A Pertussis Toxin-sensitive G-protein Mediates Some Aspects of Insulin Action in B&H-l Murine Myocytes*


The involvement of G-proteins in the insulin signal transduction system has been studied in detail using the murine B&H-l 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 (~~185 and ~~42-45). In vitro ADP-ribosylation and immunoblotting studies suggest that the major PT substrate is a 40-kDa protein of the G, 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) (GTPrS) binding to B&H1 membranes was also significantly increased in the presence of insulin. The effects of insulin on GTP hydrolysis and GTPrS binding were found to be dependent on the concentration of insulin. These effects were not detected in plasma membranes prepared from PT-pretreated B&H-l 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 [‘261]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 be-

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@inproceedings{Luttrell2001APT, title={A Pertussis Toxin-sensitive G-protein Mediates Some Aspects of Insulin Action in B&H-l Murine Myocytes*}, author={Louis M. Luttrell and Elaine E Kilgour and Joseph Lamer and Guillermo G{\'a}lvez Romero}, year={2001} }