Mast cell degranulation requires N-ethylmaleimide-sensitive factor-mediated SNARE disassembly.
Mast cells possess specialized granules that, upon stimulation of surface FcR with IgE, fuse with the plasma membrane, thereby releasing inflammatory mediators. A family of membrane fusion proteins called SNAREs, which are present on both the granule and the plasma membrane, plays a role in the fusion of these granules with the plasma membrane of mast cells. In addition to the SNAREs themselves, it is likely that the SNARE accessory protein, N-ethylmaleimide-sensitive factor (NSF), affects the composition and structure of the SNARE complex. NSF is a cytoplasmic ATPase that disassembles the SNARE complexes. To investigate the role of NSF in mast cell degranulation, we developed an assay to measure secretion from transiently transfected RBL (rat basophilic leukemia)-2H3 mast cells (a tumor analog of mucosal mast cells). RBL-2H3 cells were cotransfected with a plasmid encoding a human growth hormone secretion reporter along with either wild-type NSF or an NSF mutant that lacks ATPase activity. Human growth hormone was targeted to and released from secretory granules in RBL-2H3 cells, and coexpression with mutant NSF dramatically inhibited regulated exocytosis from the transfected cells. Biochemical analysis of SNARE complexes in these cells revealed that overexpression of the NSF mutant decreased disassembly and resulted in an accumulation of SNARE complexes. These data reveal a role for NSF in mast cell exocytosis and highlight the importance of SNARE disassembly, or priming, in regulated exocytosis from mast cells.
Puri
Niti
N
Experimental Immunology Branch, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892, USA.
Kruhlak
Michael J
MJ
Whiteheart
Sidney W
SW
Roche
Paul A
PA
D000251:Adenosine Triphosphatases / Q000172:deficiency / Q000235:genetics; D000818:Animals; D001426:Bacterial Proteins / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism; D002352:Carrier Proteins / Q000096:biosynthesis / Q000235:genetics / Q000502:physiology*; D015550:Cell Degranulation / Q000235:genetics / Q000502:physiology*; D045744:Cell Line, Tumor; D015536:Down-Regulation / Q000235:genetics; D005033:Ethylmaleimide / Q000494:pharmacology; D005089:Exocytosis / Q000235:genetics / Q000502:physiology; D017930:Genes, Reporter; D006367:HeLa Cells; D006801:Humans; D008164:Luminescent Proteins / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism; D008407:Mast Cells / Q000166:cytology / Q000201:enzymology / Q000378:metabolism*; D008565:Membrane Proteins / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism* / Q000502:physiology; D052066:N-Ethylmaleimide-Sensitive Proteins; D011499:Protein Processing, Post-Translational / Q000235:genetics / Q000502:physiology*; D050765:Qa-SNARE Proteins; D050683:R-SNARE Proteins; D051381:Rats; D050600:SNARE Proteins; D022142:Secretory Vesicles / Q000235:genetics / Q000378:metabolism; D014162:Transfection; D033921:Vesicular Transport Proteins
D001426:Bacterial Proteins; D002352:Carrier Proteins; D008164:Luminescent Proteins; D008565:Membrane Proteins; D050765:Qa-SNARE Proteins; D050683:R-SNARE Proteins; D050600:SNARE Proteins; C121241:VAMP7 protein, human; C496926:Vamp7 protein, rat; D033921:Vesicular Transport Proteins; C054562:yellow fluorescent protein, Bacteria; D000251:Adenosine Triphosphatases; D052066:N-Ethylmaleimide-Sensitive Proteins; C498737:Nsf protein, rat; D005033:Ethylmaleimide