STIMULATION OF CA2-DEPENDENT EXOCYTOSIS AND ARACHIDONIC-ACID RELEASE IN CULTURED MAST-CELLS (RBL-2H3) BY A GTPASE-DEFICIENT MUTANT OF G-ALPHA-I(3)()

Gi,, a member of the Gi family of heterotrimeric GTP-binding proteins, regulates vesicle trafficking along both the constitutive and regulat ed pathways. In mast cells, specialized secretory cells which secrete a variety of inflammatory mediators by regulated exocytosis, activatio n of Gi(3) provides a sufficient signal for exocytosis [Aridor, M., Ra jmilevich, G., Beaven, M. A. & Sagi-Eisenberg, R. (1993) Science 262, 1569-1572]. Such activation can be achieved in patch-clamped or strept olysin-O (SLO)-permeabilized mast cells by a combination of Ca2+ and n onhydrolyzable analogs of GTP. In contrast, Ca2+-activated exocytosis in intact cells is Gi(3) independent, We show here that overexpression of a GTPase-deficient mutant (G alpha i(3)Q204L), but not of the wild -type form of G alpha i(3), in rat basophilic leukemia cells (RBL-2H3) , a tumor analog of mucosal mast cells, resulted in marked potentiatio n of exocytosis and release of arachidonic acid in intact cells activa ted by a Ca2+ ionophore alone or in combination with the phorbol ester 12-O-tetradecanoylphorbol-13-acetate. In contrast, exocytosis and ara chidonic acid release stimulated by aggregation of the cell surface re ceptors for immunoglobulin E (IgE) were unaffected. These results stro ngly suggest that the intracellular receptor, responsible for the acti vation of Gi(3), is a low-affinity Ca2+-binding protein that can only be activated during Ca2+ ionophore stimulation. Moreover, these result s also suggest that the propagation of the Ca2+ activated and Gi(3)-me diated signaling pathway requires the blocking of Gi(3) GTPase activit y. Finally, our results indicate that release of arachidonic acid is a t least one of the downstream effecters of Gi(3).