title: Crucial role of the hydrophobic pocket region of Munc18 protein in mast cell degranulation.
abstract: The function of the Munc18-1 protein hydrophobic pocket, which interacts with the syntaxin-1 N-terminal peptide, has been highly controversial in neurosecretion. Recent analysis of patients with familial hemophagocytic lymphohistiocytosis type 5 has identified the E132A mutation in the hydrophobic pocket of Munc18-2, prompting us to examine the role of this region in the context of immune cell secretion. Double knockdown of Munc18-1 and Munc18-2 in RBL-2H3 mast cells eliminates both IgE-dependent and ionomycin-induced degranulation and causes a significant reduction in syntaxin-11 without altering expressions of the other syntaxin isoforms examined. These phenotypes were effectively rescued on reexpression of wild-type Munc18-1 or Munc18-2 but not the mutants (F115E, E132A, and F115E/E132A) in the hydrophobic pocket of Munc18. In addition, these mutants show that they are unable to directly interact with syntaxin-11, as tested through protein interaction experiments. Our results demonstrate the crucial roles of the hydrophobic pocket of Munc18 in mast cell degranulation, which include the regulation of syntaxin-11. We also suggest that the functional importance of this region is significantly different between neuronal and immune cell exocytosis.
authors.affiliation: Division of Fundamental Neurobiology, University Health Network, Toronto, ON, Canada M5T 2S8.
authors.forename: Na-Ryum
authors.initials: NR
authors.lastname: Bin
authors.forename: Chang Hun
authors.initials: CH
authors.lastname: Jung
authors.forename: Christopher
authors.initials: C
authors.lastname: Piggott
authors.forename: Shuzo
authors.initials: S
authors.lastname: Sugita
mesh_terms: D019943:Amino Acid Substitution; D000818:Animals; D061207:Calcium Ionophores / Q000494:pharmacology; D015550:Cell Degranulation; D057809:HEK293 Cells; D006801:Humans; D057927:Hydrophobic and Hydrophilic Interactions; D007073:Immunoglobulin E / Q000378:metabolism / Q000494:pharmacology; D015759:Ionomycin / Q000494:pharmacology; D051359:Lymphohistiocytosis, Hemophagocytic / Q000235:genetics / Q000378:metabolism* / Q000473:pathology; D008407:Mast Cells / Q000378:metabolism* / Q000473:pathology; D051379:Mice; D051938:Munc18 Proteins / Q000235:genetics / Q000378:metabolism*; D020125:Mutation, Missense; D016716:PC12 Cells; D017434:Protein Structure, Tertiary; D050765:Qa-SNARE Proteins / Q000235:genetics / Q000378:metabolism; D051381:Rats
chemical_list: D061207:Calcium Ionophores; D051938:Munc18 Proteins; D050765:Qa-SNARE Proteins; C541732:STX11 protein, human; C497940:STXBP2 protein, human; C497939:Stxbp1 protein, rat; C497942:Stxbp2 protein, rat; D007073:Immunoglobulin E; D015759:Ionomycin