To investigate the role of phospholipase D (PLD) in Fc⑀RI signaling, the wild-type or the catalytically inactive forms of PLD1 or PLD2 were stably overexpressed in RBL-2H3 mast cells. Fc⑀RI stimulation resulted in the activation of both PLD1 and PLD2. However, PLD1 was the source of most of the receptor-induced PLD activity. There was enhanced Fc⑀RI-induced degranulation only in cells that overexpressed the catalytically inactive PLD1. This dominant-negative PLD1 enhanced Fc⑀RI-induced tyrosine phosphorylations of early signaling molecules such as the receptor subunits, Syk and phospholipase C-␥ which resulted in faster release of Ca 2؉ from intracellular sources. Therefore, PLD1 negatively regulates signals upstream of the Ca 2؉ response. However, Fc⑀RI-induced PLD activation required Syk and was downstream of the Ca 2؉ response, suggesting that basal PLD1 activity rather than that activated by cell stimulation controlled these early signaling events. Dominant-negative PLD1 reduced the basal phosphatidic acid formation in unstimulated cells, which was accompanied by an increase in Fc⑀RI within the lipid rafts. These results indicate that constitutive basal PLD1 activity by regulating phosphatidic acid formation controls the early signals initiated by Fc⑀RI aggregation that lead to mast cell degranulation. ( IntroductionPhospholipase D (PLD) cleaves the terminal phosphodiester bond of phosphatidylcholine (PC) to yield phosphatidic acid (PA) and choline. The PA can then form important lipid mediators, including lysoPA, arachidonic acid, and diacylglycerol, which act as second messengers in many cellular responses. 1,2 Two mammalian PLD genes, PLD1 and PLD2, have been identified. Although both PLD enzymes require phosphatidylinositol 4,5-bisphosphate for activity, PLD1 is regulated by protein kinase C (PKC), and Arf and Rho small G protein families, whereas PLD2 can be activated by PKC, Arf, and phosphatidylinositol 4-phosphate 5-kinase ␣. 3-7 PLD activation may be involved in many cellular responses such as actin stress fiber formation, 8 exocytosis, 9,10 and endocytosis. 11 PLD is activated by many cell surface receptors; in immune cells, it is activated by antigen receptors present on T and B cells, monocytes, and mast cells. 12,13 Aggregation of the high-affinity immunoglobulin E (IgE) receptor (Fc⑀RI) on basophils and mast cells results in the rapid phosphorylation of the tyrosine residues in the immunoreceptor tyrosine-based activation motif of the  and ␥ subunits of the receptor. 14 This then recruits the protein tyrosine kinase Syk that is essential for phospholipase C-␥ (PLC-␥) activation and the increase in intracellular Ca 2ϩ ([Ca 2ϩ ] i ) and degranulation. [15][16][17][18] The mediators that are released cause allergic inflammation and anaphylactic reactions. [19][20][21] By mRNA analysis PLD1b and PLD2 are present in the RBL-2H3 cells that have been used as an in vitro model for mast cells. 22 In RBL-2H3 cells, PLD1b is predominantly in secretory granules and lysosomes, 23 whereas PLD2 is in the p...
The role of the mast cell–specific gangliosides in the modulation of the endocytic pathway of FcεRI was investigated in RBL-2H3 cells and in the ganglioside-deficient cell lines, E5 and D1. MAb BC4, which binds to the α subunit of FcεRI, was used in the analysis of receptor internalization. After incubation with BC4-FITC for 30 min, endocytic vesicles in RBL-2H3 and E5 cells were dispersed in the cytoplasm. After 1 hr, the endocytic vesicles of the RBL-2H3 cells had fused and formed clusters, whereas in the E5 cells, the fusion was slower. In contrast, in D1 cells, the endocytic vesicles were smaller and remained close to the plasma membrane even after 3 hr of incubation. When incubated with BC4-FITC and subsequently imunolabeled for markers of various endocytic compartments, a defect in the endocytic pathway in the E5 and D1 cells became evident. In the D1 cells, this defect was observed at the initial steps of endocytosis. Therefore, the ganglioside derivatives from GD1b are important in the endocytosis of FcεRI in mast cells. Because gangliosides may play a role in mast cell–related disease processes, they provide an attractive target for drug therapy and diagnosis.
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