Ligand-dependent autophosphorylation of the insulin receptor is positively regulated by Gi-proteins.
Previously, we have shown that the human insulin receptor (IR) interacts with G(i)2, independent of tyrosine kinase activity and stimulates NADPH oxidase via the Galpha subunit of G(i)2. We have now investigated the regulatory role of G(i)2-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 GDPbetaS (guanosine 5'-[beta-thio]disphosphate). Consistently, activation of G-proteins by micromolar concentrations of GTPgammaS (guanosine 5'-[gamma-thio]triphosphate) induced receptor autophosphorylation 5-fold over baseline and increased insulin-induced autophosphorylation by 3-fold. In the presence of 10 microM GTPgammaS, 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 GTPgammaS-induced autophosphorylation, but did not disrupt the IR-G(i)2 complex. The functional nature of the IR-G(i)2 complex was made evident by insulin's ability to increase association of G(i)2 with the IR. This leads to an augmentation of maximal receptor autophosphorylation induced by insulin and GTPgammaS. The specificity of this mechanism was further demonstrated by the use of isolated preactivated G-proteins. Addition of G(i)2alpha and Gbetagamma mimicked maximal response of insulin, whereas Galphas or Galphao 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.
Kreuzer
J
J
Innere Medizin III, Universität Heidelberg, Bergheimer Str. 58, D-69115, Heidelberg, Germany.
Nürnberg
B
B
Krieger-Brauer
H I
HI
D017667:Adipocytes / Q000737:chemistry / Q000201:enzymology; D019206:GTP-Binding Protein alpha Subunits, Gi-Go / Q000378:metabolism / Q000502:physiology*; D044387:GTP-Binding Protein beta Subunits / Q000378:metabolism; D044388:GTP-Binding Protein gamma Subunits / Q000378:metabolism; D006150:Guanine Nucleotides / Q000494:pharmacology; D006801:Humans; D008024:Ligands; D037342:Pertussis Toxin / Q000494:pharmacology; D010766:Phosphorylation / Q000187:drug effects; D020794:Receptor Protein-Tyrosine Kinases / Q000378:metabolism; D011972:Receptor, Insulin / Q000378:metabolism*; D011994:Recombinant Proteins / Q000378:metabolism; D015398:Signal Transduction / Q000187:drug effects / Q000502:physiology
D044387:GTP-Binding Protein beta Subunits; D044388:GTP-Binding Protein gamma Subunits; D006150:Guanine Nucleotides; D008024:Ligands; D011994:Recombinant Proteins; D037342:Pertussis Toxin; D020794:Receptor Protein-Tyrosine Kinases; D011972:Receptor, Insulin; D019206:GTP-Binding Protein alpha Subunits, Gi-Go