Activation of exocytosis by cross-linking of the IgE receptor is dependent on ADP-ribosylation factor 1-regulated phospholipase D in RBL-2H3 mast cells: evidence that the mechanism of activation is via regulation of phosphatidylinositol 4,5-bisphosphate synthesis.
The physiological stimulus to exocytosis in mast cells is the cross-linking of the high-affinity IgE receptor, FcepsilonR1, with antigen. We demonstrate a novel function for ADP-ribosylation factor 1 (ARF1) in the regulation of antigen-stimulated secretion using cytosol-depleted RBL-2H3 mast cells for reconstitution of secretory responses. When antigen is used as the stimulus, ARF1 also reconstitutes phospholipase D activation. Using ethanol to divert the phosphatidic acid (the product of phospholipase D activity) to phosphatidylethanol causes inhibition of ARF1-reconstituted secretion. In addition. ARF1 causes an increase in phosphatidylinositol 4,5-bisphosphate (PIP(2)) levels at the expense of phosphatidylinositol 4-monophosphate. The requirement for PIP(2) in exocytosis was confirmed by using phosphatidylinositol transfer protein (PITPalpha) to increase PIP(2) levels. Exocytosis, restored by either ARF1 or PITPalpha, was inhibited when PIP(2) levels were depleted by phospholipase Cdelta1. We conclude that the function of ARF1 and PITPalpha is to increase the local synthesis of PIP(2), the function of which in exocytosis is likely to be linked to lipid-protein interactions, whereby recruitment of key components of the exocytotic machinery are targeted to the appropriate membrane compartment.
Way
G
G
Department of Physiology, Rockefeller Building, University College London, University St., London WC1E 6JJ, U.K.
O'luanaigh
N
N
Cockcroft
S
S
D020823:ADP-Ribosylation Factor 1 / Q000378:metabolism*; D000818:Animals; D000941:Antigens / Q000276:immunology / Q000494:pharmacology; D002352:Carrier Proteins / Q000378:metabolism; D002462:Cell Membrane / Q000737:chemistry / Q000187:drug effects / Q000378:metabolism; D002463:Cell Membrane Permeability; D003600:Cytosol / Q000187:drug effects / Q000378:metabolism; D004789:Enzyme Activation / Q000187:drug effects; D000431:Ethanol / Q000494:pharmacology; D005089:Exocytosis / Q000187:drug effects; D020404:Glycerophospholipids / Q000378:metabolism; D016244:Guanosine 5'-O-(3-Thiotriphosphate) / Q000494:pharmacology; D007527:Isoenzymes / Q000378:metabolism; D008407:Mast Cells / Q000166:cytology* / Q000187:drug effects / Q000201:enzymology / Q000378:metabolism; D008565:Membrane Proteins; D010537:Peritoneum / Q000166:cytology / Q000276:immunology; D010712:Phosphatidic Acids / Q000378:metabolism; D019269:Phosphatidylinositol 4,5-Diphosphate / Q000096:biosynthesis*; D054803:Phospholipase C delta; D010739:Phospholipase D / Q000378:metabolism*; D049294:Phospholipid Transfer Proteins; D051381:Rats; D017455:Receptors, IgE / Q000378:metabolism*; D014407:Tumor Cells, Cultured; D010738:Type C Phospholipases / Q000378:metabolism
D000941:Antigens; D002352:Carrier Proteins; D020404:Glycerophospholipids; D007527:Isoenzymes; D008565:Membrane Proteins; D010712:Phosphatidic Acids; D019269:Phosphatidylinositol 4,5-Diphosphate; D049294:Phospholipid Transfer Proteins; D017455:Receptors, IgE; C051521:phosphatidylethanol; D016244:Guanosine 5'-O-(3-Thiotriphosphate); D000431:Ethanol; D010738:Type C Phospholipases; D054803:Phospholipase C delta; D010739:Phospholipase D; D020823:ADP-Ribosylation Factor 1