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

Ariella Zussman, Sylvie Hermuet, Ronit Sagi-Eisenberg

Research output: Contribution to journalArticlepeer-review

Abstract

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.

Original languageEnglish
Pages (from-to)144-149
Number of pages6
JournalEuropean Journal of Biochemistry
Volume258
Issue number1
DOIs
StatePublished - 15 Nov 1998

Keywords

  • Arachidonic acid
  • Exocytosis
  • GTP-binding protein
  • Mast cells
  • RBL-2H3

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