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2.
Mol Immunol ; 38(16-18): 1277-82, 2002 Sep.
Article in English | MEDLINE | ID: mdl-12217395

ABSTRACT

Phospholipase D (PLD) catalyses the hydrolysis of phosphatidylcholine to generate the lipid second messenger, phosphatidate (PA). Two mammalian phospholipase Ds (PLD1 and PLD2) have been cloned and both are present in RBL-2H3 mast cells. PLD1 is localised to secretory granules whilst PLD2 is localised to the plasma membrane, and the activity of both enzymes is increased upon antigen stimulation. Primary alcohols specifically interfere with the production of PLD-derived PA and are found to be potent inhibitors of antigen-stimulated exocytosis. One major intracellular regulator for PLD activity and exocytosis is ARF proteins, as depletion by permeabilisation leads to loss of both antigen-mediated PLD activation and exocytosis. Both responses can be restored in depleted cells by re-addition of ARF1 or ARF6. ARF proteins and PLD-derived PA synergistically regulate the activity of a Type I PIP 5-kinasealpha. It is suggested that ARF, by activating PLD and PIP 5-kinase activities regulate PA and PI(4,5)P(2) levels, and both are critical components of the exocytosis machinery in mast cells.


Subject(s)
ADP-Ribosylation Factors/physiology , Mast Cells/immunology , Phospholipase D/physiology , Receptors, IgE/metabolism , ADP-Ribosylation Factor 1/physiology , ADP-Ribosylation Factor 6 , Animals , Exocytosis , Mast Cells/cytology , Mast Cells/enzymology , Phosphatidic Acids/physiology , Phosphatidylinositol 4,5-Diphosphate/metabolism , Rats , Signal Transduction
3.
Mol Biol Cell ; 13(9): 3344-54, 2002 Sep.
Article in English | MEDLINE | ID: mdl-12221138

ABSTRACT

After permeabilization with the pore-forming toxin streptolysin-O mast cells can be triggered to secrete by addition of both calcium and a GTP analogue. If stimulation is delayed after permeabilization, there is a progressive decrease in the extent of secretion upon stimulation, eventually leading to a complete loss of the secretory response. This loss of secretory response can be retarded by the addition of cytosol from other secretory tissues, demonstrating that the response is dependent on a number of cytosolic proteins. We have used this as the basis of a bioassay to purify Secernin 1, a novel 50-kDa cytosolic protein that appears to be involved in the regulation of exocytosis from peritoneal mast cells. Secernin 1 increases both the extent of secretion and increases the sensitivity of mast cells to stimulation with calcium.


Subject(s)
Chromatography, Gel/methods , Cytosol/metabolism , Mast Cells/metabolism , Nerve Tissue Proteins/chemistry , Nerve Tissue Proteins/isolation & purification , Proteins/chemistry , Proteins/isolation & purification , Sepharose/analogs & derivatives , Amino Acid Sequence , Animals , Calcium/pharmacology , Cattle , Chromatography , Dose-Response Relationship, Drug , Durapatite/pharmacology , Exocytosis , Glutathione Transferase/metabolism , Humans , Male , Mass Spectrometry , Molecular Sequence Data , Plasmids/metabolism , Rats , Rats, Sprague-Dawley , Recombinant Fusion Proteins/metabolism , Sepharose/pharmacology , Sequence Homology, Amino Acid , Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization , Time Factors
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