Activation of exocytosis by cross-linking of the IgE receptor is dependenton 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 crosslinking of the high-affinity IgE receptor, Fc epsilon R1, with antigen. We demonstr ate 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 stim ulus, ARF1 also reconstitutes phospholipase D activation. Using ethanol to divert the phosphatidic acid (the product of phospholipase D activity) to p hosphatidylethanol causes inhibition of ARF1-reconstituted secretion. In ad dition. ARF1 causes an increase in phosphatidylinositol 4,5-bisphosphate (P IP2) levels at the expense of phosphatidylinositol 4-monophosphate. The req uirement for PIP, in exocytosis was confirmed by using phosphatidylinositol transfer protein (PITP alpha) to increase PIP2 levels. Exocytosis, restore d by either ARF1 or PITP alpha was inhibited when PIP, levels were depleted by phospholipase C delta 1. We conclude that the function of ARF1 and PITP alpha is to increase the local synthesis of PIP2, the function of which in exocytosis is likely to be linked to lipid-protein interactions, whereby r ecruitment of key components of the exocytotic machinery are targeted to th e appropriate membrane compartment.