Stimulation of Ca²⁺-dependent exocytosis and arachidonic acid release in cultured mast cells (RBL-2H3) by a GTPase-deficient mutant of Gαi₃

Gi₃, 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 Gi₃ provides a sufficient signal for exocytosis [Aridor, M., Rajmilevich, G., Beaven, M. A. and 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 Ca²⁺ and nonhydrolyzable analogs of GTP. In contrast, Ca²⁺-activated exocytosis in intact cells is Gi₃ independent. We show here that overexpression of a GTPase-deficient mutant (Gαi₃Q204L), but not of the wild-type form of Gαi₃, 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 Ca²⁺ 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 Gi₃, is a low-affinity Ca²⁺binding protein that can only be activated during Ca²⁺ ionophore stimulation. Moreover, these results also suggest that the propagation of the Ca²⁺-activated and Gi₃-mediated signaling pathway requires the blocking of Gi₃ GTPase activity. Finally, our results indicate that release of arachidonic acid is at least one of the downstream effectors of Gi₃.