Insulin receptor substrate 3 (IRS-3) and IRS-4 impair IRS-1- and IRS-2-mediated signaling

To investigate the roles of insulin receptor substrate 3 (IRS-3) and IRS-4 in the insulin-like growth factor 1 (IGF-1) signaling cascade, we introduce d these proteins into 3T3 embryonic fibroblast cell lines prepared from wil d-type (WT) and IRS-1 knockout (KO) mice by using a retroviral system, Foll owing transduction of IRS-3 or IRS-4, the cells showed a significant decrea se in IRS-2 mRNA and protein levels without any change in the IRS-1 protein level. In these cell lines, IGF-1 caused the rapid tyrosine phosphorylatio n of all four IRS proteins. However, IRS-3- or IRS-4-expressing cells also showed a marked decrease in IRS-1 and IRS-2 phosphorylation compared to the host cells, This decrease was accounted for in part by a decrease in the l evel of IRS-2 protein but occurred with no significant change in the IRS-1 protein level. IRS-3- or IRS-4-overexpressing cells showed an increase in b asal phosphatidylinositol 3-kinase activity and basal Akt phosphorylation, while the IGF-1-stimulated levels correlated well with total tyrosine phosp horylation level of all IRS proteins in each cell line. IRS-3 expression in WT cells also caused an increase in IGF-1-induced mitogen-activated protei n kinase phosphorylation and egr-1 expression (similar to1.8- and similar t o2.4-fold with respect to WT). In the IRS-1 KO cells, the impaired mitogeni c response to IGF-1 was reconstituted with IRS-1 to supranormal levels and was returned to almost normal by IRS-2 or IRS-3 but was not improved by ove rexpression of IRS-4. These data suggest that IRS-3 and IRS-4 may act as ne gative regulators of the IGF-1 signaling pathway by suppressing the functio n of other IRS proteins at several steps.