Phospholipase Ds (PLDs) are regulated enzymes that generate phosphatidic acid (PA), a putative second messenger implicated in the regulation of vesicular trafficking and cytoskeletal reorganization. Mast cells, when stimulated with antigen, show a dramatic alteration in their cytoskeleton and also release their secretory granules by exocytosis. Butan-1-ol, which diverts the production of PA generated by PLD to the corresponding phosphatidylalcohol, was found to inhibit membrane ruffling when added together with antigen or when added after antigen. Inhibition by butan-1-ol was completely reversible because removal of butan-1-ol restored membrane ruffling. Measurements of PLD activation by antigen indicate a requirement for continual PA production during membrane ruffling, which was maintained for at least 30 min. PLD1 and PLD2 are both expressed in mast cells and green fluorescent protein-tagged proteins were used to identify PLD2 localizing to membrane ruffles of antigen-stimulated mast cells together with endogenous ADP ribosylation factor 6 (ARF6). In contrast, green fluorescent protein-PLD1 localized to intracellular vesicles and remained in this location after stimulation with antigen. Membrane ruffling was independent of exocytosis of secretory granules because phorbol 12-myristate 13-acetate increased membrane ruffling in the absence of exocytosis. Antigen or phorbol 12-myristate 13-acetate stimulation increased both PLD1 and PLD2 activity when expressed individually in RBL-2H3 cells. Although basal activity of PLD2-overexpressing cells is very high, membrane ruffling was still dependent on antigen stimulation. In permeabilized cells, antigen-stimulated phosphatidylinositol(4,5)bisphosphate synthesis was dependent on both ARF6 and PA generated from PLD. We conclude that both activation of ARF6 by antigen and a continual PLD2 activity are essential for local phosphatidylinositol(4,5)bisphosphate generation that regulates dynamic actin cytoskeletal rearrangements.

All integrin ␣ subunits contain a highly conserved KXGFFKR motif in their cytoplasmic domains that plays a crucial role in the regulation of integrin affinity for their ligands. We show that a lipid-modified peptide corresponding to the cytoplasmic region, 989 -995, of the platelet integrin subunit glycoprotein GpIIb (␣IIb), palmitoyl-KVGFFKR (Ppep; 10 M), but not a similarly modified scrambled peptide (palmitoyl-FKFVRGK), can specifically induce platelet activation and aggregation equivalent to that of strong agonists such as thrombin. Ppep-induced aggregation is also associated with indices of platelet activation including thromboxane A 2 (TXA 2 ) synthesis (EC 50 ‫؍‬ 45 ؎ 5 M), secretion of ␣-granules detected as enhanced surface expression of P-selectin (EC 50 ‫؍‬ 52 ؎ 8 M), and conformational changes in GpIIb/IIIa measured by the monoclonal antibody, PAC-1 (EC 50 ‫؍‬ 3.7 ؎ 1 M). The TXA 2 receptor antagonist, SQ29548, PGE 1 , and the ADP scavenger, apyrase, differentially inhibit the aggregation response and TXA 2 synthesis in response to Ppep. Similarly, GpIIb/IIIa antagonists (RO-449883 and integrelin), which inhibit aggregation by greater than 90%, have little effect on peptide-induced TXA 2 synthesis, suggesting that this event is independent of fibrinogen binding to GpIIb/IIIa. Alanine-stepping of the Ppep sequence identifies GFFK(991-994) as the critical residues in all peptidemediated events. We conclude that this peptide can imitate the cytoplasmic domain of GpIIb and initiate parallel but independent signaling pathways, one leading to ligand binding and platelet aggregation and the other to intracellular signaling events such as TXA 2 synthesis and secretion.Integrins are a family of cell adhesion molecules composed of two subunits, ␣ and ␤, which form a complex on the cell surface. Ligand recognition by integrins may be modulated by intracellular signals that interact with the cytoplasmic tails of the subunits. This has been demonstrated most clearly for the platelet glycoprotein (Gp) 1 IIb/IIIa (␣IIb␤3), the most abundant platelet integrin, which acts as a receptor for fibrinogen, fibronectin, and other RGD-containing macromolecules (1). Under resting conditions, this receptor has a low affinity for its ligands (2). However, when platelets are stimulated by agonists such as thrombin or ADP, GpIIb/IIIa undergoes a conformational change (3) detected by the appearance of neoepitopes for monoclonal antibodies such as PAC-1 (4) and acquires a high affinity binding for its ligands, principally fibrinogen (5, 6). The binding of fibrinogen results in platelet aggregation, an early step in the generation of a thrombus. Deletion or mutation of the cytoplasmic domains of the integrin subunits can produce a constitutively active or inactive receptor (7-9), suggesting that signals resulting from cell activation interact with the intracellular components of GpIIb/IIIa to modify ligand recognition (inside-out signaling).The cytoplasmic domains are also important for events occurring as a consequence of ligand...

The physiological stimulus to exocytosis in mast cells is the crosslinking 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 (PIP # ) levels at the expense of phosphatidylinositol 4-monophosphate. The requirement for

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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.