Mutant RBL mast cells defective in Fc epsilon RI signaling and lipid raft biosynthesis are reconstituted by activated Rho-family GTPases

Characterization of defects in a variant subline of RBL mast cells has revealed a biochemical event proximal to IgE receptor (Fc epsilon RI)-stimulated tyrosine phosphorylation that is required for multiple functional responses. This cell line, designated B6A4C1, is deficient in both Fc epsilon RI-mediated degranulation and biosynthesis of several lipid raft components. Agents that bypass receptor-mediated Ca(2+) influx stimulate strong degranulation responses in these variant cells. Cross-linking of IgE-Fc epsilon RI on these cells stimulates robust tyrosine phosphorylation but fails to mobilize a sustained Ca(2+) response. Fc epsilon RI-mediated inositol phosphate production is not detectable in these cells, and failure of adenosine receptors to mobilize Ca(2+) suggests a general deficiency in stimulated phospholipase C activity. Antigen stimulation of phospholipases A(2) and D is also defective. Infection of B6A4C1 cells with vaccinia virus constructs expressing constitutively active Rho family members Cdc42 and Rac restores antigen-stimulated degranulation, and active Cdc42 (but not active Rac) restores ganglioside and GPI expression. The results support the hypothesis that activation of Cdc42 and/or Rac is critical for Fc epsilon RI-mediated signaling that leads to Ca(2+) mobilization and degranulation. Furthermore, they suggest that Cdc42 plays an important role in the biosynthesis and expression of certain components of lipid rafts.     

Molecular perspective of antigen-mediated mast cell signaling

Antigen-mediated cross-linking of the high affinity receptor for IgE (Fc epsilon RI), in the plasma membrane of mast cells, is the first step in the allergic immune response. This event triggers the phosphorylation of specific tyrosines in the cytoplasmic segments of the beta and gamma subunits of Fc epsilon RI by the Src tyrosine kinase Lyn, which is anchored to the inner leaflet of the plasma membrane. Lyn-induced phosphorylation of Fc epsilon RI occurs in a cholesterol-dependent manner, leading to the hypothesis that cholesterol-rich domains, or "lipid rafts," may act as functional platforms for IgE receptor signaling. Testing this hypothesis under physiological conditions remains challenging because of the notion that these functional domains are likely transient and much smaller than the diffraction limit of optical microscopy. Here we use ultrafast fluorescence dynamics to investigate the correlation between nanostructural changes in the plasma membrane (labeled with 1,1'-dioctadecyl-3,3,3',3'-tetramethyl-indocarbocyanine (diI-C18)) and IgE-Fc epsilon RI cross-linking in adherent RBL mast cells stimulated with multivalent antigen. Time-dependent two-photon fluorescence lifetime imaging microscopy of diI-C18 shows changes in lifetime that agree with the kinetics of stimulated tyrosine phosphorylation of Fc epsilon RI, the first identifiable biochemical step of the allergic response, under the same conditions. In addition, two-photon fluorescence lifetime imaging microscopy of Alexa Fluor 488-labeled IgE indicates that F?rster resonance energy transfer occurs with diI-C18 in the plasma membrane. Our live cell studies provide direct evidence for the association of IgE-Fc epsilon RI with specialized cholesterol-rich domains within approximately 4-nm proximity and with an energy transfer efficiency of 0.22 +/- 0.01 at maximal association during IgE receptor signaling.     

Investigation of early events in Fc epsilon RI-mediated signaling using a detailed mathematical model

Aggregation of Fc epsilon RI on mast cells and basophils leads to autophosphorylation and increased activity of the cytosolic protein tyrosine kinase Syk. We investigated the roles of the Src kinase Lyn, the immunoreceptor tyrosine-based activation motifs (ITAMs) on the beta and gamma subunits of Fc epsilon RI, and Syk itself in the activation of Syk. Our approach was to build a detailed mathematical model of reactions involving Fc epsilon RI, Lyn, Syk, and a bivalent ligand that aggregates Fc(epsilon)RI. We applied the model to experiments in which covalently cross-linked IgE dimers stimulate rat basophilic leukemia cells. The model makes it possible to test the consistency of mechanistic assumptions with data that alone provide limited mechanistic insight. For example, the model helps sort out mechanisms that jointly control dephosphorylation of receptor subunits. In addition, interpreted in the context of the model, experimentally observed differences between the beta- and gamma-chains with respect to levels of phosphorylation and rates of dephosphorylation indicate that most cellular Syk, but only a small fraction of Lyn, is available to interact with receptors. We also show that although the beta ITAM acts to amplify signaling in experimental systems where its role has been investigated, there are conditions under which the beta ITAM will act as an inhibitor.     

Cross-correlation analysis of inner-leaflet-anchored green fluorescent protein co-redistributed with IgE receptors and outer leaflet lipid raft components

To investigate the structural basis for membrane interactions that occur between Lyn tyrosine kinase and IgE-Fc(epsilon)RI or other components of lipid rafts, we prepared a green fluorescent protein analog of Lyn (PM-EGFP) and used cross-correlation analysis to quantify co-redistributions of aggregates that occur after IgE-Fc(epsilon)RI is cross-linked on the cell surface. PM-EGFP, which contains minimally the palmitoylation and myristoylation sites on Lyn, was compared with another inner leaflet probe, EGFP-GG, which contains a prenylation site and a polybasic sequence similar to K-ras. Confocal fluorescence microscopy was used to examine co-redistributions of these inner leaflet components with IgE-Fc(epsilon)RI and outer leaflet raft components, ganglioside GD1b and glycosylphosphotidylinositol-linked Thy-1, under conditions where the latter were cross-linked externally to form large patches at the cell surface. The cross-correlation analysis was developed and characterized with simulations representing cell surface distributions, and parameters from the cross-correlation curves, rho(o) (peak height) and A (peak area), were shown to be reliable measures of the extent of co-redistributed aggregates and their size. Cross-correlation analysis was then applied to quantify co-redistributions of the fluorescently labeled inner and outer leaflet components on RBL-2H3 cells. As visually observed and parameterized in this manner, PM-EGFP was found to co-redistribute with lipid rafts significantly more than EGFP-GG or an endogenous prenylated protein, Cdc42. These quantitative results are consistent with previous analyses of Lyn co-redistributions and support the hypothesis that the functionally important interaction of Lyn with cross-linked IgE- Fc(epsilon)RI is due to their mutual co-association with lipid rafts.     

Reconstitution of interactions between tyrosine kinases and the high affinity IgE receptor which are controlled by receptor clustering.

High affinity IgE receptor (Fc epsilon RI) signaling after contact with antigen occurs in response to receptor clustering. This paper describes methodology, based on vaccinia virus driven protein expression, for probing signaling pathways and its application to Fc epsilon RI interactions with the lyn and syk tyrosine kinases. Reconstitution of the complete tetrameric Fc epsilon RI receptor, lyn and syk in a non-hematopoietic 'null' cell line is sufficient to reconstruct clustering-controlled receptor tyrosine phosphorylation and activation of syk, without apparent requirement for hematopoietic specific phosphatases. The src family kinase lyn phosphorylates Fc epsilon RI in response to receptor clustering, resulting in syk binding to the phosphorylated Fc epsilon RI. Lyn also participates in the tyrosine phosphorylation and activation of syk in a manner which is dependent on phosphorylated Fc epsilon RI. Using overexpression of active and dominant negative syk proteins in a mast cell line which naturally expresses Fc epsilon RI, we corroborate syk's role downstream of receptor phosphorylation, and demonstrate that syk SH2 domains protect receptor ITAMs from ongoing dephosphorylation. Based on these results, we propose that receptor clustering controls lyn-mediated Fc epsilon RI tyrosine phosphorylation by shifting a balance between phosphorylation and dephosphorylation towards accumulation of tyrosine phosphorylated Fc epsilon RI. Fc epsilon RI tyrosine phosphorylation functions to bring syk into a microenvironment where it becomes tyrosine phosphorylated and activated, thereby allowing clustering to indirectly control syk activity.     

Inactivation of c-Cbl or Cbl-b differentially affects signaling from the high affinity IgE receptor

The Cbl family of proteins negatively regulate signaling from tyrosine kinase-coupled receptors. Among the three members of this family, only c-Cbl and Cbl-b are expressed in hemopoietic cells. To examine the role of c-Cbl and Cbl-b in Fc epsilon RI signaling, mast cell cultures from wild-type, c-Cbl(-/-), and Cbl-b(-/-) mice were generated. Cell growth rates and cell surface expression of Fc epsilon RI were similar in the different cell populations. Compared with control cells, Cbl-b inactivation resulted in increases in Fc epsilon RI-induced Ca(2+) response and histamine release. Fc epsilon RI-induced tyrosine phosphorylation of total cellular proteins, Syk, and phospholipase C-gamma was also enhanced by Cbl-b deficiency, whereas receptor-initiated phosphorylation of Vav, JNK, and p38 kinases was not changed in these cells. In contrast to Cbl-b, c-Cbl deficiency had no detectable effect on Fc epsilon RI-induced histamine release or on the phosphorylation of total cellular proteins or Syk. The absence of c-Cbl increased the phosphorylation of ERK after receptor stimulation, but resulted in slightly reduced p38 phosphorylation and Ca(2+) response. These results suggest that Cbl-b and c-Cbl have divergent effects on Fc epsilon RI signal transduction and that Cbl-b, but not c-Cbl, functions as a negative regulator of Fc epsilon RI-induced degranulation.     

How does the plasma membrane participate in cellular signaling by receptors for immunoglobulin E?

Accumulating evidence strongly supports the view that the plasma membrane participates in transmembrane signaling by IgE-receptors (IgE-Fc epsilon RI) through the formation of lipid-based domains, also known as rafts. Ongoing biochemical and biophysical experiments investigate the composition, structure, and dynamics of the corresponding membrane components and how these are related to functional coupling between Fc epsilon RI and Lyn tyrosine kinase to initiate signaling in mast cells.     

Ligand stimulation of transfected and endogenous growth factor receptors enhances cytokine production by mast cells.

IL-3 dependent mast cell lines produce cytokines in response to Fc receptor cross-linkage or to ionomycin. In this study we have observed that cells pre-cultured in IL-3 produce 10-100 times more cytokine after receptor cross-linkage in comparison with IL-4 pre-cultured cells. Although several hematopoietin receptors, including those for IL-3, IL-4 and EPO, do not contain tyrosine kinase domains, their occupancy with ligand causes tyrosine phosphorylation of specific cellular substrates. Therefore, the contribution of tyrosine kinase activation to the ability of an IL-3 dependent mast cell line, CFTL-15, to produce cytokines was analyzed. The CFTL-15 cells were transfected with growth factor receptors containing ligand-inducible tyrosine kinase domains (EGFR and PDGFR, and CSF-IR) or with the EPOR. All of the transfectants were able to proliferate in response to IL-3 or to their respective growth factor and to produce IL-3 in response to IgE receptor cross-linkage. Stimulation of the EGFR and PDGFR transfectants with their respective ligands resulted in the production of IL-3, IL-6, and GM-CSF. Stimulation of the CSF-1R or EPOR transfectants with growth factor alone failed to induce cytokine production. However, in co-stimulation assays each of the growth factors enhanced the amount of cytokine produced in response to Fc epsilon RI cross-linkage. The ability of these stimuli to induce tyrosine phosphorylation in the transfectants was analyzed. Fc epsilon RI cross-linkage in the transfectants routinely induced the tyrosine phosphorylation of 145, 86 and 72 kDa proteins, with occasional phosphorylation of 55, 52, and 40 kDa proteins.(ABSTRACT TRUNCATED AT 250 WORDS)     

Trivalent ligands with rigid DNA spacers reveal structural requirements for IgE receptor signaling in RBL mast cells.

Antigen-mediated cross-linking of IgE bound to its receptor, FcRI, stimulates degranulation, phospholipid metabolism, and cytokine production in mast cells and basophils to initiate inflammatory and allergic responses. Previous studies suggested that spatial organization of the clustered receptors affects the assembly of the transmembrane signaling complexes. To investigate systematically the structural constraints in signal initiation, we utilized rigid double-stranded DNA scaffolds to synthesize ligands with tunable lengths. We characterized a series of symmetric trivalent DNA ligands with rigid spacing between 2,4-dinitrophenyl (DNP) haptenic groups in the range of 5-15 nm. These ligands all bind to anti-DNP IgE on RBL mast cells with similar avidity, and they all cross-link IgE-FcRI complexes effectively. We observe length-dependent stimulation of tyrosine phosphorylation of FcRI beta and gamma subunits and the adaptor protein LAT: the shortest ligand is approximately 5-10-fold more potent than the longest. Stimulated Ca2+ mobilization and degranulation also exhibits kinetics and magnitudes that differ as a function of ligand length. In contrast, tyrosine phosphorylation of phospholipase Cgamma1 and consequent Ca2+ release from intracellular stores do not show this dependence on ligand length. Our results with these rigid, DNA-based ligands provide direct support for receptor transphosphorylation as a key step in amplified signaling leading to degranulation, and they further reveal branching of pathways in signaling events.     

Protein tyrosine phosphorylation induced via the IgG receptors Fc gamma Ri and Fc gamma RII in the human monocytic cell line THP-1.

We have investigated the role of protein tyrosine phosphorylation in transmembrane signaling via the IgG receptors Fc gamma RI and Fc gamma RII in the human monocytic cell line THP-1. Fc gamma RI and Fc gamma RII were selectively engaged using the anti-Fc gamma RI mAb 197 (IgG2a) and the anti-Fc gamma RII mAb IV.3 (IgG2b). Addition to cells of mAb 197, but not addition of IgG2a mAb of irrelevant specificity, resulted in the rapid induction of cytoplasmic protein tyrosine phosphorylation as assessed by antiphosphotyrosine immunoblotting. A similar pattern of tyrosine phosphorylation was induced by mAb IV.3, but not by control IgG2b mAb. The induction of tyrosine phosphorylation by anti-Fc gamma R mAb was not dependent on antibody Fc region-FcR interactions, because tyrosine phosphorylation was also induced by cross-linked anti-Fc gamma RI F(ab')2 fragments and by cross-linked anti-Fc gamma RII Fab fragments. To investigate the relationship of Fc gamma R-induced tyrosine phosphorylation and activation of phospholipase C, which is known to follow Fc gamma R engagement, we assessed the effect of the tyrosine kinase inhibitor herbimycin A on Fc gamma R-induced Ca2+ flux. Herbimycin A strongly inhibited cellular Ca2+ flux induced by mAb 197, but did not inhibit Ca2+ flux induced by aluminum fluoride, suggesting that tyrosine phosphorylation may be important in regulating Fc gamma R-mediated activation of phospholipase C. Consistent with this, mAb 197 induced rapid phosphorylation of the gamma-1 isoform of phospholipase C. Finally, herbimycin A strongly inhibited the induction of TNF-alpha mRNA accumulation by Fc gamma R cross-linking. These results suggest that protein tyrosine phosphorylation may play an important role in the activation of phospholipase C and in the induction of monokine gene expression that follows engagement of Fc gamma R in human monocytes.     

Kinetics of ligand binding to the type 1 Fc epsilon receptor on mast cells

Rates of association and dissociation of several specific monovalent ligands to and from the type I Fc epsilon receptor (Fc epsilon RI) were measured on live mucosal type mast cells of the rat line RBL-2H3. The ligands employed were a monoclonal murine IgE and Fab fragments prepared from three different, Fc epsilon RI-specific monoclonal IgG class antibodies. These monoclonals (designated H10, J17, and F4) were shown previously to trigger mediator secretion by RBL-2H3 mast cells upon binding to and dimerization of the Fc epsilon RI. Analysis of the kinetics shows that the minimal mechanism to which all data can be fitted involves two consecutive steps: namely, ligand binding to a low-affinity state of the receptor, followed by a conformational transition into a second, higher affinity state h of the receptor-ligand complex. These results resolve the recently noted discrepancy between the affinity of IgE binding to the Fc epsilon RI as determined by means of binding equilibrium measurements [Ortega et al. (1988) EMBO J. 7, 4101] and the respective parameter derived from the ratio of the rate constant of rat IgE dissociation and the initial rate of rat IgE association [Wank et al. (1983) Biochemistry 22, 954]. The probability of undergoing the conformational transition differs for the four different Fc epsilon RI-ligand complexes: while binding of Fab-H10 and IgE favors the h state, binding of Fab-J17 and Fab-F4 preferentially maintains the low-affinity 1 state (at 25 degrees C). The temperature dependence of the ligand interaction kinetics with the Fc epsilon RI shows that the activation barrier for ligand association is determined by positive enthalpic and entropic contributions. The activation barrier of the 1----h transition, however, has negative enthalpic contributions counteracted by a decrease in activation entropy. The h----1 transition encounters a barrier that is predominantly entropic and similar for all ligands employed, thus suggesting that the Fc epsilon RI undergoes a similar conformational transition upon binding any of the ligands.     

Tyrosine phosphorylation is required for mast cell activation by Fc epsilon RI cross-linking.

We investigated the possible role of tyrosine phosphorylation in the activation process of mast cells by cross-linking of cell-bound IgE antibodies. Bone marrow-derived mouse mast cells (BMMC) were sensitized with mouse IgE antiDNP mAb and then challenged with multivalent Ag DNP conjugates of human serum albumin. Analysis of phosphotyrosine-containing proteins in their lysates by SDS-PAGE and immunoblotting revealed that cross-linking of cell-bound IgE antibodies induced a marked increase in tyrosine phosphorylation of several proteins. To obtain direct evidence for activation of protein-tyrosine kinases (PTK), phosphotyrosine-containing proteins in lysates of mast cells were affinity purified, and kinase activity of the immunoprecipitates was assessed by an in vitro kinase assay. The results clearly showed activation of PTK upon cross-linking of Fc epsilon RI. Activation of PTK was not detected by the same assay when the sensitized BMMC were challenged with monovalent DNP-lysine. Treatment of sensitized BMMC with either Ca2+ ionophore or PMA failed to induce the activation of PTK. A representative IgE-independent secretagogue, thrombin, induced histamine release from BMMC but failed to induce activation of PTK. The results excluded the possibility that PTK activation is the consequence of an increase in intracellular Ca2+ or activation of protein kinase C. Addition of genistein, a PTK inhibitor, to sensitized BMMC before Ag challenge inhibited not only Ag-induced PTK activation, but also inositol 1,4,5-trisphosphate production, and histamine release in a similar dose-response relationship. Other PTK inhibitors, such as lavendustin A and tyrphostin RG50864, also inhibited the Ag-induced activation of PTK and histamine release. The results collectively suggest that activation of PTK is an early event upstream of the activation of phospholipase C, and is involved in transduction of IgE-dependent triggering signals to mediator release.     

Downstream of kinase, p62(dok), is a mediator of Fc gamma IIB inhibition of Fc epsilon RI signaling

The low-affinity receptor for IgG, Fc gamma RIIB, is expressed widely in the immune system and functions to attenuate Ag-induced immune responses. In mast cells, coaggregation of Fc gamma RIIB with the high-affinity IgE receptor, Fc epsilon RI, leads to inhibition of Ag-induced degranulation and cytokine production. Fc gamma RIIB inhibitory activity requires a conserved motif within the Fc gamma RIIB cytoplasmic domain termed the immunoreceptor tyrosine-based inhibition motif. When coaggregated with an activating receptor (e.g., Fc epsilon RI, B cell Ag receptor), Fc gamma RIIB is rapidly phosphorylated on tyrosine and recruits the SH2 domain-containing inositol 5-phosphatase (SHIP). However, the mechanisms by which SHIP mediates Fc gamma RIIB inhibitory function in mast cells remain poorly defined. In this report we demonstrate that Fc gamma RIIB coaggregation with Fc epsilon RI stimulates enhanced SHIP tyrosine phosphorylation and association with Shc and p62(dok). Concurrently, enhanced p62(dok) tyrosine phosphorylation and association with RasGAP are observed, suggesting that SHIP may mediate Fc gamma RIIB inhibitory function in mast cells via recruitment of p62(dok) and RasGAP. Supporting this hypothesis, recruitment of p62(dok) to Fc epsilon RI is sufficient to inhibit Fc epsilon RI-induced calcium mobilization and extracellular signal-regulated kinase 1/2 activation. Interestingly, both the amino-terminal pleckstrin homology and phosphotyrosine binding domains and the carboxyl-terminal proline/tyrosine-rich region of p62(dok) can mediate inhibition, suggesting activation of parallel downstream signaling pathways that converge at extracellular signal-regulated kinase 1/2 activation. Finally, studies using gene-ablated mice indicate that p62(dok) is dispensable for Fc gamma RIIB inhibitory signaling in mast cells. Taken together, these data suggest a role for p62(dok) as a mediator of Fc gamma RIIB inhibition of Fc epsilon RI signal transduction in mast cells.     

Cross-linking of the high affinity Fc receptor for human immunoglobulin G1 triggers transient activation of NADPH oxidase activity. Continuous oxidase activation requires continuous de novo receptor cross-linking

Cross-linking of the high affinity Fc receptor for human immunoglobulin G1 (Fc gamma RI) on U937 cells triggered superoxide anion (O-2) release. This was accomplished by the binding of an Fc gamma RI-specific monoclonal antibody, mAb 32, followed by cross-linking of the mAb on the cell with anti-mouse IgG F(ab')2 by Fc gamma RI-specific mAbs 32 and 22 used as an equimolar mixture or by Fc gamma RI-specific mAb 197 (a murine IgG2a and thus a multivalent ligand for Fc gamma RI) alone. At subsaturating concentrations of the Fc gamma RI-cross-linking ligands, O2- generation was continuous over relatively long intervals. However, saturating concentrations triggered an often substantial but always transient O2- burst. This transient burst of oxidase activity ceased with maximal ligand accumulation on the cell. Cells in which oxidase activity had ceased could be restimulated using phorbol 12-myristate 13-acetate or aggregated human IgG1, indicating that cessation of O2- generation was not due to a generalized exhaustion or inhibition of the NADPH oxidase pathway. Cells incubated in subsaturating concentrations of cross-linking antibodies continued to release O2- until binding of the ligand ceased. In addition, the rates of O2- production and ligand accumulation were the same. Thus, continuous O2- production appeared to be dependent upon continuous de novo formation of cross-linked and activated Fc gamma RI. Furthermore, the mol of O2- released in response to Fc gamma RI cross-linking by the multivalent ligand mAb 197 were directly proportional to the mol of mAb bound over a range of saturating and subsaturating concentrations. This evidence suggests a quantal relationship between each Fc gamma RI activated (cross-linked) and the resultant oxidase activity and supports a "rate" model for the activation of this response. Thus, each Fc gamma RI entering the pool of activated receptors probably makes a unitary contribution to the signal. An additional finding showed that cross-linked Fc gamma RI became associated with the cell cytoskeleton and that this association was also transient. Dissociation of Fc gamma RI from its cytoskeletal attachment occurred well after cessation of O2- production.     

Activation of the high-affinity immunoglobulin E receptor Fc epsilon RI in RBL-2H3 cells is inhibited by Syk SH2 domains.

Antigen-mediated aggregation of the high-affinity receptor for immunoglobulin E, Fc epsilon RI, results in the activation of multiple signaling pathways, leading to the release of mediators of the allergic response. One of the earliest responses to receptor stimulation is the tyrosine phosphorylation of the beta and gamma subunits of Fc epsilon RI and the association of the tyrosine kinase Syk with the phosphorylated receptor. This association is mediated by the SH2 domains of Syk and is believed to be critical for activating signaling pathways resulting in mediator release. To examine the importance of the interaction of Syk with Fc epsilon RI in signaling events following receptor activation, we introduced a protein containing the SH2 domains of Syk into streptolysin O-permeabilized RBL-2H3 cells. The Syk SH2 domains completely inhibited degranulation and leukotriene production following receptor aggregation, and they blocked the increase in protein tyrosine phosphorylation observed after receptor activation. Inhibition was specific for Fc epsilon RI-mediated signaling, since degranulation of cells activated by alternative stimuli was not blocked by the Syk SH2 domains. A protein containing a point mutation in the carboxy-terminal SH2 domain which abolishes phosphotyrosine binding was not inhibitory. In addition, inhibition of degranulation was reversed by pretreatment of the SH2 domains with a tyrosine phosphorylated peptide corresponding to the tyrosine-based activation motif found in the gamma subunit of Fc epsilon RI, the nonphosphorylated peptide had no effect. The association of Syk with the tyrosine-phosphorylated gamma subunit of the activated receptor was blocked by the Syk SH2 domains, and deregulation in cells activated by clustering of Syk directly without Fc epsilon RI aggregation was not affected by the Syk SH2 domains. These results demonstrate that the association of Syk with the activated Fc epsilon RI is critical for both early and late events following receptor activation and confirm the key role Syk plays in signaling through the high-affinity IgE receptor.     

Tyrosine phosphorylation and activation of Bruton tyrosine kinase upon Fc epsilon RI cross-linking.

Tyrosine phosphorylation of several cellular proteins is one of the earliest signaling events induced by cross-linking of the high-affinity receptor for immunoglobulin E (Fc epsilon RI) on mast cells or basophils. Tyrosine kinases activated during this process include the Src family kinases, Lyn, c-Yes, and c-Src, and members of another subfamily, Syk and PTK72 (identical or highly related to Syk). Recently, some of us described two novel tyrosine kinases, Emb and Emt, whose expression was limited to subsets of hematopoietic cells, including mast cells. Emb turned out to be identical to Btk, a gene product defective in human X-linked agammaglobulinemia and in X-linked immunodeficient (xid) mice. Here we report that Fc epsilon RI cross-linking induced rapid phosphorylation on tyrosine, serine, and threonine residues and activation of Btk in mouse bone marrow-derived mast cells. A small fraction of Btk translocated from the cytosol to the membrane compartment following receptor cross-linking. Tyrosine phosphorylation of Btk was not induced by either a Ca2+ ionophore (A23187), phorbol 12-myristate 13-acetate, or a combination of the two reagents. Co-immunoprecipitation between Btk and receptor subunit beta or gamma was not detected. The data collectively suggest that Btk is not associated with Fc epsilon but that its activation takes place prior to protein kinase C activation and plays a novel role in the Fc epsilon RI signaling pathway.     

Peroxisome proliferator-activated receptor ligands negatively regulate the expression of the high-affinity IgE receptor Fc epsilon RI in human basophilic KU812 cells

The high-affinity IgE receptor Fc epsilon RI is expressed on the cell surface of mast cells and basophils, and plays a central role in IgE-mediated inflammatory reactions. Recently, peroxisome proliferator-activated receptors (PPARs) have been implicated in the anti-inflammatory response. To investigate a possible role for PPAR in human basophils, the effect of PPAR ligands on Fc epsilon RI expression in human basophilic KU812 cells was studied. The PPARalpha ligand, leukotriene B(4), did not affect the cell surface expression of Fc epsilon RI. However, prostaglandin (PG) A(1) and 15-deoxy-Delta(12,14) PGJ(2) (15d-PGJ(2)), which are PPARbeta and gamma ligands, respectively, were both able to decrease Fc epsilon RI expression. Treatment with PGA(1) or 15d-PGJ(2) separately also reduced histamine release from KU812 cells in response to cross-linkage of Fc epsilon RI. In addition, RT-PCR analysis showed that KU812 cells expressed the mRNA for PPARalpha, beta, and gamma, indicating that PPARbeta or gamma may negatively regulate the cell activation via Fc epsilon RI. Cells treated with 15d-PGJ(2) expressed lower levels of Fc epsilon RI alpha and gamma mRNA, and PGA(1) treatment decreased the level of Fc epsilon RI gamma mRNA. These results suggest that the suppression of Fc epsilon RI expression by PPARs may be due to the down-regulation of Fc epsilon RI alpha or gamma mRNA.     

Forced expression of the Fc receptor gamma-chain renders human T cells hyperresponsive to TCR/CD3 stimulation

High level expression of Fc epsilon RI gamma chain replaces the deficient TCR zeta-chain and contributes to altered TCR/CD3-mediated signaling abnormalities in T cells of patients with systemic lupus erythematosus. Increased responsiveness to Ag has been considered to lead to autoimmunity. To test this concept, we studied early signaling events and IL-2 production in fresh cells transfected with a eukaryotic expression vector encoding the Fc epsilon RI gamma gene. We found that the overexpressed Fc epsilon RI gamma chain colocalizes with the CD3 epsilon chain on the surface membrane of T cells and that cross-linking of the new TCR/CD3 complex leads to a dramatic increase of intracytoplasmic calcium concentration, protein tyrosine phosphorylation, and IL-2 production. We observed that overexpression of Fc epsilon RI gamma is associated with increased phosphorylation of Syk kinase, while the endogenous TCR zeta-chain is down-regulated. We propose that altered composition of the CD3 complex leads to increased T cell responsiveness to TCR/CD3 stimulation and sets the biochemical grounds for the development of autoimmunity.     

Signal transduction by the cytoplasmic domains of Fc epsilon RI-gamma and TCR-zeta in rat basophilic leukemia cells.

The gamma subunit of the high affinity IgE receptor, Fc epsilon RI, is a member of a family of proteins which form disulfide-linked dimers. This family also includes the zeta- and eta-chains of the T cell receptor. Engagement of Fc epsilon RI activates src-related protein tyrosine kinases in basophils and mast cells. However, the role of individual subunits of Fc epsilon RI in this activation is still not known. In an effort to determine the function of Fc epsilon RI-gamma, we used chimeric proteins containing the extracellular and transmembrane domains of the alpha chain of the human interleukin 2 receptor (Tac) and the cytoplasmic domains of either T cell receptor-zeta or Fc epsilon RI-gamma. We show that while cross-linking of the Tac chimeras in the rat basophilic leukemia cell line RBL-2H3 resulted in the tyrosine phosphorylation of a subset of proteins and a portion of the degranulation normally observed after Fc epsilon RI-mediated stimulation, no detectable activation of p56lyn or pp60c-src was observed. In contrast, an apparent transient deactivation of these two kinases was observed after Tac chimera cross-linking. These observations suggest that Fc epsilon RI-gamma is responsible for some, but not all, of the signaling that occurs after engagement of its receptor, and that other receptor subunits may also play important roles in this signaling process.     

High resolution mapping of mast cell membranes reveals primary and secondary domains of Fc(epsilon)RI and LAT

In mast cells, cross-linking the high-affinity IgE receptor (Fc(epsilon)RI) initiates the Lyn-mediated phosphorylation of receptor ITAMs, forming phospho-ITAM binding sites for Syk. Previous immunogold labeling of membrane sheets showed that resting Fc(epsilon)RI colocalize loosely with Lyn, whereas cross-linked Fc(epsilon)RI redistribute into specialized domains (osmiophilic patches) that exclude Lyn, accumulate Syk, and are often bordered by coated pits. Here, the distribution of Fc(epsilon)RI beta is mapped relative to linker for activation of T cells (LAT), Grb2-binding protein 2 (Gab2), two PLCgamma isoforms, and the p85 subunit of phosphatidylinositol 3-kinase (PI3-kinase), all implicated in the remodeling of membrane inositol phospholipids. Before activation, PLCgamma1 and Gab2 are not strongly membrane associated, LAT occurs in small membrane clusters separate from receptor, and PLCgamma2, that coprecipitates with LAT, occurs in clusters and along cytoskeletal cables. After activation, PLCgamma2, Gab2, and a portion of p85 colocalize with Fc(epsilon)RI beta in osmiophilic patches. LAT clusters enlarge within 30 s of receptor activation, forming elongated complexes that can intersect osmiophilic patches without mixing. PLCgamma1 and another portion of p85 associate preferentially with activated LAT. Supporting multiple distributions of PI3-kinase, Fc(epsilon)RI cross-linking increases PI3-kinase activity in anti-LAT, anti-Fc(epsilon)RIbeta, and anti-Gab2 immune complexes. We propose that activated mast cells propagate signals from primary domains organized around Fc(epsilon)RIbeta and from secondary domains, including one organized around LAT.