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

Abstract

The physiological stimulus to exocytosis in mast cells is the cross-linking of the high-affinity IgE receptor, FcϵR1, 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 (PIP2) levels at the expense of phosphatidylinositol 4-monophosphate. The requirement for PIP2 in exocytosis was confirmed by using phosphatidylinositol transfer protein (PITPα) to increase PIP2 levels. Exocytosis, restored by either ARF1 or PITPα, was inhibited when PIP2 levels were depleted by phospholipase C∆1. We conclude that the function of ARF1 and PITPα is to increase the local synthesis of PIP2, 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.