A COMPLEX OF GRB2 DYNAMIN BINDS TO TYROSINE-PHOSPHORYLATED INSULIN-RECEPTOR SUBSTRATE-1 AFTER INSULIN-TREATMENT

Insulin drives the formation of a complex between tyrosine-phosphoryla ted IRS-1 and SH2-containing proteins. The SH2-containing protein Grb2 also possesses adjacent SH3 domains, which bind the Ras guanine nucle otide exchange factor Sos. In this report, we examined the involvement of another SH3 binding protein, dynamin, in insulin signal transducti on. SH3 domains of Grb2 as GST fusion proteins bound dynamin from lysa tes of CHO cells expressing wild-type insulin receptor (IR) (CHO-IR ce lls) in a cell-free system (in vitro). Immunoprecipitation studies usi ng specific antibodies against Grb2 revealed that Grb2 was co-immunopr ecipitated with dynamin from unstimulated CHO-IR cells. After insulin treatment of CHO-IR cells, anti-dynamin antibodies co-immunoprecipitat ed the IR beta-subunit and IRS-1, as tyrosine-phosphorylated proteins and PI 3-kinase activity. However, purified rat brain dynamin did not bind directly to either the IR, IRS-1 or the p85 subunit of PI 3-kinas e in vitro. Together, these results suggest that in CHO-IR cells, insu lin stimulates the binding of dynamin to tyrosine-phosphorylated IRS-1 via Grb2 and that IRS-1 also associates with PI 3-kinase in response to insulin. This complex formation was reconstituted in vitro using re combinant baculovirus-expressed IRS-1, GST-Grb2 fusion proteins and dy namin peptides containing proline-rich sequences. Furthermore, dynamin GTPase activity was found to be stimulated when an IRS-1-derived phos phopeptide, containing the Grb2 binding site, was added to the dynamin -Grb2 complex in vitro. These findings provide evidence that dynamin i s complexed with Grb2 in CHO-IR cells and, after insulin stimulation, the IFS-1 molecule is able to bind this Grb2-dynamin complex and may r egulate dynamin GTPase activity in the complex in intact cells (in viv o).