Comparison of the insulin and insulin-like growth factor 1 mitogenic intracellular signaling pathways

We compared the intracellular insulin-like growth factor-11 (IGF-1) and insulin signaling pathways in Rat1 fibroblasts expressing the equivalent number of insulin receptors and endogenous IGF-1 receptors. Insulin and IGF-1 stimulated tyrosine phosphorylation of IRS-1 and Shc in a similar dose- and time-dependent manner. The time course of Shc phosphorylation by both IGF-1 and insulin was slower than that of IRS-1. Both phosphorylated IRS-1 and Shc associated with Grb2 . Sos complexes, leading to p2l(ras) activation. To compare the functional importance of p21(ras) for IGF-1- and insulin-induced DNA synthesis, single cell microinjection studies were performed. BrdU incorporation into newly synthesized DNA was measured by immunofluorescence microscopy to assess the functional importance of p21(ras). Both IGF-1 and insulin stimulated BrdU incorporation, but the effect of IGF-1 was greater. Microinjection of anti-p21(ras) antibody completely inhibited both IGF-1- and insulin-induced DNA synthesis, indicating the central role of p21(ras) in signaling by both hormones. Signal transduction from these receptors to Grb2 . Sos complexes can occur through IRS-1 and/or Shc. To assess these two possible pathways, we performed Western blots for Grb2 in anti-Shc and anti-IRS-1 immunoprecipitates and found that 5-fold more Grb2 was associated with Shc than with IRS-1 after either IGF-1 or insulin stimulation. Microinjection of anti-Shc antibody inhibited IGF-1 and insulin stimulation of DNA synthesis by 78% and 74%, respectively. By microinjecting Shc subdomains of GST fusion proteins, we found that Shc N-terminus, but not the Shc SH2, was the functionally important domain through which Shc interacts with IGF-1 and insulin receptors. Insulin stimulation caused hyperphosphorylation and decreased electrophoretic mobility of Sos, and a similar effect was seen with IGF-1, although the time course was delayed compared with insulin. Finally, IGF-1 activated mitogen-activated proten kinase activity more effectively than insulin. These data indicate that Shc, rather than IRS-1, appears to be the predominant functional link to Grb2 . Sos complexes from the IGF-1 receptor, as it is from the insulin receptor. Although IGF-1 and insulin stimulate cell cycle progression with similar coupling mechanisms from the receptor to Shc, to Grb2 . Sos, to p21(ras), the delayed IGF-1 induced, mobility shift of Sos could lead to, at least in part, more efficient coupling to mitogen-activated protein kinase. These findings might explain the greater mitogenic activity of IGF-1 compared with insulin.