Functional characterization of hybrid receptors composed of a truncated insulin receptor and wild type insulin-like growth factor 1 or insulin receptors.
To assess the characteristics of hybrid receptors composed of one kinase-inactive alpha beta-insulin half-receptor and one endogenous alpha beta-insulin-like growth factor 1 (IGF-1) or insulin half-receptor, a cell line expressing an insulin receptor truncated by 365 amino acids (HIR delta 978) was studied, which lacks most of the cytoplasmic beta-subunit. Analysis by sodium dodecyl sulfate polyacrylamide gel electrophoresis under nonreducing conditions revealed four distinct receptor species: endogenous receptors, the more rapidly migrating HIR delta 978 homodimer, and two intermediate species representing HIR delta 978/IGF-1 hybrid receptors and HIR delta 978/IR hybrid receptors. In vivo ligand-binding affinity of the hybrid receptors was studied by receptor-ligand cross-linking, and the delta 978/IGF-1R hybrid receptor was found to have a high affinity for IGF-1, whereas its affinity for insulin was low. Autophosphorylation studies of lectin-purified receptors revealed that neither the HIR delta 978 holoreceptor nor the hybrid receptors underwent autophosphorylation in response to either ligand, despite the presence of intact IGF-1 or insulin half-receptors in the hybrids. Neither hybrid receptor underwent ligand-induced endocytosis, as assessed with the bioactive photoaffinity probes B2(2-nitro-4-azidophenylacetyl)-des-PheB1-insulin and N-epsilon B28-monoazidobenzoyl-IGF-1. In conclusion, the HIR delta 978/IGF-1R hybrid receptor has a high in vivo affinity for IGF-1 but not for insulin. Neither the delta 978/IGF-1R nor the delta 978/IR hybrids undergo autophosphorylation or ligand-induced endocytosis in response to either ligand, indicating that intramolecular trans-, rather than cis-, signal transduction is important in mediating autophosphorylation and endocytosis.
Langlois
W J
WJ
Department of Medicine, University of California, San Diego, La Jolla 92093, USA.
Sasaoka
T
T
Yip
C C
CC
Olefsky
J M
JM
D000818:Animals; D001667:Binding, Competitive; D006801:Humans; D007328:Insulin / Q000378:metabolism; D007334:Insulin-Like Growth Factor I / Q000378:metabolism; D007457:Iodine Radioisotopes; D007700:Kinetics; D055503:Protein Multimerization; D011869:Radioligand Assay; D051381:Rats; D017526:Receptor, IGF Type 1 / Q000096:biosynthesis / Q000302:isolation & purification / Q000378:metabolism*; D011972:Receptor, Insulin / Q000096:biosynthesis / Q000302:isolation & purification / Q000378:metabolism*; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000378:metabolism; D014162:Transfection
D007328:Insulin; D007457:Iodine Radioisotopes; D011994:Recombinant Proteins; D007334:Insulin-Like Growth Factor I; D017526:Receptor, IGF Type 1; D011972:Receptor, Insulin