Signaling-competent receptor chimeras allow mapping of major insulin receptor binding domain determinants.
Chimeric receptors were generated in which structurally defined subdomains of the insulin receptor (IR) and insulin growth factor-I receptor (IGF-1R) alpha-subunits were exchanged between their respective receptor backbone structures. Upon expression in human fibroblasts, nine IR/IGF-1R chimeras were transported to the cell surface, where they formed binding sites with differential properties. One IGF-1R/IR chimera (C3') exhibited to some extent high insulin specificity, demonstrating the presence of major insulin binding determinants within the amino acid 325-524 region of the IR alpha-subunit. Complementation of this region with subdomain 1 (amino acids 1-137) reconstituted full insulin binding potential within an IGF-1R framework. In addition, both the IGF-1R/IR C3' chimera and another chimera (C13') displayed high affinity binding properties for IGF-1, which suggests distinct locations for major insulin and IGF-1 binding determinants in their respective receptors, in agreement with our previous findings (Schumacher, R., Mosthaf, L., Schlessinger, J., Brandenburg, D., and Ullrich, A. (1991) J. Biol. Chem. 266, 19288-19295). The binding characteristics of all receptor chimeras correlated directly with the ability of the ligands to regulate their tyrosine kinase activity in intact cells. These results demonstrate direct coupling of ligand binding affinity and capacity for tyrosine kinase activation.
Schumacher
R
R
Department of Molecular Biology, Max-Planck-Institut für Biochemie, Martinsried, Germany.
Soos
M A
MA
Schlessinger
J
J
Brandenburg
D
D
Siddle
K
K
Ullrich
A
A
D000906:Antibodies; D000911:Antibodies, Monoclonal; D001483:Base Sequence; D001665:Binding Sites; D002460:Cell Line; D004247:DNA / Q000235:genetics / Q000378:metabolism; D000939:Epitopes / Q000032:analysis; D005347:Fibroblasts / Q000378:metabolism; D006801:Humans; D007328:Insulin / Q000378:metabolism*; D007334:Insulin-Like Growth Factor I / Q000378:metabolism*; D007700:Kinetics; D008024:Ligands; D008969:Molecular Sequence Data; D016296:Mutagenesis; D009838:Oligodeoxyribonucleotides; D017526:Receptor, IGF Type 1 / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism*; D011972:Receptor, Insulin / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000378:metabolism*; D011994:Recombinant Proteins / Q000378:metabolism; D015183:Restriction Mapping; D015398:Signal Transduction; D013462:Sulfur Radioisotopes; D014162:Transfection
D000906:Antibodies; D000911:Antibodies, Monoclonal; D000939:Epitopes; D007328:Insulin; D008024:Ligands; D009838:Oligodeoxyribonucleotides; D011993:Recombinant Fusion Proteins; D011994:Recombinant Proteins; D013462:Sulfur Radioisotopes; D007334:Insulin-Like Growth Factor I; D004247:DNA; D017526:Receptor, IGF Type 1; D011972:Receptor, Insulin