Stimulation of Ca 2+ ‐dependent exocytosis and arachidonic acid release in cultured mast cells (RBL‐2H3) by a GTPase‐deficient mutant of Gαi 3

Gi 3 , a member of the Gi family of heterotrimeric GTP‐binding proteins, regulates vesicle trafficking along both the constitutive and regulated pathways. In mast cells, specialized secretory cells which secrete a variety of inflammatory mediators by regulated exocytosis, activation of Gi 3 provides a sufficient signal for exocytosis [Aridor, M., Rajmilevich, G., Beaven, M. A. & Sagi‐Eisenberg, R. (1993) Science 262 , 1569−1572]. Such activation can be achieved in patch‐clamped or streptolysin‐O (SLO)‐permeabilized mast cells by a combination of Ca 2+ and nonhydrolyzable analogs of GTP. In contrast, Ca 2+ ‐activated exocytosis in intact cells is Gi 3 independent. We show here that overexpression of a GTPase‐deficient mutant (Gαi 3 Q204L), but not of the wild‐type form of Gαi 3 , in rat basophilic leukemia cells (RBL‐2H3), a tumor analog of mucosal mast cells, resulted in marked potentiation of exocytosis and release of arachidonic acid in intact cells activated by a Ca 2+ ionophore alone or in combination with the phorbol ester 12‐ O ‐tetradecanoylphorbol‐13‐acetate. In contrast, exocytosis and arachidonic acid release stimulated by aggregation of the cell surface receptors for immunoglobulin E (IgE) were unaffected. These results strongly suggest that the intracellular receptor, responsible for the activation of Gi 3 , is a low‐affinity Ca 2+ ‐binding protein that can only be activated during Ca 2+ ionophore stimulation. Moreover, these results also suggest that the propagation of the Ca 2+ ‐activated and Gi 3 ‐mediated signaling pathway requires the blocking of Gi 3 GTPase activity. Finally, our results indicate that release of arachidonic acid is at least one of the downstream effectors of Gi 3 .