Relationship between alpha subunit ligand occupancy and beta subunit autophosphorylation in insulin/insulin-like growth factor-1 hybrid receptors.
Insulin receptor beta subunit autophosphorylation occurs in an intramolecular trans-reaction in which one beta subunit phosphorylates the adjacent beta subunit within an alpha 2 beta 2 holoreceptor complex (Frattali, A. L., Treadway, J. L., and Pessin, J. E. (1992) J. Biol. Chem. 267, 19521-19528). To determine the spatial relationship between alpha subunit occupancy and beta subunit autophosphorylation, the vaccinia virus/bacteriophage T7 transient expression system was used to generate insulin/insulin-like growth factor (IGF)-1 hybrid receptors. The extent of hybrid receptor formation was proportional to the molar ratio of the insulin and IGF-1 receptor expression plasmids used for transfection of cultured fibroblasts. Insulin/IGF-1 hybrid receptors displayed high affinity binding for insulin and IGF-1 similar to that observed for homotypic insulin and IGF-1 receptors, respectively. As expected, insulin poorly competed for 125I-IGF-1 binding to the insulin/IGF-1 hybrid receptors compared with IGF-1. IGF-1, however, competed more efficiently than insulin for 125I-insulin binding, indicating interactions between the alpha subunit binding sites. Furthermore, insulin or IGF-1 stimulated the autophosphorylation of both beta subunits within wild type insulin/IGF-1 hybrid receptors. Ligand-stimulated autophosphorylation of two different mutant/wild type insulin/IGF-1 hybrid receptors also resulted in the labeling of each beta subunit independent of which alpha subunit was occupied with ligand. These data demonstrate that insulin/IGF-1 hybrid receptors bind both ligands with high affinity and that occupancy of either alpha subunit results in a series of intramolecular trans-autophosphorylation reactions between beta subunits.
Frattali
A L
AL
Department of Physiology and Biophysics, University of Iowa, Iowa City 52242.
Pessin
J E
JE
D000818:Animals; D001483:Base Sequence; D002460:Cell Line; D003001:Cloning, Molecular; D006224:Cricetinae; D004247:DNA; D004591:Electrophoresis, Polyacrylamide Gel; D006367:HeLa Cells; D006801:Humans; D007328:Insulin / Q000378:metabolism*; D007334:Insulin-Like Growth Factor I / Q000378:metabolism*; D008969:Molecular Sequence Data; D010766:Phosphorylation; D017526:Receptor, IGF Type 1 / Q000235:genetics / Q000378:metabolism*; D011972:Receptor, Insulin / Q000235:genetics / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism
D007328:Insulin; D011993:Recombinant Fusion Proteins; D007334:Insulin-Like Growth Factor I; D004247:DNA; D017526:Receptor, IGF Type 1; D011972:Receptor, Insulin