THE ADAPTER PROTEIN GRB10 ASSOCIATES PREFERENTIALLY WITH THE INSULIN-RECEPTOR AS COMPARED WITH THE IGF-I RECEPTOR IN MOUSE FIBROBLASTS

To identify receptor-associated proteins that may contribute to the sp ecificity of insulin and IGF-I signaling responses, a mouse embryo lib rary was screened using the yeast two-hybrid system. Multiple receptor -interactive clones encoding the SH2 domain of the adapter protein Grb 10 were isolated, Subsequent cloning of the full-length Grb10 sequence from a mouse fat cDNA library defined a previously unknown Grb10 vari ant, that appears to be the predominant isoform in mouse tissues, Rece ptor-deficient R- cells (fibroblasts from mice with homologous disrupt ion of the IGF-I receptor gene) and transfected R- cells expressing ei ther insulin receptors (R-IR cells) or IGF-I receptors (R+ cells) were used to investigate the specificity of Grb10 interaction with the two related receptors. Hormone-activated insulin receptors in R-IR cells coprecipitated with three species, all recognized as Grb 10 isoforms b y specific Grb10 antibody, Under the same conditions, Grb10 was essent ially undetectable in IGF-I receptor immunoprecipitates from stimulate d R+ cells, Grb10 association with insulin receptors was maximal at 10 nM insulin stimulation and sustained from 5-10 min after hormone stim ulation in R-IR cells, In conclusion, Grb10 interacts preferentially w ith insulin vs. IGF-I receptors in intact cells and, thus, may have a role in mediating insulin receptor-specific cellular responses.