Ligand-dependent autophosphorylation of the insulin receptor is positively regulated by Gi-proteins Available to Purchase

Previously, we have shown that the human insulin receptor (IR) interacts with G_i2, independent of tyrosine kinase activity and stimulates NADPH oxidase via the Gα subunit of G_i2. We have now investigated the regulatory role of G_i2-proteins in IR function. For the experiments, isolated IRs from plasma membranes of human fat cells were used. The activation of IR autophosphorylation by insulin was blocked by G-protein inactivation through GDPβS (guanosine 5´-[β-thio]disphosphate). Consistently, activation of G-proteins by micromolar concentrations of GTPγS (guanosine 5´-[γ-thio]triphosphate) induced receptor autophosphorylation 5-fold over baseline and increased insulin-induced autophosphorylation by 3-fold. In the presence of 10 µM GTPγS, insulin was active at picomolar concentrations, indicating that insulin acted via its cognate receptor. Pretreatment of the plasma membranes with pertussis toxin prevented insulin- and GTPγS-induced autophosphorylation, but did not disrupt the IR–G_i2 complex. The functional nature of the IR–G_i2 complex was made evident by insulin's ability to increase association of G_i2 with the IR. This leads to an augmentation of maximal receptor autophosphorylation induced by insulin and GTPγS. The specificity of this mechanism was further demonstrated by the use of isolated preactivated G-proteins. Addition of G_i2α and Gβγ mimicked maximal response of insulin, whereas Gαs or Gαo had no stimulatory effect. These results define a novel mechanism by which insulin signalling mediates tyrosine kinase activity and autophosphorylation of the IR through recruitment of G_i-proteins.