Insulin receptor substrates form high-molecular-mass complexes that modulate their availability to insulin/insulin-like growth factor-I receptor tyrosine kinases

► We examine protein complexes containing insulin receptor substrates (IRSs). ► IRS-1 and IRS-2 form high-molecular-mass complexes with other proteins. ► Different complexes are formed depending on IRS-isoform, cell-type or stimulus. ► The complexes can modulate availability of IRSs to receptor tyrosine kinases. Insulin receptor substrates (IRSs) are phosphorylated by activated insulin/insulin-like growth factor (IGF)-I receptor tyrosine kinases. Phosphotyrosyl IRSs are recognized by signaling molecules possessing src homology region 2 (SH2) domains, which mediate various insulin/IGF bioactivities. However, we have shown that IRSs are also associated with other proteins by a phosphotyrosine-independent mechanism. Here, we demonstrated that IRSs form high-molecular-mass complexes (we named these complexes IRSomes) with various proteins and we elucidated their possible roles. Blue native-polyacrylamide gel electrophoresis of cell lysates revealed IRSome formation. Some proteins associated with IRSs in IRS-isoform-, cell-type-, or stimulus-specific manners. Results of the in vitro tyrosine phosphorylation assay indicated that tyrosine phosphorylation of IRS-1 by insulin receptor was decreased when IRS-1 was contained in IRSomes prepared from 3T3-L1 adipocytes treated with TNF-α. Also, tyrosine phosphorylation of IRS-2 by IGF-I receptor was increased when IRS-2 was contained in IRSomes prepared from FRTL-5 thyrocytes treated with dibutyryl cAMP. These results demonstrated that cytokine/hormone-induced formation of IRSomes modulates availability of IRSs to receptor tyrosine kinases.