Regulation of the Tyrosine Phosphorylation of Phospholipid Scramblase 1 in Mast Cells That Are Stimulated through the High-Affinity IgE Receptor

Engagement of high-affinity immunoglobulin E receptors (FcεRI) activates two signaling pathways in mast cells. The Lyn pathway leads to recruitment of Syk and to calcium mobilization whereas the Fyn pathway leads to phosphatidylinositol 3-kinase recruitment. Mapping the connections between both pathways remains an important task to be completed. We previously reported that Phospholipid Scramblase 1 (PLSCR1) is phosphorylated on tyrosine after cross-linking FcεRI on RBL-2H3 rat mast cells, amplifies mast cell degranulation, and is associated with both Lyn and Syk tyrosine kinases. Here, analysis of the pathway leading to PLSCR1 tyrosine phosphorylation reveals that it depends on the FcRγ chain. FcεRI aggregation in Fyn-deficient mouse bone marrow-derived mast cells (BMMC) induced a more robust increase in FcεRI-dependent tyrosine phosphorylation of PLSCR1 compared to wild-type cells, whereas PLSCR1 phosphorylation was abolished in Lyn-deficient BMMC. Lyn association with PLSCR1 was not altered in Fyn-deficient BMMC. PLSCR1 phosphorylation was also dependent on the kinase Syk and significantly, but partially, dependent on detectable calcium mobilization. Thus, the Lyn/Syk/calcium axis promotes PLSCR1 phosphorylation in multiple ways. Conversely, the Fyn-dependent pathway negatively regulates it. This study reveals a complex regulation for PLSCR1 tyrosine phosphorylation in FcεRI-activated mast cells and that PLSCR1 sits at a crossroads between Lyn and Fyn pathways.     

FcεRI-mediated mast cell migration: Signaling pathways and dependence on cytosolic free Ca concentration

IgE-sensitized rat basophilic leukemia (RBL)-2H3 mast cells have been shown to migrate towards antigen. In the present study we tried to identify the mechanism by which antigen causes mast cell migration. Antigen caused migration of RBL-2H3 cells at the concentration ranges of 1000-fold lower than those required for degranulation and the dose response was biphasic. This suggests that mast cells can detect very low concentration gradients of antigen (pg/ml ranges), which initiate migration until they degranulate near the origin of antigen, of which concentration is in the ng/ml ranges. Similar phenomenon was observed in human mast cells (HMCs) derived from CD34⁺ progenitors. As one mechanism of mast cell migration, we tested the involvement of sphingosine 1-phosphate (S1P). FcεRI-mediated cell migration was dependent on the production of S1P but independent of a S1P receptor or its signaling pathways as determined with S1P receptor antagonist VPC23019 and Gi protein inhibitor pertussis toxin (PTX). This indicated that the site of action of S1P produced by antigen stimulation was intracellular. However, S1P-induced mast cell migration was dependent on S1P receptor activation and inhibited by both VPC23019 and PTX. Cell migration towards antigen or extracellular S1P was dependent on the activation of the phosphatidylinositol 3-kinase (PI3K) and mitogen-activated protein kinase (MAPK) pathways, while only migration towards antigen was inhibited by the inhibitors of sphingosine kinase and phospholipase C (PLC) and intracellular calcium chelator BAPTA. In summary, our data suggest that the high affinity receptor for IgE (FcεRI)-mediated mast cell migration is dependent on the production of S1P but independent of S1P receptors. Cell migration mediated by either FcεRI or S1P receptors involves activation of both PI3K and MAPK.     

Temporally resolved interactions between antigen-stimulated IgE receptors and Lyn kinase on living cells

Upon cross-linking by antigen, the high affinity receptor for immunoglobulin E (IgE), FcepsilonRI, is phosphorylated by the Src family tyrosine kinase Lyn to initiate mast cell signaling, leading to degranulation. Using fluorescence correlation spectroscopy (FCS), we observe stimulation-dependent associations between fluorescently labeled IgE-FcepsilonRI and Lyn-EGFP on individual cells. We also simultaneously measure temporal variations in the lateral diffusion of these proteins. Antigen-stimulated interactions between these proteins detected subsequent to the initiation of receptor phosphorylation exhibit time-dependent changes, suggesting multiple associations between FcepsilonRI and Lyn-EGFP. During this period, we also observe a persistent decrease in Lyn-EGFP lateral diffusion that is dependent on Src family kinase activity. These stimulated interactions are not observed between FcepsilonRI and a chimeric EGFP that contains only the membrane-targeting sequence from Lyn. Our results reveal real-time interactions between Lyn and cross-linked FcepsilonRI implicated in downstream signaling events. They demonstrate the capacity of FCS cross-correlation analysis to investigate the mechanism of signaling-dependent protein-protein interactions in intact, living cells.     

Differential Effects of the Toll-Like Receptor 2 Agonists,PGN and Pam3CSK4 on Anti-IgE Induced Human Mast Cell Activation

Mast cells are pivotal in the pathogenesis of allergy and inflammation. In addition to the classical IgE-dependent mechanism involving crosslinking of the high-affinity receptor for IgE (FcεRI), mast cells are also activated by Toll-like receptors (TLRs) which are at the center of innate immunity. In this study, we demonstrated that the response of LAD2 cells (a human mast cell line) to anti-IgE was altered in the presence of the TLR2 agonists peptidoglycan (PGN) and tripalmitoyl-S-glycero-Cys-(Lys)4 (Pam3CSK4). Pretreatment of PGN and Pam3CSK4 inhibited anti-IgE induced calcium mobilization and degranulation without down-regulation of FcεRI expression. Pam3CSK4 but not PGN acted in synergy with anti-IgE for IL-8 release when the TLR2 agonist was added simultaneously with anti-IgE. Studies with inhibitors of key enzymes implicated in mast cell signaling revealed that the synergistic release of IL-8 induced by Pam3CSK4 and anti-IgE involved ERK and calcineurin signaling cascades. The differential modulations of anti-IgE induced mast cell activation by PGN and Pam3CSK4 suggest that dimerization of TLR2 with TLR1 or TLR6 produced different modulating actions on FcεRI mediated human mast cell activation.     

Compartmentalized IgE Receptor–mediated Signal Transduction in Living Cells

Several receptor-mediated signal transduction pathways, including EGF and IgE receptor pathways, have been proposed to be spatially restricted to plasma membrane microdomains. However, the experimental evidence for signaling events in these microdomains is largely based on biochemical fractionation and immunocytochemical studies and only little is known about their spatial dynamics in living cells. Here we constructed green fluorescent protein–tagged SH2 domains to investigate where and when IgE receptor (FcεRI)–mediated tyrosine phosphorylation occurs in living tumor mast cells. Strikingly, within minutes after antigen addition, tandem SH2 domains from Syk or PLC-γ1 translocated from a uniform cytosolic distribution to punctuate plasma membrane microdomains. Colocalization experiments showed that the microdomains where tyrosine phosphorylation occurred were indistinguishable from those stained by cholera toxin B, a marker for glycosphingolipids. Competitive binding studies with coelectroporated unlabeled Syk, PLC-γ1, and other SH2 domains selectively suppressed the induction of IgE receptor–mediated calcium signals as well as the binding of the fluorescent SH2 domains. This supports the hypothesis that PLC-γ1 and Syk SH2 domains selectively bind to Syk and IgE receptors, respectively. Unlike the predicted prelocalization of EGF receptors to caveolae microdomains, fluorescently labeled IgE receptors were found to be uniformly distributed in the plasma membrane of unstimulated cells and only transiently translocated to glycosphingolipid rich microdomains after antigen addition. Thus, these in vivo studies support a plasma membrane signaling mechanism by which IgE receptors transiently associate with microdomains and induce the spatially restricted activation of Syk and PLC-γ1.     

Protein Tyrosine Phosphatase Inhibitors in FcγRI-Induced Myeloid Oxidant Signaling

Fc-receptor stimulation in myeloid cells results in increased oxygen consumption, termed the respiratory burst, which is coupled to a rapid and transient increase in tyrosine phosphorylation of cellular proteins. In a previous paper in this journal we showed that the protein tyrosine phosphatase (PTPase) inhibitors sodium orthovanadate and phenylarsine oxide (PAO) block the FcγRI-induced respiratory burst in interferon-γ-differentiated U937 cells (U937IF) while augmenting the FcγRI-induced tyrosine phosphorylation of cellular proteins. Herein we examine the effects of PTPase inhibitors on specific molecules involved in FcγRI signaling. We show that orthovanadate and PAO augmented the FcγRI-induced tyrosine phosphorylation of the adaptor protein CBL. CBL interactions with other phosphoproteins, among them SHC and CRKL, were also augmented in response to pretreatment with the PTPase inhibitors. SHC was tyrosine phosphorylated in response to FcγRI stimulation of U937IF cells and bound to the SH2 domain of GRB2 in a stimulation-dependent manner. In fusion protein pull-down experiments the interaction of SHC with the SH2 domain of GRB2 was increased in PTPase inhibitor pretreated U937IF cells in response to FcγRI stimulation. Our data support the hypothesis that a tyrosine dephosphorylation event is required for effective transmission of the FcγRI signal to result in activation of the myeloid respiratory burst response.     

Characterization of Cbl-Nck and Nck-Pak1 Interactions in Myeloid FcγRII Signaling

Fc receptors modulate inflammatory processes, including phagocytosis, serotonin and histamine release, superoxide production, and secretion of cytokines. Aggregation of FcγRIIa, the low-affinity receptor for monomeric IgG, activates nonreceptor protein tyrosine kinases such as Lyn, Hck, and Syk, potentially driving the phosphorylation of the downstream adaptor proteins, including Cbl and/or Nck. Previous work from our laboratory using interferon-γ-differentiated U937 (U937IF) myeloid cells investigated mechanisms which regulate Fcγ receptor-induced assembly of adaptor complexes. Herein we report that FcγRII receptor signaling in U937IF and HEL cells involves Cbl and Nck, suggesting that Cbl–Nck interactions may link FcγRII to downstream activation of Pak kinase. FcγRII crosslinking induced the phosphorylation of Cbl and Nck on tyrosine. The αCbl immunoprecipitations revealed constitutive binding of Nck and Grb2 to Cbl and FcγRII-inducible binding of CrkL to Cbl. The interactions of Cbl with Nck and CrkL were phosphorylation dependent since dephosphorylation of cellular proteins with potato acid phosphatase abrogated binding. GST–Nck fusion protein pulldown experiments show that Cbl and Pak1 bind to the second SH3 domain of Nck. A specific Src inhibitor, PP1, was shown to completely abrogate the FcγR-induced superoxide response, correlating with a decrease in Cbl and Nck tyrosine phosphorylation. Our results provide the first evidence that Src is required for FcγR activation of the respiratory burst in myeloid cells and suggest that Cbl–Nck, Cbl–Pak1, and Nck–Pak1 interactions may regulate this response.     

Ethanol Inhibits High-Affinity Immunoglobulin E Receptor (FcεRI) Signaling in Mast Cells by Suppressing the Function of FcεRI-Cholesterol Signalosome

Ethanol has multiple effects on biochemical events in a variety of cell types, including the high-affinity immunoglobulin E receptor (FcεRI) signaling in antigen-activated mast cells. However, the underlying molecular mechanism remains unknown. To get better understanding of the effect of ethanol on FcεRI-mediated signaling we examined the effect of short-term treatment with non-toxic concentrations of ethanol on FcεRI signaling events in mouse bone marrow-derived mast cells. We found that 15 min exposure to ethanol inhibited antigen-induced degranulation, calcium mobilization, expression of proinflammatory cytokine genes (tumor necrosis factor-α, interleukin-6, and interleukin-13), and formation of reactive oxygen species in a dose-dependent manner. Removal of cellular cholesterol with methyl-β-cyclodextrin had a similar effect and potentiated some of the inhibitory effects of ethanol. In contrast, exposure of the cells to cholesterol-saturated methyl-β-cyclodextrin abolished in part the inhibitory effect of ethanol on calcium response and production of reactive oxygen species, supporting lipid-centric theories of ethanol action on the earliest stages of mast cell signaling. Further studies showed that exposure to ethanol and/or removal of cholesterol inhibited early FcεRI activation events, including tyrosine phosphorylation of the FcεRI β and γ subunits, SYK kinases, LAT adaptor protein, phospholipase Cγ, STAT5, and AKT and internalization of aggregated FcεRI. Interestingly, ethanol alone, and particularly in combination with methyl-β-cyclodextrin, enhanced phosphorylation of negative regulatory tyrosine 507 of LYN kinase. Finally, we found that ethanol reduced passive cutaneous anaphylactic reaction in mice, suggesting that ethanol also inhibits FcεRI signaling under in vivo conditions. The combined data indicate that ethanol interferes with early antigen-induced signaling events in mast cells by suppressing the function of FcεRI-cholesterol signalosomes at the plasma membrane.     

Relationships between Levels of Serum IgE,Cell-Bound IgE,and IgE-Receptors on Peripheral Blood Cells in a Pediatric Population

Background

Elevated serum immunoglobulin (Ig) E is a diagnostic marker of immediate-type allergic reactions. We hypothesize that serum IgE does not necessarily reflect total body IgE because in vivo IgE can be bound to cell surface receptors such as FcεRI and FcεRII (CD23). The aim of this study was to analyze the relationships between levels of serum IgE, cell-bound IgE, and IgE-receptors on peripheral blood cells in a pediatric population.

Methodology

Whole blood samples from 48 children (26 boys, 22 girls, mean age 10,3±5,4 years) were analyzed by flow cytometry for FcεRI, CD23, and cell-bound IgE on dendritic cells (CD11c+MHC class II+), monocytes (CD14+), basophils (CD123+MHC class II-) and neutrophils (myeloperoxidase+). Total serum IgE was measured by ELISA and converted into z-units to account for age-dependent normal ranges. Correlations were calculated using Spearman rank correlation test.

Principal Findings

Dendritic cells, monocytes, basophils, and neutrophils expressed the high affinity IgE-receptor FcεRI. Dendritic cells and monocytes also expressed the low affinity receptor CD23. The majority of IgE-receptor positive cells carried IgE on their surface. Expression of both IgE receptors was tightly correlated with cell-bound IgE. In general, cell-bound IgE on FcεRI+ cells correlated well with serum IgE. However, some patients carried high amounts of cell-bound IgE despite low total serum IgE levels.

Conclusion/Significance

In pediatric patients, levels of age-adjusted serum IgE, cell-bound IgE, and FcεRI correlate. Even in the absence of elevated levels of serum IgE, cell-bound IgE can be detected on peripheral blood cells in a subgroup of patients.     

Disease associated cellular machinery in anaphylaxis – And the de novo paradigm shift

Anaphylaxis is a sudden immune reaction against an allergen that can potentially lead to Anaphylactic Shock (AS). This immune reaction is characterized by an increase in Immunoglobulin-E (IgE) type of antibodies that bind with FcεRI receptors on mast cells to release inflammatory mediators. Various intracellular signaling molecules downstream of IgE/ FcεRI axis play a potential role in cytokine, chemokine and eicosanoid secretion as well as degranulation of immune cells causing vasodilation, vascular permeability, and reduction of intravascular volume leading to cardiovascular collapse. Here, we discuss the cellular machinery of anaphylaxis and the de novo paradigm shift in the cellular aspects of AS.     

Mapping of the CD23 Binding Site on Immunoglobulin E (IgE) and Allosteric Control of the IgE-Fc{epsilon}RI Interaction

IgE, the antibody that mediates allergic responses, acts as part of a self-regulating protein network. Its unique effector functions are controlled through interactions of its Fc region with two cellular receptors, FcεRI on mast cells and basophils and CD23 on B cells. IgE cross-linked by allergen triggers mast cell activation via FcεRI, whereas IgE-CD23 interactions control IgE expression levels. We have determined the CD23 binding site on IgE, using a combination of NMR chemical shift mapping and site-directed mutagenesis. We show that the CD23 and FcεRI interaction sites are at opposite ends of the Cε3 domain of IgE, but that receptor binding is mutually inhibitory, mediated by an allosteric mechanism. This prevents CD23-mediated cross-linking of IgE bound to FcεRI on mast cells and resulting antigen-independent anaphylaxis. The mutually inhibitory nature of receptor binding provides a degree of autonomy for the individual activities mediated by IgE-FcεRI and IgE-CD23 interactions.     

IgE by Itself Affects Mature Rat Mast Cell Preformed and De Novo-Synthesized Mediator Release and Amplifies Mast Cell Migratory Response

Background

Immunoglobulin E (IgE) binds to high affinity receptor FcεRI numerously expressed on mast cells. Recent findings have revealed that IgE by itself may regulate various aspects of mast cell biology, however, detailed data is still limited.

Methodology/Findings

Here, we have examined the influence of IgE alone, used at different concentrations, on mast cell activity and releasability. For the study we have employed in vivo differentiated mature tissue mast cells isolated from rat peritoneal cavity. Mast cells were exposed to IgE alone and then the release of preformed and de novo-synthesized mediators, surface FcεRI expression and mast cell migratory response were assessed. IgE by itself was found to up-regulate FcεRI expression and activate mast cells to degranulation, as well as de novo synthesis and release of cysteinyl leukotrienes and TNF. We have provided evidence that IgE alone also amplified spontaneous and CCL5- or TNF-induced migration of mast cells. Importantly, IgE was effective only at concentrations ≥ 3 µg/mL. A molecular basis investigation using an array of specific inhibitors showed that Src kinases, PLC/PLA₂, MAP kinases (ERK and p38) and PI3K were entirely or partially involved in IgE-induced mast cell response. Furthermore, IgE alone stimulated the phosphorylation of MAP kinases and PI3K in rat mast cells.

Conclusion

Our results clearly demonstrated that IgE by itself, at higher concentrations, influences mast cell activity and releasability. As there are different conditions when the IgE level is raised it might be supposed that in vivo IgE is one of the important factors modulating mast cell biology within tissues.     

Regulation of Fc?RI Signaling in Mast Cells by G Protein-coupled Receptor Kinase 2 and Its RH Domain

Agonist-induced phosphorylation of G protein-coupled receptors (GPCRs) by GPCR kinases (GRKs) promotes their desensitization and internalization. Here, we sought to determine the role of GRK2 on FcϵRI signaling and mediator release in mast cells. The strategies utilized included lentiviral shRNA-mediated GRK2 knockdown, GRK2 gene deletion (GRK2^flox/flox/cre recombinase) and overexpression of GRK2 and its regulator of G protein signaling homology (RH) domain (GRK2-RH). We found that silencing GRK2 expression caused ∼50% decrease in antigen-induced Ca²⁺ mobilization and degranulation but resulted in ablation of cytokine (IL-6 and IL-13) generation. The effect of GRK2 on cytokine generation does not require its catalytic activity but is mediated via the phosphorylation of p38 and Akt. Overexpression of GRK2 or its RH domain (GRK2-RH) enhanced antigen-induced mast cell degranulation and cytokine generation without affecting the expression levels of any of the FcϵRI subunits (α, β, and γ). GRK2 or GRK2-RH had no effect on antigen-induced phosphorylation of FcϵRIγ or Src but enhanced tyrosine phosphorylation of Syk. These data demonstrate that GRK2 modulates FcϵRI signaling in mast cells via at least two mechanisms. One involves GRK2-RH and modulates tyrosine phosphorylation of Syk, and the other is mediated via the phosphorylation of p38 and Akt.     

Wortmannin-Sensitive Phosphorylation, Translocation, and Activation of PLCγ1, but Not PLCγ2, in Antigen-stimulated RBL-2H3 Mast Cells

In RBL-2H3 tumor mast cells, cross-linking the high affinity IgE receptor (FcεRI) with antigen activates cytosolic tyrosine kinases and stimulates Ins(1,4,5)P₃ production. Using immune complex phospholipase assays, we show that FcεRI cross-linking activates both PLCγ1 and PLCγ2. Activation is accompanied by the increased phosphorylation of both PLCγ isoforms on serine and tyrosine in antigen-treated cells. We also show that the two PLCγ isoforms have distinct subcellular localizations. PLCγ1 is primarily cytosolic in resting RBL-2H3 cells, with low levels of plasma membrane association. After antigen stimulation, PLCγ1 translocates to the plasma membrane where it associates preferentially with membrane ruffles. In contrast, PLCγ2 is concentrated in a perinuclear region near the Golgi and adjacent to the plasma membrane in resting cells and does not redistribute appreciably after FcεRI cross-linking. The activation of PLCγ1, but not of PLCγ2, is blocked by wortmannin, a PI 3-kinase inhibitor previously shown to block antigen-stimulated ruffling and to inhibit Ins(1,4,5)P₃ synthesis. In addition, wortmannin strongly inhibits the antigen-stimulated phosphorylation of both serine and tyrosine residues on PLCγ1 with little inhibition of PLCγ2 phosphorylation. Wortmannin also blocks the antigen-stimulated translocation of PLCγ1 to the plasma membrane. Our results implicate PI 3-kinase in the phosphorylation, translocation, and activation of PLCγ1. Although less abundant than PLCγ2, activated PLCγ1 may be responsible for the bulk of antigen-stimulated Ins(1,4,5)P₃ production in RBL-2H3 cells.     

Contributions of F-BAR and SH2 Domains of Fes Protein Tyrosine Kinase for Coupling to the Fc?RI Pathway in Mast Cells

This study investigates the roles of Fer-CIP4 homology (FCH)-Bin/amphiphysin/Rvs (F-BAR) and SH2 domains of Fes protein tyrosine kinase in regulating its activation and signaling downstream of the high-affinity immunoglobulin G (IgE) receptor (FcɛRI) in mast cells. Homology modeling of the Fes F-BAR domain revealed conservation of some basic residues implicated in phosphoinositide binding (R113/K114). The Fes F-BAR can bind phosphoinositides and induce tubulation of liposomes in vitro. Mutation of R113/K114 to uncharged residues (RK/QQ) caused a significant reduction in phosphoinositide binding in vitro and a more diffuse cytoplasmic localization in transfected COS-7 cells. RBL-2H3 mast cells expressing full-length Fes carrying the RK/QQ mutation show defects in FcɛRI-induced Fes tyrosine phosphorylation and degranulation compared to cells expressing wild-type Fes. This correlated with reduced localization to Lyn kinase-containing membrane fractions for the RK/QQ mutant compared to wild-type Fes in mast cells. The Fes SH2 domain also contributes to Fes signaling in mast cells, via interactions with the phosphorylated FcɛRI β chain and the actin regulatory protein HS1. We show that Fes phosphorylates C-terminal tyrosine residues in HS1 implicated in actin stabilization. Thus, coordinated actions of the F-BAR and SH2 domains of Fes allow for coupling to FcɛRI signaling and potential regulation the actin reorganization in mast cells.Mast cells reside in connective and mucosal tissues and play a key protective role in the immune response to helminth infection (13, 21), sepsis (39), and snake or bee venoms (42). Mast cells express FcɛRI, which becomes sensitized to antigens or allergens upon immunoglobulin E (IgE) binding. Aggregation of FcɛRI by multivalent antigens causes the release of preformed mediators by degranulation and the de novo production of lipid mediators and cytokines (1, 52). Release of these mediators causes increased vascular permeability, leukocyte recruitment and activation, and inflammation (41). Aberrant mast cell activation is implicated in IgE-mediated type I hypersensitivity reactions including anaphylaxis, allergic rhinitis, and asthma (20). FcɛRI is a tetrameric receptor composed of an IgE-binding α chain and of β and γ chains containing immunoreceptor tyrosine-based activation motifs that become phosphorylated following multivalent antigen-mediated clustering of FcɛRI and activation of Src family protein tyrosine kinases (PTKs), primarily involving Lyn (51). Lyn phosphorylates and activates both positive effectors of FcɛRI signaling (e.g., Syk PTK) and key negative regulators (e.g., Shp-1 and SHIP) that serve to limit mast cell activation (28, 46, 69).Fes (the mammalian orthologue of the v-Fps and v-Fes oncoproteins from avian [57, 58] and feline [15, 56] retroviruses) and Fer are closely related PTKs that become activated following FcɛRI aggregation in mast cells (10). Surprisingly, FcɛRI-induced tyrosine phosphorylation of Fes and Fer does not require their kinase activities (55) and is almost entirely dependent on Lyn (67). Through the use of transgenic mouse models, evidence for both unique and redundant functions for Fes and Fer has been described in regulating hematopoiesis (55) and limiting the innate immune response (22, 40, 50, 72). In mast cells, we have shown that Fer promotes activation of p38 mitogen-activated protein kinase and chemotaxis of mast cells (10). We also found that Fer and Fes PTKs contribute to FcɛRI-evoked phosphorylation of platelet-endothelial cell adhesion molecule 1 (PECAM-1) (67).Each of the Fes and Fer PTKs is composed of an N-terminal regulatory domain containing a Fer-CIP4 homology (FCH) domain followed by several predicted coiled-coils (CC), a central SH2 domain, and C-terminal PTK domain (19). It is worth noting that early studies pointed toward an important role for the N-terminal domain of v-Fps for its transforming activity and membrane localization (5, 63). Several recent studies have defined the FCH and first CC domain (amino acids 1 to 300) in Fer, CIP4, and other pombe Cdc15 homology (PCH) family adaptor proteins as an F-BAR domain (also termed extended FCH or EFC domain) (reviewed in references 3 and 9). The F-BAR domain was found to constitute a novel phosphoinositide-binding domain that can promote tubulation of liposomes in vitro and membranes in vivo (27, 33, 66). The crystal structures of F-BAR domains from several PCH adaptors were recently solved (27, 59). The F-BAR module was shown to consist of a triple helical bundle that forms a homodimer, with a concave surface rich in basic residues that have recently been shown to contact phospholipids in curved membranes (16). In vitro studies using the Fer F-BAR domain have shown that the F-BAR domain binds strongly to phosphatidylinositol-4,5-bisphosphate [PI(4,5)P₂]; however, the Fer F-BAR is relatively weak compared with the adaptor protein FBP17 at inducing membrane tubulation (66). Liposome sedimentation assays have identified several conserved basic residues required for F-BAR domain binding to PI(4,5)P₂(66). The substitution of R113/K114 to glutamines (RK/QQ) in FBP17 reduced phosphoinositide binding by 80% (66). A recent electron cryoelectron microscopy study provided insights into binding of F-BAR dimers to flat and curved membranes via different binding faces (16). This study also confirmed that R113/K114 residues (in CIP4) constitute a site of direct interaction with the liposomes. Interestingly, microdomains of the plasma membrane rich in PI(4,5)P₂ are sites of dynamic actin assembly (47) and endocytosis (4, 31). Previous studies have described Fes localization to a variety of subcellular structures, including endocytic vesicles (71), the trans-Golgi apparatus (71), microtubules (37), and focal adhesions (44). The rapid activation of Fes and Fer PTKs upon FcɛRI aggregation on mast cells (10) would suggest that there is a mechanism by which Fes localizes at or near the plasma membrane. Phosphoinositide-binding via the F-BAR domain of Fes and Fer PTKs may promote their recruitment to the plasma membrane prior to their activation by cell surface receptors such as FcɛRI. The potential colocalization with endocytosis and actin assembly regulators may allow for regulation of receptor endocytosis or chemotaxis of mast cells by Fes/Fer PTKs. A recent study implicates Rab5 GTPase and its exchange factor RabGEF1/Rabex-5 in promoting internalization of FcɛRI following clustering by antigens (34). It is worth noting that defects in internalization of Toll-like receptor 4 and transferrin receptor were observed in Fes-deficient macrophages (48), and there is a potential role for Fes in regulating internalization of mast cell receptors.In this study, we provide novel insights into the phospholipid binding and liposome tubulating properties of the Fes F-BAR domain. Mutation of two conserved basic residues within the Fes F-BAR domain (RK/QQ) reduced phospholipid binding in vitro, and membrane localization in vivo. In transfected RBL-2H3 mast cells, the Fes harboring the RK/QQ mutation (Fes^RK/QQ) displayed reduced FcɛRI-evoked tyrosine phosphorylation compared to wild-type Fes (Fes^WT), which correlated with reduced localization to Lyn-containing membranes in mast cells. The SH2 domain of Fes was found to interact with several phosphoproteins in mast cells, including FcɛRI and HS1, an actin regulator and cortactin homologue. We found that Fes contributes to HS1 phosphorylation at C-terminal residues implicated in actin branch stabilization, and we present a model for how F-BAR-containing adaptor proteins and PTKs may coordinate actin-driven endocytosis in mast cells.     

Flotillin-1 regulates IgE receptor-mediated signaling in rat basophilic leukemia (RBL-2H3) cells

Cross-linking of high-affinity IgE receptors by multivalent Ag on mast cells (rat basophilic leukemia (RBL)-2H3) induces the phosphorylation of ITAM motifs of an IgE receptor by Src family tyrosine kinase, Lyn. The phosphorylation of IgE receptors is followed by a series of intracellular signals, such as Ca(2+) mobilization, MAPK activation, and degranulation. Therefore, Lyn is a key molecule in the activation of mast cells, but the molecular mechanisms for the activation of Lyn are still unclear. Recently, it is suggested that the localization of Lyn in lipid rafts is critical for its activation in several cell lines, although the precise mechanism is still unknown. In this study, we found that flotillin-1, which is localized in lipid rafts, is involved in the process of Lyn activation. We obtained flotillin-1 knockdown (KD)(2) rat basophilic leukemia (RBL)-2H3 cells, which express a low level of flotillin-1. In the flotillin-1 KD cells, we observed a significant decrease in Ca(2+) mobilization, the phosphorylation of ERKs, tyrosine phosphorylation of the gamma-subunit of IgE receptor, and IgE receptor-mediated degranulation. We also found that flotillin-1 is constitutively associated with Lyn in lipid rafts in RBL-2H3 cells, and Ag stimulation induced the augmentation of flotillin-1 binding to Lyn, resulting in enhancement of kinase activity of Lyn. These results suggest that flotillin-1 is an essential molecule in IgE receptor-mediated mast cell activation, and regulates the kinase activity of Lyn in lipid rafts.     

Cross-talk between Tetraspanin CD9 and Transmembrane Adaptor Protein Non-T Cell Activation Linker (NTAL) in Mast Cell Activation and Chemotaxis

Chemotaxis, a process leading to movement of cells toward increasing concentrations of chemoattractants, is essential, among others, for recruitment of mast cells within target tissues where they play an important role in innate and adaptive immunity. Chemotaxis is driven by chemoattractants, produced by various cell types, as well as by intrinsic cellular regulators, which are poorly understood. In this study we prepared a new mAb specific for the tetraspanin CD9. Binding of the antibody to bone marrow-derived mast cells triggered activation events that included cell degranulation, Ca²⁺ response, dephosphorylation of ezrin/radixin/moesin (ERM) family proteins, and potent tyrosine phosphorylation of the non-T cell activation linker (NTAL) but only weak phosphorylation of the linker for activation of T cells (LAT). Phosphorylation of the NTAL was observed with whole antibody but not with its F(ab)₂ or Fab fragments. This indicated involvement of the Fcγ receptors. As documented by electron microscopy of isolated plasma membrane sheets, CD9 colocalized with the high-affinity IgE receptor (FcϵRI) and NTAL but not with LAT. Further tests showed that both anti-CD9 antibody and its F(ab)₂ fragment inhibited mast cell chemotaxis toward antigen. Experiments with bone marrow-derived mast cells deficient in NTAL and/or LAT revealed different roles of these two adaptors in antigen-driven chemotaxis. The combined data indicate that chemotaxis toward antigen is controlled in mast cells by a cross-talk among FcϵRI, tetraspanin CD9, transmembrane adaptor proteins NTAL and LAT, and cytoskeleton-regulatory proteins of the ERM family.     

Lipid Raft-Dependent FcεRI Ubiquitination Regulates Receptor Endocytosis through the Action of Ubiquitin Binding Adaptors

The best characterized role for ubiquitination of membrane receptors is to negatively regulate signaling by targeting receptors for lysosomal degradation. The high affinity receptor for IgE (FcεRI) expressed on mast cells and basophils is rapidly ubiquitinated upon antigen stimulation. However, the nature and the role of this covalent modification are still largelly unknown. Here, we show that FcεRI subunits are preferentially ubiquitinated at multiple sites upon stimulation, and provide evidence for a role of ubiquitin as an internalization signal: under conditions of impaired receptor ubiquitination a decrease of receptor entry is observed by FACS analysis and fluorescence microscopy. We also used biochemical approaches combined with fluorescence microscopy, to demonstrate that receptor endocytosis requires the integrity of specific membrane domains, namely lipid rafts. Additionally, by RNA interference we demonstrate the involvement of ubiquitin-binding endocytic adaptors in FcεRI internalization and sorting. Notably, the triple depletion of Eps15, Eps15R and Epsin1 negatively affects the early steps of Ag-induced receptor endocytosis, whereas Hrs depletion retains ubiquitinated receptors into early endosomes and partially prevents their sorting into lysosomes for degradation. Our results are compatible with a scenario in which the accumulation of engaged receptor subunits into lipid rafts is required for receptor ubiquitination, a prerequisite for efficient receptor internalization, sorting and delivery to a lysosomal compartment.     

SH2 domain-containing inositol polyphosphate 5'-phosphatase is the main mediator of the inhibitory action of the mast cell function-associated antigen.

The mast cell function-associated Ag (MAFA) is a type II membrane glycoprotein originally found on the plasma membrane of rat mucosal-type mast cells (RBL-2H3 line). A C-type lectin domain and an immunoreceptor tyrosine-based inhibitory motif (ITIM) are located in the extracellular and intracellular domains of MAFA, respectively. MAFA clustering has previously been shown to suppress the secretory response of these cells to the FcepsilonRI stimulus. Here we show that the tyrosine of the ITIM undergoes phosphorylation, on MAFA clustering, that is markedly enhanced on pervanadate treatment of the cells. Furthermore, the Src homology 3 domain of the protein tyrosine kinase Lyn binds directly to a peptide containing nonphosphorylated MAFA ITIM and PAAP motif. Results of both in vitro and in vivo experiments suggest that Lyn is probably responsible for this ITIM phosphorylation, which increases the Src homology domain 2 (SH2) affinity of Lyn for the peptide. In vitro measurements established that tyrosine-phosphorylated MAFA ITIM peptides also bind the SH2 domains of inositol 5'-phosphatase (SHIP) as well as protein tyrosine phosphatase-2. However, the former single domain is bound 8-fold stronger than both of the latter. Further support for the role of SHIP in the action of MAFA stems from in vivo experiments in which tyrosine-phosphorylated MAFA was found to bind primarily SHIP. In RBL-2H3 cells overexpressing wild-type SHIP, MAFA clustering causes markedly stronger inhibition of the secretory response than in control cells expressing normal SHIP levels or cells overexpressing either wild-type protein tyrosine phosphatase-2 or its dominant negative form. In contrast, on overexpression of the SH2 domain of SHIP, the inhibitory action of MAFA is essentially abolished. Taken together, these results suggest that SHIP is the primary enzyme responsible for mediating the inhibition by MAFA of RBL-2H3 cell response to the FcepsilonRI stimulus.