The SNARE Machinery in Mast Cell Secretion

Abstract

Mast cells are known as inflammatory cells which exert their functions in allergic and anaphylactic reactions by secretion of numerous inflammatory mediators. During an allergic response, the high-affinity IgE receptor, FcεRI, becomes cross-linked by receptor-bound IgE and antigen resulting in immediate release of pre-synthesized mediators - stored in granules - as well as in de novo synthesis of various mediators like cytokines and chemokines. Soluble N-ethylmaleimide-sensitive factor attachment protein (SNAP) receptors (SNARE) proteins were found to play a central role in regulating membrane fusion events during exocytosis. In addition, several accessory regulators like Munc13, Munc18, Rab GTPases, secretory carrier membrane proteins, complexins, or synaptotagmins were found to be involved in membrane fusion. In this review we summarize our current knowledge about the SNARE machinery and its mechanism of action in mast cell secretion.

Keywords: SNARE proteins; exocytosis; mast cell.

Figure 1

May–Grünwald/Giemsa stain of a resting human intestinal mast cell and a mast cell following activation induced degranulation. Note the increase in size and loss of granule staining.

Figure 2

SNARE catalyzed granule fusion in mast cells. (A) Secretion of mediators requires fusion of vesicle and plasma membranes. Upon activation through FcεRI secretory granules translocate to and dock at the plasma membrane where the t-SNAREs SNAP-23 and STX4 together with the v-SNARE VAMP8 form stable tetrameric complexes of bundled helices bringing the lipid bilayers into a close distance to catalyze membrane fusion. The SNARE motifs of SNAP-23, STX4, and VAMP8, which become highly organized in the four helical bundle during the formation of the trans-SNARE complex are highlighted in color. (B) The primary structure of human SNAP-23, STX4, and VAMP8 as adapted from Hong (2005) is shown with SNARE motifs for each protein in like colors. STX4 and VAMP8 have C-terminal transmembrane domains (TM), whereas the linker domain of SNAP-23, which connects the two SNARE motifs, has a membrane anchor domain, consisting of palmitoylated cysteine residues (M). Numbers indicate protein or domain boundaries, arrows indicate potential phosphorylation sites (http://www.phosphosite.org). Phosphorylation of mouse SNAP-23 on Ser⁹⁵ and Ser¹²⁰ was found to modulate regulated mast cell exocytosis (Hepp et al., 2005), whereas phosphorylation of STX4 was not altered during secretion in RBL cells (Pombo et al., 2001).

Figure 3

Possible cytokine secretion pathways in mast cells. Cytokine secretion may occur constitutively through small vesicular carriers or through recycling endosomes (ER) as described for TNF in macrophages. Moreover, TNF could get re-endocytosed from the plasma membrane and transported into secretory granules (SG) and then rapidly released upon stimulation. Another possible pathway of regulated exocytosis in mast cells may be piecemeal degranulation as reported for eosinophils.

Figure 4

Domain structure of Munc18-1 (A) and Munc13-1 (B) as adapted from Koch et al. (2000); Misura et al. (2000); Li et al. (2011). Numbers above indicate protein or domain boundaries. For Munc18-1 domain boundaries are also directly included in the inset. The Munc18-1 protein contains three domains (with domain 3 being divided in D3a and D3b). According to crystal structure analysis the molecule adopts a horseshoe like structure that holds the STX-bound molecule in its closed conformation. Contact surfaces reside in D1 and D3a, which form the bottom ends of the horseshoe, while D2 and D3b form the upper end. Domain 3b includes the so-called Sly 1 homology domain containing the residue homologous to the Sly1-20 mutant in the yeast Sly1 protein, which bypasses the requirement for a Rab effector protein in yeast vesicular transport (Dascher et al., 1991). Above the domains are also indicated protein kinase C (Ser³⁰⁶ and Ser³¹³) and Cyclin dependent kinase (Thr⁵⁷⁴) phosphorylation sites that may regulate Munc18-1 effector functions. (B) The Munc13-1 protein contains several calcium-binding C2 domains, a Diacylglycerol (DAG) C1 domain making it responsive to stimulation with PKC as well as a calmodulin binding domain. Note that the ubiquitouisly expressed isoform 13-4, which is important in mast cell exocytosis lacks the N-terminal C2a, CaMb, and C1 domains. The Mun domain shows structural similarity to thethering proteins and may play a role in the transition of the closed conformation of STX molecules by contacting either Munc18 or STX molecules (Li et al., ; Sudhof and Rizo, 2011).

Figure 5

The fusion machinery in mast cell secretion. In resting cells membrane fusion is blocked likely by molecules such as STX3-bound Munc18-2. Upon activation through FcεRI secretory granules translocate to and dock at the plasma membrane where the SNARE proteins assemble into tetrameric complexes. Multiple accessory proteins such as Munc18-2, Munc13-4, Rab27, Doc2α, complexin II, synaptotagmin II, or SCAMPs, are involved as “gatekeepers” of the fusion reaction holding effectors in an inactive state, as tethering factors controlling docking and priming, or as regulators of the SNARE complexes triggering the calcium-dependent membrane fusion.