We previously identified and molecularly cloned a substrate that becomes tyrosine phosphorylated in response to interleukin (IL)-4 and insulin stimulation, designated insulin receptor substrate (IRS)-2, which possesses over 50% homology with IRS-1. While IL-3-dependent 32D cells do not endogenously express IRS molecules, exogenous expression of IRS-1 or IRS-2 restored their capacity to respond mitogenically to IL-4 and insulin. Tyrosine phosphorylation of IRS molecules creates binding sites for multiple SH2-containing proteins that are proposed to be involved in modulating IRS function. Expression of tyrosine to phenylalanine mutants of IRS-1 in 32 cells indicate that IRS-1 contains phosphotyrosine-independent elements that weakly mediate mitogenesis and restoration of tyrosines within phosphotidylinositol-3 kinase binding motifs enhances DNA synthesis. However, additional tyrosines are essential for full mitogenic sensitivity to insulin. We have utilized the yeast two-hybrid system and GST fusion protein interaction assays to demonstrate that two domains of IRS-2 interact directly with the insulin and insulin-like growth factor I (IGF-I) receptors in a phosphotyrosine-dependent manner. One of these regions does not appear to possess SH2 or PTB domains. We conclude that IRS-2 can interact with tyrosine-phosphorylated receptors via independent binding motifs and suggest the existence of a previously unidentified phosphotyrosine-dependent binding domain. The Crk proto-oncogene product is an SH2 and SH3 domain-containing adaptor protein which was previously shown to become tyrosine phosphorylated in response to stimulation with IGF-I in NIH-3T3 cells. In order to further characterize the role of Crk in IGF-I signaling, NIH-3T3 and 293 cells were stably transfected with an expression vector containing crk cDNA. In these cells, IRS-1 and IRS-2 both associated with Crk, and this association was decreased by IGF-I treatment, whereas the association of Grb2 with IRS-1 and 4PS was enhanced by IGF-I. Overexpression of Crk also enhanced IGF-I-induced mitogenesis of NIH-3T3 cells. The levels of IGF-I-induced mitogenesis were proportional to the level of Crk expression. These results suggest that Crk is a positive effector of IGF-I signaling and may mediate its effects via a unique interaction with IRS-1 and/or IRS-2.
