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. 1997 Apr 15;323(Pt 2):321–328. doi: 10.1042/bj3230321

Rat basophilic leukaemia (RBL) cells overexpressing Rab3a have a reversible block in antigen-stimulated exocytosis.

J Smith 1, N Thompson 1, J Thompson 1, J Armstrong 1, B Hayes 1, A Crofts 1, J Squire 1, C Teahan 1, L Upton 1, R Solari 1
PMCID: PMC1218322  PMID: 9163319

Abstract

The rat basophilic leukaemia (RBL) cell line has been widely used as a convenient model system to study regulated secretion in mast cells. Activation of these cells through the high-affinity receptor for IgE (Fcepsilon-RI) results in degranulation and the extracellular release of mediators. There is good evidence of a role for GTPases in mast cell degranulation, and a number of studies with peptides derived from the Rab3a effector domain have suggested that Rab3a may function in this process. However, in neuroendocrine cells, overexpression of Rab3a can act as a negative regulator of stimulated exocytosis [Holz, Brondyk, Senter, Kuizon and Macara (1994) J. Biol. Chem. 269, 10229-10234; Johanes, Lledo, Roa, Vincent, Henry and Darchen (1994) EMBO J. 13, 2029-2037]. In order to study the function of Rab3a in RBL degranulation, we have generated clones of RBL cells stably expressing Rab3a, and show that in these haematopoietic cells Rab3a can also function as a negative regulator of exocytosis. Overexpression of a mutant form of Rab3a (Asn-135 to Ile), which is predicted to be predominantly GTP-bound, also inhibited degranulation. However, overexpression of a mutant form of Rab3a that was truncated at the C-terminus to remove the sites for geranylgeranylation failed to inhibit degranulation. The effect of Rab3a is specific to secretion, and we observe no effect of Rab3a on receptor-mediated endocytosis. The Rab3a-induced block in degranulation can be bypassed by stimulation of streptolysin-O-permeabilized cells with guanosine 5'-[gamma-thio]triphosphate. We conclude from these studies that Rab3a is implicated in an early stage of granule targeting, whereas fusion of granules with the plasma membrane is regulated by a distinct downstream GTP-binding protein or proteins.

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Selected References

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