Mast cells upon stimulation through high affinity IgE receptors massively release inflammatory mediators by the fusion of specialized secretory granules (related to lysosomes) with the plasma membrane. Using the RBL-2H3 rat mast cell line, we investigated whether granule secretion involves components of the soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) machinery. Several isoforms of each family of SNARE proteins were expressed. Among those, synaptosome-associated protein of 23 kDa (SNAP23) was central in SNARE complex formation. Within the syntaxin family, syntaxin 4 interacted with SNAP23 and all vesicle-associated membrane proteins (VAMPs) examined, except tetanus neurotoxin insensitive VAMP (TI-VAMP). Overexpression of syntaxin 4, but not of syntaxin 2 nor syntaxin 3, caused inhibition of FcεRI-dependent exocytosis. Four VAMP proteins, i.e., VAMP2, cellubrevin, TI-VAMP, and VAMP8, were present on intracellular membrane structures, with VAMP8 residing mainly on mediator-containing secretory granules. We suggest that syntaxin 4, SNAP23, and VAMP8 may be involved in regulation of mast cell exocytosis. Furthermore, these results are the first demonstration that the nonneuronal VAMP8 isoform, originally localized on early endosomes, is present in a regulated secretory compartment.

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Following activation through high affinity IgE receptors (Fc⑀RI), mast cells release, within a few minutes, their granule content of inflammatory and allergic mediators. Fc⑀RI-induced degranulation is a SNARE (soluble N-ethylmaleimide attachment protein receptors)-dependent fusion process. It is regulated by Rab3D, a subfamily member of Rab GTPases. Evidence exists showing that Rab3 action is calcium-regulated although the molecular mechanisms remain unclear. To obtain an understanding of Rab3D function we have searched for Rab3D-associated effectors that respond to allergic triggering through Fc⑀RI. Using the RBL-2H3 mast cell line we detected a Ser/Thr kinase activity, termed here Rak3D (from Rab3D-associated kinase), because it was specifically co-immunoprecipitated with anti-Rab3D antibody. Rak3D activity, as measured by its auto-or transphosphorylation, was maximal in resting cells and decreased upon stimulation. The down-regulation of the observed activity was blocked with EGTA, but not with other degranulation inhibitors, suggesting that its activity functions downstream of calcium influx. We found that Rak3D phosphorylates the NH 2 -terminal regulatory domain of the t-SNARE syntaxin 4, but not syntaxin 2 or 3. The phosphorylation of syntaxin 4 decreased its binding to its partner SNAP23. Thus, we propose a novel phosphorylation-dependent mechanism by which Rab3D controls SNARE assembly in a calcium-dependent manner.Mast cells are specialized immune cells able to secrete a variety of mediators stored in cytoplasmic granules and involved in inflammatory and allergic responses (1). Upon activation, they can discharge almost their entire granular content by compound exocytosis (2, 3). A potent physiological stimulus for degranulation is the antigen-dependent aggregation of IgE bound to high affinity IgE receptors (Fc⑀RI).1 This activates both membrane-attached and cytoplasmic tyrosine kinases that pave the way for the intracellular calcium increase that is necessary for secretion (4 -6).The accumulated evidence demonstrates that during regulated exocytosis calcium can act directly on the molecular machinery involved in membrane fusion. An essential part of this machinery are SNARE proteins (7). During neurotransmitter release at the synapse, SNAREs assemble into an extremely stable multimeric core complex composed of one v-SNARE 2 (synaptobrevin or vesicular-associated membrane protein ϭ VAMP) and two t-SNAREs 2 (syntaxin 1 and SNAP25) (8 -10). Complex formation is thought to provide the necessary energy to bring together the two opposing membranes and drive bilayer mixing. SNARE complex formation is regulated by additional positive and negative effectors (8, 10). These include small GTPases of the Rab3 family (8,11,12). In mast cells, degranulation also depends on SNARE-mediated fusion but instead utilizes SNAP23, a SNAP25 homolog and syntaxin 4 (13, 14) as well as the calcium sensor synaptotagmin (15). Rab3D also plays an important role in regulating the fusion process in mast cells (16,17) but the mechani...

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