A pertussis toxin-sensitive G-protein mediates some aspects of insulin action in BC3H-1 murine myocytes.
The involvement of G-proteins in the insulin signal transduction system has been studied in detail using the murine BC3H-1 myocyte system. Pertussis toxin (PT) treatment, previously shown to attenuate some of the metabolic effects of insulin in this cell line (Luttrell, L.M., Hewlett, E.L., Romero, G., and Rogol, A.D. (1988) J. Biol. Chem. 263, 6134-6141), abolished insulin-induced generation of diacylglycerol and inositolglycan mediators with no effects on either the autophosphorylation of the insulin receptor or the phosphorylation of the major endogenous substrates for insulin-stimulated tyrosine kinase activity (pp185 and pp42-45). In vitro ADP-ribosylation and immunoblotting studies suggest that the major PT substrate is a 40-kDa protein of the G alpha family. This protein band did not exhibit detectable tyrosine phosphorylation upon stimulation of either intact cells or cell membranes with insulin. In the presence of low concentrations of GTP, insulin treatment of isolated myocyte plasma membranes resulted in a small (30-40%) but significant stimulation of GTP hydrolysis. This effect was best observed in the presence of small concentrations of sodium dodecyl sulfate. The rate of guanosine 5'-O-(3-thiotriphosphate) (GTP gamma S) binding to BC3H-1 membranes was also significantly increased in the presence of insulin. The effects of insulin on GTP hydrolysis and GTP gamma S binding were found to be dependent on the concentration of insulin. These effects were not detected in plasma membranes prepared from PT-pretreated BC3H-1 myocytes. In contrast, pretreatment with the B (inactive) subunit of PT did not alter the response of myocyte membranes to insulin. High affinity binding of [125I]iodoinsulin to myocyte plasma membranes was reduced by 60-70% in the presence of guanine nucleotides. Similar effects on insulin binding were produced by PT pretreatment of the cells. In contrast, adenine nucleotides had no effect on insulin binding. Scatchard analysis of the binding data showed that the observed effects of guanine nucleotides and PT on insulin binding resulted either from a reduction in the number of high affinity insulin binding sites or from a significant reduction of the affinity of insulin for its receptor. Low affinity binding sites did not appear to be affected by either guanine nucleotides nor PT pretreatment. These results provide substantial evidence suggestive of a noncovalent interaction between the insulin receptor and a regulatory G-protein system during the process of insulin signaling.
Luttrell
L
L
Department of Pharmacology, University of Virginia Health Sciences Center, Charlottesville 22908.
Kilgour
E
E
Larner
J
J
Romero
G
G
D000818:Animals; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism; D019204:GTP-Binding Proteins / Q000502:physiology*; D016244:Guanosine 5'-O-(3-Thiotriphosphate) / Q000378:metabolism; D006160:Guanosine Triphosphate / Q000378:metabolism; D007328:Insulin / Q000494:pharmacology*; D007545:Isoproterenol / Q000494:pharmacology; D007700:Kinetics; D046911:Macromolecular Substances; D051379:Mice; D009132:Muscles / Q000187:drug effects / Q000378:metabolism*; D037342:Pertussis Toxin; D010750:Phosphoproteins / Q000302:isolation & purification; D010766:Phosphorylation; D019000:Phosphotyrosine; D011972:Receptor, Insulin / Q000302:isolation & purification / Q000378:metabolism; D015398:Signal Transduction; D014443:Tyrosine / Q000031:analogs & derivatives / Q000302:isolation & purification; D010566:Virulence Factors, Bordetella / Q000494:pharmacology*
D007328:Insulin; D046911:Macromolecular Substances; D010750:Phosphoproteins; D010566:Virulence Factors, Bordetella; D019000:Phosphotyrosine; D016244:Guanosine 5'-O-(3-Thiotriphosphate); D014443:Tyrosine; D006160:Guanosine Triphosphate; D037342:Pertussis Toxin; D011972:Receptor, Insulin; D019204:GTP-Binding Proteins; D007545:Isoproterenol