have constructed seven chimeric receptor cDNAs and stably expressed them in Chinese hamster ovary cells. Clonal cell lines expressing high levels of each receptor chimera were analyzed for insulin and insulin-like growth factor I (IGFI) binding activity. Measurements of hormone binding and immunoprecipitation of metabolically labeled receptors showed that all chimeras were properly processed and expressed at the cell surface. The binding data indicate that 56 amino acids of the IR and 52 amino acids of the IGFIR located in corresponding regions of the cysteine-rich domains are the primary determinants of hormone binding specificity. These regions are located between amino acids Asn-230 and Ile-285 on the IR and between His-223 and Met-274 on the IGFIR. In addition, the alpha IR-3 antibody, which competes for IGFI binding, was found to interact with the same 52 amino acids of the IGFIR which determines hormone specificity. Other antibodies which interfere with insulin binding (5D9, MC51, and MA20) interact with epitopes in the COOH-terminal 288 amino acids of the alpha-subunit. We conclude that 56 and 52 amino acids of the cysteine-rich domains of the IR and IGFIR contain the major determinants of hormone binding specificity although other more COOH-terminal regions of both receptors contribute to hormone binding.
Gustafson
T A
TA
Hormone Research Institute, University of California, San Francisco 94143-0534.
Rutter
W J
WJ
D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal; D001483:Base Sequence; D001665:Binding Sites; D003001:Cloning, Molecular; D006224:Cricetinae; D003545:Cysteine; D066298:In Vitro Techniques; D007328:Insulin / Q000378:metabolism*; D008969:Molecular Sequence Data; D009841:Oligonucleotides; D011233:Precipitin Tests; D011972:Receptor, Insulin / Q000737:chemistry / Q000276:immunology / Q000378:metabolism*; D011956:Receptors, Cell Surface / Q000737:chemistry / Q000276:immunology / Q000378:metabolism*; D017451:Receptors, Somatomedin; D011993:Recombinant Fusion Proteins; D013002:Somatomedins / Q000378:metabolism*; D013329:Structure-Activity Relationship
D000911:Antibodies, Monoclonal; D007328:Insulin; D009841:Oligonucleotides; D011956:Receptors, Cell Surface; D017451:Receptors, Somatomedin; D011993:Recombinant Fusion Proteins; D013002:Somatomedins; D011972:Receptor, Insulin; D003545:Cysteine