Phospholipases D1 and D2 regulate different phases of exocytosis in mast cells.
The rat mast cell line RBL-2H3 contains both phospholipase D (PLD)1 and PLD2. Previous studies with this cell line indicated that expressed PLD1 and PLD2 are both strongly activated by stimulants of secretion. We now show by use of PLDs tagged with enhanced green fluorescent protein that PLD1, which is largely associated with secretory granules, redistributes to the plasma membrane in stimulated cells by processes reminiscent of exocytosis and fusion of granules with the plasma membrane. These processes and secretion of granules are suppressed by expression of a catalytically inactive mutant of PLD1 or by the presence of 50 mM 1-butanol but not tert-butanol, an indication that these events are dependent on the catalytic activity of PLD1. Of note, cholera toxin induces translocation of PLD1-labeled granules to the plasma membrane but not fusion of granules with plasma membrane or secretion. Subsequent stimulation of calcium influx with Ag or thapsigargin leads to rapid redistribution of PLD1 to the plasma membrane and accelerated secretion. Also of note, PLD1 is recycled from plasma membrane back to granules within 4 h of stimulation. PLD2, in contrast, is largely confined to the plasma membrane, but it too participates in the secretory process, because expression of catalytically inactive PLD2 also blocks secretion. These data indicate a two-step process: translocation of granules to the cell periphery, regulated by granule-associated PLD1, and a calcium-dependent fusion of granules with the plasma membrane, regulated by plasma membrane-associated PLD2 and possibly PLD1.
Choi
Wahn Soo
WS
Laboratory of Molecular Immunology and Light Microscopy Core Facility, National Heart Lung and Blood Institute, National Institutes of Health, Bethesda, MD 20892, USA.
Kim
Young Mi
YM
Combs
Christian
C
Frohman
Michael A
MA
Beaven
Michael A
MA
D000818:Animals; D002459:Cell Fusion; D002460:Cell Line; D003594:Cytoplasmic Granules / Q000201:enzymology; D005089:Exocytosis; D008407:Mast Cells / Q000378:metabolism*; D010739:Phospholipase D / Q000502:physiology*; D011493:Protein Kinase C / Q000502:physiology; D051381:Rats; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology
D011493:Protein Kinase C; C105606:phospholipase D2; D010739:Phospholipase D; C106801:phospholipase D1; D013755:Tetradecanoylphorbol Acetate