REGULATION OF EXOCYTOSIS IN MAST CELLS

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Mast cells are specialized for responding to immunoglobulin E and associated allergens and play a key role in initiating allergic inflammation. They respond to stimulation by secreting a variety of inflammatory mediators and cytokines that alter vascular permeability, remodel extracellular matrix, and recruit other host defense cells that amplify the inflammatory response. Many of the secretory products are stored in membrane-bounded granules within the cytoplasm, and their release occurs by compound exocytosis, a massive cascade of granule-plasma membrane and granule-granule fusions involving most if not all of the storage granules. While much progress has been made in understanding the structure and early signaling of the IgE receptor (FceR), much less is known about the regulation and mechanisms of downstream events in the cascade that links stimulation to compound exocytosis. Understanding these events is of great medical interest as early occurrence in the hierarchical inflammatory response suggests an attractive site for intervention in developing therapies that control asthma, anaphylactic shock, and other acute host reactions to allergens and active peptides. Studies forming the basis of this proposal have shown that compound exocytosis in mast cells is regulated by a novel mechanism involving stimulus-dependent relocation within the cell of the protein SNAP-23 that is thought to comprise part of the fusion machinery. SNAP-23, one of the SNARE family of membrane fusion proteins, relocates in response to secretory stimulation from lamellipodia-like plasma membrane folds along the plasma membrane and intracellularly to granule surfaces. Relocation of SNAP-23 is essential for compound exocytosis and is hypothesized to involve distinct steps of mobilization, cytoskeletally-assisted relocation, and engagement with other SNARE proteins to promote membrane fusion. The overall goal of this proposal is to characterize the molecular mechanisms comprising each of these steps. Streptolysin-O permeabilized mast cells and rat basophilic leukemia (RBL-2H3) cells will be used to address how phosphorylation of SNAP-23 regulates mobilization; what proteins interact with SNAP-23 preceding and following mobilization; how Rho family GTPases and F-actin promote relocation; and how assembly of SNAP-23-containing SNARE complexes relates to engagement and secretion. In addition, a newly discovered inhibitor of compound exocytosis, a peptide derived from one of the secretory carrier membrane proteins (SCAMPs), will be used to analyze its effects on the relocation and function of SNAP-23.