High-affinity IgE Receptor Fc Research Articles

Accelerated disassembly of IgE-receptor complexes by a disruptive macromolecular inhibitor.

IgE antibodies bind the high affinity IgE Fc receptor (FcεRI), found primarily on mast cells and basophils, and trigger inflammatory cascades of the allergic response1,2. Inhibitors of IgE:FcεRI binding have been identified and an anti-IgE therapeutic antibody (omalizumab) is used to treat severe allergic asthma3,4. However, preformed IgE:FcεRI complexes that prime cells prior to allergen exposure dissociate extremely slowly5 and cannot be disrupted by strictly competitive inhibitors. IgE-Fc conformational flexibility indicated that inhibition could be mediated by allosteric or other non-classical mechanisms6–8. Here we demonstrate that an engineered protein inhibitor, DARPin E2_799–11, acts through a non-classical inhibition mechanism, not only blocking IgE:FcεRI interactions, but actively stimulating the dissociation of preformed ligand-receptor complexes. The structure of the E2_79:IgE-Fc3-4 complex predicts the presence of two non-equivalent E2_79 sites in the asymmetric IgE:FcεRI complex, with Site 1 distant from the receptor and Site 2 exhibiting partial steric overlap. While the structure is suggestive of an allosteric inhibition mechanism, mutational studies and quantitative kinetic modeling indicate that E2_79 acts through a facilitated dissociation mechanism at Site 2 alone. These results demonstrate that high affinity IgE:FcεRI complexes can be actively dissociated to block the allergic response and suggest that protein:protein complexes may be more generally amenable to active disruption by macromolecular inhibitors.

An Engineered Disulfide Bond Reversibly Traps the IgE-Fc3–4 in a Closed, Nonreceptor Binding Conformation

IgE antibodies interact with the high affinity IgE Fc receptor, FcεRI, and activate inflammatory pathways associated with the allergic response. The IgE-Fc region, comprising the C-terminal domains of the IgE heavy chain, binds FcεRI and can adopt different conformations ranging from a closed form incompatible with receptor binding to an open, receptor-bound state. A number of intermediate states are also observed in different IgE-Fc crystal forms. To further explore this apparent IgE-Fc conformational flexibility and to potentially trap a closed, inactive state, we generated a series of disulfide bond mutants. Here we describe the structure and biochemical properties of an IgE-Fc mutant that is trapped in the closed, non-receptor binding state via an engineered disulfide at residue 335 (Cys-335). Reduction of the disulfide at Cys-335 restores the ability of IgE-Fc to bind to its high affinity receptor, FcεRIα. The structure of the Cys-335 mutant shows that its conformation is within the range of previously observed, closed form IgE-Fc structures and that it retains the hydrophobic pocket found in the hinge region of the closed conformation. Locking the IgE-Fc into the closed state with the Cys-335 mutation does not affect binding of two other IgE-Fc ligands, omalizumab and DARPin E2_79, demonstrating selective blocking of the high affinity receptor binding.

FcεRI-induced mast cell cytokine production critically involves an aspartic acid residue (D234) in the C-terminal intracellular domain of the FcεRIβ chain

The high affinity IgE Fc receptor (FcεRI) β chain is well implicated as a signal amplifier through the immunoreceptor tyrosine-based activation motif (ITAM) in its C-terminal intracellular region. Our previous study, however, demonstrated that mutation in all of the three tyrosine residues within the FcεRIβ ITAM did not impair FcεRI-induced cytokine production, suggesting a possible functional region other than the ITAM. To investigate the ITAM-independent mechanism by which FcεRIβ regulates FcεRI-induced cytokine production, mouse mast cells expressing various FcεRIβ mutants were generated. We observed that truncation of the FcεRIβ C-terminus downstream of the ITAM resulted in a considerable decrease in FcεRI-induced IL-6 production but not degranulation. Furthermore, mutagenesis of a single C-terminal aspartic acid (D234) to alanine (β-D234A) also significantly impaired IL-6 production. In addition, the similarity between the circular dichroism (CD) spectra of the wild type and β-D234A suggests that the secondary structure of the FcεRIβ C-terminus was not affected by the D234A mutation. Consistently, we did not observe any effect of this mutation on FcεRI-induced tyrosine phosphorylation of FcεRIβ. These observations strongly suggest a novel signaling pathway mediated by the cytoplasmic tail downstream of the FcεRIβ ITAM.

Anti-IgE monoclonal antibodies that bind to IgE bound by CD23 but not to IgE bound by IgE Fc receptors on basophils (86.10)

Abstract IgE is a central mediator responsible for immediate-type hypersensitivity reactions. The anti-IgE monoclonal antibody (mAb), omalizumab, has been shown in numerous clinical trials to be efficacious in the treatment of severe allergic asthma and other allergic diseases. Omalizumab was designed to bind to free IgE in blood and membrane-bound IgE (mIgE) on B cells, but not to IgE bound by the high-affinity IgE Fc receptors (FcϵRI) on basophils and mast cells and by the low-affinity IgE Fc receptors (CD23) on B cells and many other cell types. Additionally, omalizumab can bind to IgE and prevent it from binding to both FcϵRI and CD23. Studies by other groups have also shown that CD23 is involved in the synthesis of IgE and that interfering CD23 function, such as by cross-linking CD23 on B cell surface, can cause the down-regulation of IgE production. We have developed anti-IgE mAbs that mimic omalizumab in various aspects except their ability to bind to IgE on CD23. These newly developed mAbs bind to linear epitopes on IgE. They do not induce the degranulation of IgE-pulsed rat basophilic leukemic cells (RBL SX-38), which had been transfected with the genes of human FcϵRI. The ability of the mAbs to cross-link IgE-occupied CD23 on the cell surface of B cell lines is being tested. The results suggest that these anti-IgE mAbs may render a set of pharmacological mechanisms, which are somewhat different from that of omalizumab, for controlling IgE in patients with allergic diseases.

The High Affinity IgE Receptor FcεRI Is Expressed by Human Intestinal Epithelial Cells

BackgroundIgE antibodies play a paramount role in the pathogenesis of various intestinal disorders. To gain insights in IgE-mediated pathophysiology of the gut, we investigated the expression of the high affinity IgE receptor FcεRI in human intestinal epithelium.Methodology/Principal FindingsFcεRI α-chain, as detected by immunohistochemistry, was positive in epithelial cells for eight of eleven (8/11) specimens from colon cancer patients and 5/11 patients with inflammation of the enteric mucosa. The FcεRIα positive epithelial cells co-expressed FcεRIγ, whereas with one exception, none of the samples was positive for the β-chain in the epithelial layer. The functionality of FcεRI was confirmed in situ by human IgE binding. In experiments with human intestinal tumor cell lines, subconfluent Caco-2/TC7 and HCT-8 cells were found to express the α- and γ-chains of FcεRI and to bind IgE, whereas confluent cells were negative for γ-chains.Conclusions/SignificanceOur data provide the first evidence that the components of a functional FcεRI are in vitro expressed by the human intestinal epithelial cells depending on differentiation and, more importantly, in situ in epithelia of patients with colon cancer or gastrointestinal inflammations. Thus, a contribution of FcεRI either to immunosurveillance or pathophysiology of the intestinal epithelium is suggested.

Functionality of the IgA Fc receptor (FcαR, CD89) is down-regulated by extensive engagement of FcɛRI

Besides mast cells and basophils, the high-affinity IgE Fc receptor (FcɛRI) is exclusively expressed on certain FcαR (IgA Fc receptor)-expressing immune cells such as neutrophils in allergic patients. Transfected rat basophilic leukemia cell line (RBL-2H3) co-expressing FcɛRI and FcαR was analyzed for effects of simultaneous receptor engagement by their specific antibodies on degranulation and signaling. Whereas supraoptimal FcɛRI engagement decreased degranulation, which is known as a bell-shaped dose–response curve, such inhibitory effect was not observed with FcαR engagement. However, simultaneous engagement of FcɛRI and FcαR showed that supraoptimal FcɛRI engagement down-regulates FcαR-mediated degranulation. This inhibition was associated with extensive phosphorylation of inositol polyphosphate 5′-phosphatase SHIP1 and FcɛRIβ, and reversed by adding actin-depolymerizing drug, latrunculin B. The results suggest an endogenous mechanism by which FcαR functionality is down-regulated in an ‘allergic environment’ where FcɛRI is co-expressed and extensively cross-linked on FcαR-expressing effector cells.

IgE in allergy and asthma today

The spreading epidemic of allergies and asthma has heightened interest in IgE, the central player in the allergic response. The activity of IgE is associated with a network of proteins; prominent among these are its two principal receptors, FcepsilonRI (high-affinity Fc receptor for IgE) and CD23, as well as galectin-3 and several co-receptors for CD23, notably CD21 and various integrins. Here, we review recent progress in uncovering the structures of these proteins and their complexes, and in our understanding of how IgE exerts its effects and how its expression is regulated. The information that has emerged suggests new therapeutic directions for combating allergic disease.

New developments in FcεRI regulation, function and inhibition

The high-affinity Fc receptor for IgE (FcepsilonRI), a multimeric immune receptor, is a crucial structure for IgE-mediated allergic reactions. In recent years, advances have been made in several important areas of the study of FcepsilonRI. The first area relates to FcepsilonRI-mediated biological responses that are antigen independent. The second area encompasses the biological relevance of the distinct signalling pathways that are activated by FcepsilonRI; and the third area relates to the accumulated evidence for the tight control of FcepsilonRI signalling through a broad array of inhibitory mechanisms, which are being developed into promising therapeutic approaches.

Characterization of (4-Hydroxy-3-Nitrophenyl)Acetyl (NP)-Specific Germinal Center B Cells and Antigen-Binding B220− Cells after Primary NP Challenge in Mice

Previous studies examining the primary germinal center (GC) response to SRBC in mice demonstrated a steady ratio of IgM(+) to isotype-switched GC B cells and a persistent population of GC B cells with a founder phenotype. These characteristics held true at the inductive, plateau, and dissociative phases of the GC response, suggesting a steady-state environment. To test whether these characteristics apply to the primary response of other T cell-dependent Ags, the present study examined the GC response after challenge with (4-hydroxy-3-nitrophenyl)acetyl (NP) in C57BL/6 mice. Multiparameter flow cytometric analysis was used to assess the phenotype of splenic NP-reactive cells at multiple time points after immunization. Results of these studies demonstrated the characteristics of the SRBC-induced GC reaction to be fully maintained in the NP response. In particular, there was a steady ratio of nonswitched to switched B cells, with the majority of NP-reactive GC B cells displaying IgM. In addition, a substantial frequency of B220(-) NP-binding cells was observed in the spleen at later time points after NP challenge. Although these cells were IgE(+), they were found to express both kappa and lambda L chains and display the high-affinity IgE Fc (FcepsilonRI) receptor, suggesting that this population is not of B cell origin. Adoptive transfer studies further demonstrated the B220(-) NP-binding subset to be derived from the myeloid lineage.

Structural Changes in the Lectin Domain of CD23, the Low-Affinity IgE Receptor, upon Calcium Binding

CD23, the low-affinity receptor for IgE (Fc epsilonRII), regulates IgE synthesis and also mediates IgE-dependent antigen transport and processing. CD23 is a unique Fc receptor belonging to the C-type lectin-like domain superfamily and binds IgE in an unusual, non-lectin-like manner, requiring calcium but not carbohydrate. We have solved the high-resolution crystal structures of the human CD23 lectin domain in the presence and absence of Ca2+. The crystal structures differ significantly from a previously determined NMR structure and show that calcium binding occurs at the principal binding site, but not at an auxiliary site that appears to be absent in human CD23. Conformational differences between the apo and Ca2+ bound structures suggest how IgE-Fc binding can be both calcium-dependent and carbohydrate-independent.

RabGEF1 regulates stem cell factor/c-Kit-mediated signaling events and biological responses in mast cells

We recently reported that RabGEF1 is a negative regulator of high-affinity Fc receptor for IgE (Fc epsilonRI)-dependent mast cell activation and that mice lacking RabGEF1 develop severe skin inflammation and increased numbers of dermal mast cells. To better understand how RabGEF1 can regulate signaling events and biological responses in mast cells, we examined the responses of bone marrow-derived cultured mast cells (BMCMCs) from wild-type (+/+) and Rabgef1 knockout (-/-) mice after stimulation with the c-Kit ligand, stem cell factor (SCF), an important regulator of mast cell development, survival, proliferation, and activation. We found that RabGEF1-deficient mast cells exhibited enhanced and prolonged activation of Ras and extracellular regulated kinase, and significantly elevated IL-6 secretion, after stimulation with SCF. SCF-induced activation of c-Jun N-terminal kinase was increased in Rabgef1-/- BMCMCs, but without corresponding significant increases in SCF-induced migration or adhesion. SCF-mediated activation of the survival-enhancing kinase, Akt, also was increased in Rabgef1-/- BMCMCs, and these cells had a survival advantage over their +/+ counterparts in vitro. Despite enhanced Ras activation in the absence of RabGEF1, SCF-induced proliferation was lower in Rabgef1-/- BMCMCs compared with their +/+ counterparts. Finally, we found that c-Kit internalization was delayed in the absence of RabGEF1, probably reflecting a positive role for RabGEF1 in the regulation of endocytic events, and that infection of Rabgef1-/- BMCMCs with a wild-type RabGEF1 lentiviral construct normalized c-Kit internalization to the levels seen in +/+ BMCMCs. Thus, RabGEF1 plays a critical role in the regulation of SCF/c-Kit-mediated signaling events and biological responses in mast cells.

New Insights on the Allergy-Linked β Subunit of the High Affinity IgE Receptor: Mechanisms and Clinical Implications

The high affinity IgE Fc receptor (FcεRI) on mast cells and basophils has a central role in allergy and asthma. The β subunit of this multimeric receptor is a membrane tetra-spanning protein that encodes a nonconventional immunoreceptor tyrosine-based activation motif (ITAM) that imparts it signaling competence. This subunit was shown to amplify FcεRI signaling causing increased cellular responses such as degranulation. Polymorphisms of the β subunit have been linked to atopy, asthma, and allergy. Although considerable effort has been made to understand the functional consequence(s) of expressing these polymorphisms, the outcome has not been revealing. Recent studies show a link between β subunit polymorphisms, β gene transcription, and increased FcεRI expression. Additionally, a newly described function for the β subunit suggests an important role in downregulating cytokine production. Among the molecules whose response is dampened by FcεRIb is NFkB, a central regulator of inflammation. Collectively, these studies suggest that the β subunit is important in controlling the sensitivity and extent of an elicited response upon mast cell or basophil activation. Thus the findings, described herein, provide new insights on the mechanistic link between the FcεRIb and allergic disease.

The FcϵRIβ Immunoreceptor Tyrosine-based Activation Motif Exerts Inhibitory Control on MAPK and IκB Kinase Phosphorylation and Mast Cell Cytokine Production

The high affinity IgE Fc receptor (FcepsilonRI) beta chain functions as a signal amplifier and has been linked to atopy, asthma, and allergy. Herein, we report on a previously unrecognized negative regulatory role for the nonconventional beta chain immunoreceptor tyrosine-based activation motif that contains three tyrosine residues (YX5YX3Y). Degranulation and leukotriene production was found to be impaired in cells expressing the mutated FcepsilonRIbeta immunoreceptor tyrosine-based activation motifs FYY, YYF, FYF, and FFF. In contrast, cytokine synthesis and secretion were enhanced in the YFY and FFF mutants. FcepsilonRI phosphorylation and Lyn kinase co-immunoprecipitation was intact in the YFY mutant but was lost in the FYF and FFF mutants. The phosphorylation of Syk, LAT, phospholipase gamma1/2, and Srchomology 2 domain-containing protein phosphatase 2 was intact, whereas the phosphorylation of SHIP-1 was significantly reduced in the YFY mutant cells. The FYF and FFF mutants were defective in phosphorylating all of these molecules. In contrast, the phosphorylation of ERK, p38 MAPK, IkappaB kinase beta (IKKbeta), and nuclear NFkappaB activity was enhanced in the YFY and FFF mutants. These findings show that the FcepsilonRIbeta functions to both selectively amplify (degranulation and leukotriene secretion) and dampen (lymphokine) mast cell effector responses.

Chronic idiopathic urticaria.

Chronic idiopathic urticaria has long been a demoralizing disease, baffling allergists and dermatologists alike, to the detriment of the patient. Recent findings, however, have shed light on causation in many, though not all, of these patients. The purpose of this review is to bring the reader up to date on the current position regarding aetiology and pathogenesis and the strength of the evidence. The review also seeks to point up rational approaches to diagnosis and treatment in the light of these developments. Chronic idiopathic urticaria encompasses at least two subgroups. One of these is the now well-established entity of autoimmune chronic urticaria, due to autoantibodies against either the high-affinity IgE receptor Fc epsilon R1 or, less commonly, IgE. These patients, who co-segregate with chronic idiopathic urticaria patients having an increased frequency of antithyroid autoantibodies, represent 30-50% of the patients previously designated as having chronic idiopathic urticaria. Convenient routine diagnostic tests for this subset remain elusive. The remaining 50% of patients with chronic idiopathic urticaria remain truly 'idiopathic', although the condition in some may have an autoimmune basis, autoantibodies having eluded current techniques for detection. Selected patients with autoimmune urticaria may benefit from immunotherapy. It is now known that in 30-50% of patients with chronic idiopathic urticaria, the condition has an autoimmune basis, although confirmation of the diagnosis in these patients is not straightforward. In selected patients, attempts to establish this diagnosis are worthwhile since there are important therapeutic implications.

Cysteinyl leukotrienes and uridine diphosphate induce cytokine generation by human mast cells through an interleukin 4-regulated pathway that is inhibited by leukotriene receptor antagonists.

We previously reported that interleukin (IL)-4 upregulates the expression of leukotriene C4 synthase (LTC4S) by human cord blood–derived mast cells (hMCs), augments their high-affinity Fc receptor for IgE (FcεRI)-dependent generation of eicosanoids and cytokines, and induces a calcium flux in response to cysteinyl leukotrienes (cys-LTs) and uridine diphosphate (UDP) that is blocked by cys-LT receptor antagonists. We speculated that this IL-4–dependent, receptor-mediated response to the cys-LTs and UDP might induce cytokine generation by hMCs without concomitant exocytosis. Unlike hMCs maintained in cytoprotective stem cell factor (SCF) alone, hMCs primed for 5 d with IL-4 responded to UDP (1 μM), LTC4 (100 nM), and LTD4 (100 nM) by producing IL-5, tumor necrosis factor (TNF)-α, and especially large quantities of macrophage inflammatory protein (MIP)-1β de novo at 6 h, preceded by the induced expression of the corresponding mRNAs. Cys-LT– and UDP-mediated cytokine production by the primed hMCs occurred without histamine release or PGD2 generation and was inhibited by the CysLT1 receptor antagonist MK571. Additionally, pretreatment of hMCs with MK571 or with the cys-LT biosynthetic inhibitor MK886 decreased IL-5 and TNF-α production in response to IgE receptor cross-linkage, implying a positive feedback by endogenously produced cys-LTs. Cys-LTs and UDP thus orchestrate a novel, IL-4–regulated, non-IgE–dependent hMC activation for cytokine gene induction that could be initiated by microbes, cellular injury, or neurogenic or inflammatory signals; and this pathobiologic event would not be recognized in tissue studies where hMC activation is classically defined by exocytosis.

A tea catechin suppresses the expression of the high-affinity IgE receptor Fc epsilon RI in human basophilic KU812 cells.

Human basophilic KU812 cells express the high-affinity IgE receptor Fc epsilon RI, which plays a central role in the IgE-mediated allergic response. The effect of several major tea catechins, (+)-catechin, (-)-epicatechin, (-)-epigallocatechin, (-)-epicatechin gallate, and (-)-epigallocatechin gallate (EGCg), on the cell surface expression of Fc epsilon RI in KU812 cells was studied. Flow cytometric analysis showed that only EGCg was able to decrease the cell surface expression of Fc epsilon RI after a 24 h treatment in a dose-dependent manner. Moreover, immunoblot analysis revealed that the total cellular expression of the Fc epsilon RI alpha chain decreased upon treatment with EGCg. Fc epsilon RI is a tetrameric structure comprising one alpha chain, one beta chain, and two gamma chains. The level of mRNA production of each subunit in KU812 cells was investigated. KU812 cells treated with EGCg expressed lower levels of Fc epsilon RI alpha and gamma mRNA than nontreated cells. These results suggest that EGCg has an ability to down-regulate Fc epsilon RI expression, and this suppressive effect may be due to the down-regulation of Fc epsilon RI alpha and gamma mRNA levels.

IL-4 and -5 prime human mast cells for different profiles of IgE-dependent cytokine production.

Mast cells (MC) are stem cell factor-dependent tissue-based hematopoietic cells with substantial functional heterogeneity. Cord blood-derived human MC (hMC) express functional receptors for IL-5, and IL-5 mediates stem cell factor-dependent comitogenesis of hMC in vitro. Although IL-5 is not required for normal hMC development, we considered that it might prime hMC for their high-affinity Fc receptor for IgE (FcvarepsilonRI)-dependent generation of cytokines, as previously demonstrated for IL-4. Compared with hMC maintained in stem cell factor alone, hMC primed with IL-5 expressed 2- to 4-fold higher steady-state levels of TNF-alpha, IL-5, IL-13, macrophage inflammatory protein 1alpha, and granulocyte-macrophage colony-stimulating factor transcripts 2 h after FcvarepsilonRI crosslinking and secreted 2- to 5-fold greater quantities of the corresponding cytokines, except IL-13, at 6 h. Unlike IL-4, IL-5 priming did not enhance FcvarepsilonRI-dependent histamine release. Thus, IL-5 augments cytokine production by hMC by a mechanism distinct from that of IL-4 and with a different resultant profile of cytokine production. These observations suggest a potentially autocrine effect of IL-5 on hMC for amplification of allergic immune responses, in addition to its recognized paracrine effects on eosinophils, and implicate both IL-4 and IL-5 in the modulation of the hMC phenotype.

The pharmacological basis of anti-IgE therapy

The treatment of asthma and allergic rhinitis using unique, humanized anti-IgE monoclonal antibodies with very particular binding specificities is now supported by the results of multiple phase II and III human clinical studies. The therapeutic efficacy of this approach is attributable to several pharmacological mechanisms. In addition to the expected effects of these monoclonal antibodies in neutralizing free IgE and inhibiting IgE production by B cells, several indirect biochemical and cellular effects have been uncovered during the course of the clinical trials. These include the accumulation of potentially beneficial IgE-anti-IgE immune complexes and the downregulation of the high-affinity IgE Fc receptors (FcvarepsilonRI) on basophils and mast cells. This article analyzes the structural basis of the specificity of the anti-IgE antibodies and pertinent results from in vitro experiments, animal model studies, and human clinical trials in an attempt to provide a cogent pharmacological interpretation of the therapeutic effects of anti-IgE therapy in both the near- and long term. The development of anti-IgE therapy over the past 10 years provides an interesting example of the emergence of a conceptually new, biotechnology-produced pharmaceutical.

Basis of the 1:1 stoichiometry of the high affinity receptor Fc epsilon RI-IgE complex.

A soluble fragment of the high-affinity IgE receptor Fc epsilon RI alpha-chain (sFc epsilon RI alpha) binds to the Fc fragment of IgE (IgE-Fc) as a 1:1 complex. IgE-Fc consists of a dimer of the C epsilon 2, C epsilon 3 and C epsilon 4 domains of the epsilon-heavy chain of IgE. This region of IgE has been modelled on the crystal structure of the Fc region of IgG1, which exhibits twofold rotational symmetry. This implies that IgE should be divalent with respect to its ligands. X-ray scattering studies reveal however that the twofold rotational symmetry of IgE-Fc is perturbed by a bend in the linker region between the C epsilon 2 and C epsilon 3 domains. The 1:1 stoichiometry could then arise from the conformational asymmetry or from steric occlusion of one of the sites by the overhanging C epsilon 2 domains. To test this hypothesis we have expressed a recombinant epsilon-chain fragment containing C epsilon 3 and C epsilon 4. This product, Fc epsilon 3-4, is secreted from cells as a disulphide linked dimer and binds with higher affinity than either IgE or IgE-Fc to cell surface Fc epsilon RI. Titration experiments, together with molecular mass measurements of the Fc epsilon 3-4/sFc epsilon RI alpha complex, reveal that Fc epsilon 3-4 binds only a single receptor molecule. This excludes the possibility that steric hindrance by C epsilon 2 accounts for the unexpected stoichiometry.

Tyrosine Phosphorylation of the vav Proto-oncogene Product Links FcεRI to the Rac1-JNK Pathway

Stimulation of high affinity IgE Fc receptors (FcepsilonRI) in basophils and mast cells activates the tyrosine kinases Lyn and Syk and causes the tyrosine phosphorylation of phospholipase C-gamma, resulting in the Ca2+- and protein kinase C-dependent secretion of inflammatory mediators. Concomitantly, FcepsilonRI stimulation initiates a number of signaling events resulting in the activation of mitogen-activated protein kinase (MAPK) and c-Jun NH2-terminal kinase (JNK), which, in turn, regulate nuclear responses, including cytokine gene expression. To dissect the signaling pathway(s) linking FcepsilonRI to MAPK and JNK, we reconstructed their respective biochemical routes by expression of a chimeric interleukin-2 receptor alpha subunit (Tac)-FcepsilonRI gamma chain (Tacgamma) in COS-7 cells. Cross-linking of Tacgamma did not affect MAPK in COS-7 cells, but when coexpressed with the tyrosine kinase Syk, Tacgamma stimulation potently induced Syk and Shc tyrosine phosphorylation and MAPK activation. In contrast, Tacgamma did not signal JNK activation, even when coexpressed with Syk. Ectopic expression of a hematopoietic-specific guanine nucleotide exchange factor (GEF), Vav, reconstituted the Tacgamma-induced, Syk- and Rac1-dependent JNK activation; and tyrosine-phosphorylation of Vav by Syk stimulated its GEF activity for Rac1. Thus, these data strongly suggest that Vav plays a critical role linking FcepsilonRI and Syk to the Rac1-JNK pathway. Furthermore, these findings define a novel signal transduction pathway involving a multimeric cell surface receptor acting on a cytosolic tyrosine kinase, which, in turn, phosphorylates a GEF, thereby regulating its activity toward a small GTP-binding protein and promoting the activation of a kinase cascade.