INSULIN AND INTERLEUKIN-4 INDUCE DESENSITIZATION TO THE MITOGENIC EFFECTS OF INSULIN-LIKE GROWTH-FACTOR-I - PIVOTAL ROLE FOR INSULIN-RECEPTOR SUBSTRATE-2

Insulin-induced desensitization to insulin-like growth factor-I (IGF-I ) stimulated mitogenesis in bovine fibroblasts involves steps distal t o IGF-I binding to its tyrosine kinase receptor, When quiescent cultur es of bovine fibroblasts were stimulated with 10 nM IGF-I and total ce ll lysates immunoblotted with anti-phosphotyrosine antibody, we observ ed a band at similar to 97 kDa, representing the beta-subunit of the I GF-I receptor, and a predominant tyrosyl-phosphorylated species migrat ing as a broad band between 170 and 190 kDa. The majority of proteins in this latter band were immunoprecipitated by antibodies against insu lin receptor substrate (IRS)-2 and not by antibodies against IRS-1. Pr etreatment of bovine fibroblasts with 10 nM insulin for 48 h blocked s ubsequent IGF-I-stimulated DNA synthesis and the IGF-I-induced increas e in tyrosyl-phosphorylated IRS-2. Insulin pretreatment did not alter IRS-1 or IRS-2 expression by these cells, as assessed by metabolic lab eling and direct immunoblotting with IRS antibodies. The interleukin-4 (IL-4) cytokine receptor also has IRS-2 as its major substrate for ty rosine phosphorylation. Although 10 nM IL-4 was as effective as 10 nM IGF-I in stimulating IRS-2 phosphorylation, 10 nm IL-4 did not have co mparable mitogenic power in these cells. Nonetheless, pretreatment of bovine fibroblasts with IL-4 inhibited IGF-I-stimulated DNA synthesis by 50-60%, concomitant with a decrease in IGF-I-induced IRS-2 phosphor ylation. Insulin-induced desensitization could be prevented if a speci fic inhibitor of phosphatidylinositol 3-kinase (LY294002), but not an inhibitor of mitogen-activated protein kinase (PD98059), was present d uring the preincubation period. LY294002 also prevented the shift in I RS-2 molecular mass in response to prolonged incubation of cells with insulin. These data indicate that, in a nontransformed cell system, IR S-2 plays a key role in cellular desensitization to IGF-I-stimulated m itogenesis most likely through a feedback mechanism in the phosphatidy linositol 3-kinase pathway. Furthermore, they suggest that signaling t hrough IRS-2 may provide an important point of integration for hormone , growth factor, and cytokine receptor systems that regulate critical cellular growth responses.