Interaction of insulin receptor substrate 3 with insulin receptor, insulinreceptor-related receptor, insulin-like growth factor-1 receptor, and downstream signaling proteins

Insulin receptor substrates (IRS) mediate biological actions of insulin, gr owth factors, and cytokines. All four mammalian IRS proteins contain plecks trin homology (PH) and phosphotyrosine binding (PTB) domains at their N ter mini. However, the molecules diverge in their C-terminal sequences. IRS3 is considerably shorter than IRS1, IRS2, and IRS4, and is predicted to intera ct with a distinct group of downstream signaling molecules. In the present study, we investigated interactions of IRS3 with various signaling molecule s. The PTB domain of mIRS3 is necessary and sufficient for binding to the j uxtamembrane NPXpY motif of the insulin receptor in the yeast two-hybrid sy stem. This interaction is stronger if the PH domain or the C-terminal phosp horylation domain is retained in the construct. As determined in a modified yeast two-hybrid system, mIRS3 bound strongly to the p85 subunit of phosph atidylinositol 3-kinase. Although high affinity interaction required the pr esence of at least two of the four YXXM motifs in mIRS3, there was not a re quirement for specific YXXM motifs. mIRS3 also bound to SHP2, Grb2, Nck, an d Shc, but less strongly than to p85. Studies in COS-7 cells demonstrated t hat deletion of either the PH or the PTB domain abolished insulin-stimulate d phosphorylation of mIRS3. Insulin stimulation promoted the association of mIRS3 with p85, SHP2, Nck, and Shc. Despite weak association between mIRS3 and Grb2, this interaction was not increased by insulin, and may not be me diated by the SH2 domain of Grb2. Thus, in contrast to other IRS proteins, mIRS3 appears to have greater specificity for activation of the phosphatidy linositol 3-kinase pathway rather than the Grb2/Ras pathway.