Previously, we reported that the isoprenoid pathway inhibitor, lovastatin, blocks the activation by IgE receptor cross-linking of Ca-45(2+) influx, 1,4,5-inositol trisphosphate production, secretion, and membrane changes (ruffling, spreading) in intact RBL-2H3 rat basophilic leukemia cells. These results indicated that an isoprenoid pathway intermediate, very likely an isoprenylated protein, is importantly involved in the control of IgE receptor-mediated signal transduction. Here, we show that 20 h of pretreatment with lovastatin also inhibits antigen-induced secretion and membrane responses in streptolysin O-(SLO)-permeabilized cells. However, lovastatin does not inhibit secretion stimulated by the nonhydrolyzable GTP analog, GTP-gamma-S. Furthermore, the membrane responses to GTP-gamma-S persist, although in an attenuated form, in lovastatin-treated permeabilized cells. The relative insensitivity of GTP-gamma-S-induced responses to lovastatin was one of several indications that antigen and GTP-gamma-S may activate separate pathways leading to trans-membrane responses in permeabilized cells. Further experiments showed that the beta-thio derivative of GDP, GDPBAS, inhibits the secretory and membrane responses to GTP-gamma-S, as expected for a GTP-binding protein-dependent signaling pathway, while having little effect on antigen-induced responses. Conversely, genistein blocks the secretory and membrane responses to antigen, as expected for a tyrosine kinase-dependent pathway, without altering the GTP-gamma-S-induced responses. From these results, and from additional data from cells treated with tyrphostins and sodium orthovanadate, we propose that IgE receptor-mediated secretion from permeabilized RBL-2H3 cells occurs by a tyrosine kinase-dependent pathway that requires isoprenoid pathway activity for function. We propose further that RBL-2H3 cells contain a separate GTP-binding protein-mediated signaling pathway whose direct activation by GTP-gamma-S is either independent of isoprenoid pathway activity or depends on the activity of an isoprenylated protein that is not significantly depleted after 20 h of lovastatin treatment.
