Abstract
Mast cell degranulation is regulated by the small guanosine triphosphatases (GTPases) Rab27a and Rab27b, which have distinct and opposing roles: Rab27b acts as a positive regulator through its effector protein Munc13-4, a non-neuronal isoform of the vesicle-priming Munc13 family of proteins, whereas Rab27a acts as a negative regulator through its effector protein melanophilin, by maintaining integrity of cortical filamentous actin (F-actin), a barrier to degranulation. Here we investigated the role of Rab37, one of the Rab GTPases assumed to be implicated in regulated secretion during mast cell degranulation. Using the RBL-2H3 mast cell line, we detected Rab37 on the secretory granules and found that antigen-induced degranulation was extensively increased by either knockdown of Rab37 or overexpression of a dominant-active Rab37 mutant. This hypersecretion phenotype in the Rab37-knockdown cells was suppressed by simultaneous knockdown of Rab27a and Rab27b or of Munc13-4, but not by disruption of cortical F-actin. We further found that Rab37 interacted with Munc13-4 in a GTP-independent manner and formed a Rab27-Munc13-4-Rab37 complex. These results suggest that Rab37 is a Munc13-4-binding protein that inhibits mast cell degranulation through its effector protein, by counteracting the vesicle-priming activity of the Rab27-Munc13-4 system.
Highlights
Mast cells are granulated cells that play a central role in allergic responses such as anaphylaxis and asthma, as well as certain innate and adaptive immune responses[1]
When HA-Rab37-expressing cells were stimulated with antigen in a Ca2+ -free medium, HA-Rab[37] and CD63 were translocated to the cell periphery including the rod-like protrusions (Fig. 1B, + Ag: r = 0.77 ± 0.13, n = 10). These results suggest that Rab[37] is expressed and resides on secretory granules in RBL-2H3 cells, consistent with recent global study of Rab guanosine triphosphatases (GTPases) expressed in the cells reported by Azouz et al.[34], in the course of our study
Degranulation can be directly induced by the A23187/TPA stimulation
Summary
Mast cells are granulated cells that play a central role in allergic responses such as anaphylaxis and asthma, as well as certain innate and adaptive immune responses[1]. Antigen-mediated cross-linking of the high-affinity IgE receptor Fcε RI activates a complicated signalling cascade leading to the activation of protein kinase C (PKC), elevation of intracellular Ca2+ concentration[1,4], and microtubule-dependent translocation of secretory granules towards the plasma membrane[5,6], eventually resulting in the release of granule contents This Ca2+ -dependent, regulated exocytosis in mast cells is called degranulation, which involves granule-granule and granule-plasma membrane fusion via multigranular or compound exocytosis[7]. The second and third secretory Rabs that were identified in mast cells are Rab27a and Rab27b, which share more than 70% amino acid identity[14] Based on their intracellular localisation, they are thought to regulate the movement and/or exocytosis of dense-core vesicles and lysosome-related organelles in various non-neuronal secretory cells[15,16]. We investigated the regulatory role of Rab[37] in mast cell degranulation and the functional relationship between Rab[37] and the Rab27-Munc[] positive regulatory system
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