Activation of Cdc42 is necessary for sustained oscillations of Ca and PIP2 stimulated by antigen in RBL mast cells

Antigen stimulation of mast cells via FceRI, the high-affinity receptor for IgE, triggers a signaling cascade that requires Ca mobilization for exocytosis of secretory granules during the allergic response. To characterize the role of Rho GTPases in FceRI signaling, we utilized a mutant RBL cell line, B6A4C1, that is deficient in antigen-stimulated Cdc42 activation important for these processes. Recently the importance of stimulated intracellular oscillations has emerged, and we find that B6A4C1 cells exhibit severely attenuated Ca oscillations in response to antigen, which are restored to wild-type RBL-2H3 levels by expression of constitutively active Cdc42 G12V or by a GEF for Cdc42, DOCK7, but not when the C-terminal di-arginine motif of active Cdc42 is mutated to di-glutamine. We found that antigen-stimulated FceRI endocytosis, which occurs independently of Ca mobilization, is also defective in B6A4C1 cells, and Cdc42 G12V reconstitutes this response as well. Thus, activation of Cdc42 occurs prior to and is critical for antigen-stimulated pathways leading separately to both Ca mobilization and receptor endocytosis. Accounting for these downstream functional consequences, we show that Cdc42 G12V reconstitutes antigen-stimulated oscillations of phosphatidylinositol 4,5-bisphosphate (PIP2) at the plasma membrane in mutant B6A4C1 cells, pointing to Cdc42 participation in the regulation of stimulated PIP2 synthesis.