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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.

SUBMITTER: Bin NR

PROVIDER: S-EPMC3607013 | biostudies-literature | 2013 Mar

REPOSITORIES: biostudies-literature

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Crucial role of the hydrophobic pocket region of Munc18 protein in mast cell degranulation.

Bin Na-Ryum NR Jung Chang Hun CH Piggott Christopher C Sugita Shuzo S

Proceedings of the National Academy of Sciences of the United States of America 20130304 12

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-dependen ...[more]

PMID: 23487749

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Project description:BackgroundAs the meningeally derived, fibroblast-rich, mass-produced by intrathecal morphine infusion is not produced by all opiates, but reduced by mast cell stabilizers, the authors hypothesized a role for meningeal mast cell/fibroblast activation. Using the guinea pig, the authors asked: (1) Are intrathecal morphine masses blocked by opiate antagonism?; (2) Do opioid agonists not producing mast cell degranulation or fibroblast activation produce masses?; and (3) Do masses covary with Mas-related G protein-coupled receptor signaling thought to mediate mast cell degranulation?MethodsIn adult male guinea pigs (N = 66), lumbar intrathecal catheters connected to osmotic minipumps (14 days; 0.5 µl/h) were placed to deliver saline or equianalgesic concentrations of morphine sulfate (33 nmol/h), 2',6'-dimethyl tyrosine-(Tyr-D-Arg-Phe-Lys-NH2) (abbreviated as DMT-DALDA; 10 pmol/h; μ agonist) or PZM21 (27 nmol/h; biased μ agonist). A second pump delivered subcutaneous naltrexone (25 µg/h) in some animals. After 14 to 16 days, animals were anesthetized and perfusion-fixed. Drug effects on degranulation of human cultured mast cells, mouse embryonic fibroblast activation/migration/collagen formation, and Mas-related G protein-coupled receptor activation (PRESTO-Tango assays) were determined.ResultsIntrathecal infusion of morphine, DMT-DALDA or PZM21, but not saline, comparably increased thermal thresholds for 7 days. Spinal masses proximal to catheter tip, composed of fibroblast/collagen type I (median: interquartile range, 0 to 4 scale), were produced by morphine (2.3: 2.0 to 3.5) and morphine plus naltrexone (2.5: 1.4 to 3.1), but not vehicle (1.2: 1.1 to 1.5), DMT-DALDA (1.0: 0.6 to 1.3), or PZM21 (0.5: 0.4 to 0.8). Morphine in a naloxone-insensitive fashion, but not PZM21 or DMT-DALDA, resulted in mast cell degranulation and fibroblast proliferation/collagen formation. Morphine-induced fibroblast proliferation, as mast cell degranulation, is blocked by cromolyn. Mas-related G protein-coupled receptor activation was produced by morphine and TAN67 (∂-opioid agonist), but not by PZM21, TRV130 (mu biased ligand), or DMT-DALDA.ConclusionsOpiates that activate Mas-related G protein-coupled receptor will degranulate mast cells, activate fibroblasts, and result in intrathecal mass formation. Results suggest a mechanistically rational path forward to safer intrathecal opioid therapeutics.

Dataset Information

Crucial role of the hydrophobic pocket region of Munc18 protein in mast cell degranulation.

Publications

Crucial role of the hydrophobic pocket region of Munc18 protein in mast cell degranulation.

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