High-affinity Receptor For Immunoglobulin Research Articles

CD45 exclusion- and cross-linking-based receptor signaling together broaden FcεRI reactivity.

For many years, the high-affinity receptor for immunoglobulin E (IgE) FcεRI, which is expressed by mast cells and basophils, has been widely held to be the exemplar of cross-linking (that is, aggregation dependent) signaling receptors. We found, however, that FcεRI signaling could occur in the presence or absence of receptor cross-linking. Using both cell and cell-free systems, we showed that FcεRI signaling was stimulated by surface-associated monovalent ligands through the passive, size-dependent exclusion of the receptor-type tyrosine phosphatase CD45 from plasma membrane regions of FcεRI-ligand engagement. Similarly to the T cell receptor, FcεRI signaling could also be initiated in a ligand-independent manner. These data suggest that a simple mechanism of CD45 exclusion-based receptor triggering could function together with cross-linking-based FcεRI signaling, broadening mast cell and basophil reactivity by enabling these cells to respond to both multivalent and surface-presented monovalent antigens. These findings also strengthen the case that a size-dependent, phosphatase exclusion-based receptor triggering mechanism might serve generally to facilitate signaling by noncatalytic immune receptors.

Immune system network in allergic diseases during sublingual immunotherapy: The role of oral mucosal tissue

Specific sublingual immunotherapy (SLIT) represents an approach currently available to redirect inappropriate immune response in atopic patients. Since oral mucosal tissue displays high affinity for allergens, it is conceivable that the sublingual administration route might induce immunological tolerance towards allergens involving cells and mediators specific of oral and intestinal mucosa. The presence in oral mucosa of dendritic cells (DCs) which express the high-affinity receptor for immunoglobulin (Ig)E (FceRI) seems to suggest that the generation of T regulatory cells in periphery is orchestrated by a particular subset of DCs. It seems that repeated stimulation of naive CD4 T cells with allogenic immature DCs induce Tr1 cell maturation. Nevertheless, other cells are involved in this process, such as Toll Like Receptors (TLR), Major Histocompatibility Complex I and II (MHC I and II) and costimulatory molecules, such as CD40, CD 80/B7.1 and CD 86/B7.2. An increase of serum IgG4 and IgA, a reduced number of inflammatory cells infiltrating target organs, as well as a reduction of eosinophilic cationic protein and a T cell suppression in the peripheral blood also occur with SLIT. All these molecules orchestrate the immune network within the regional immune system, recreating a favourable environment for the induction of tolerance operated by SLIT.

Detection of IgE-positive leukocytes by synthetic peptide and monoclonal antibodies

The aim: to assess the use of the synthetic peptide p137-142FcεRIα, the fragment of the FcεRI active site, in identifying the IgE+ cells, compared to monoclonal antibodies to IgE. Materials and methods. The study involved 39 children, mean age of 11 [10.0; 12.8] years. 105 blood samples were received. For phenotyping blood cells we used a synthetic hexapeptide p137-142FcεRIα, FITC-labeled (NIKP “Resan” Belarus), and monoclonal antibodies to IgE, Mouse Anti-Human (FITC), the production of Thermo Fisher Scientific Inc. USA. To count the eosinophils number general blood analysis was conducted. Results. No significant differences between the number of cells identified with the synthetic peptide and mAb were found (p = 0,2), the correlation between the IgE+-cells number identified by both methods was high (0.8). From the 105 samples in 51 (49%) – normal and in 54 (51%) - increased level of eosinophils (≥6%) was found. IgE+-cells number was higher in children with an increased level of eosinophils, than with normal eosinophils (p = 0,00). Correlation between IgE+-cells and eosinophils increased in samples with eosinophilia. In 68% of cases with a low level of eosinophils (0-3%), the level of IgE+ cells was above half of the medium: 3-3.5% at 30,000 cells. Conclusions. Determining the cells bound IgE by a synthetic peptide p137-142FcεRIα, fragment of the active center of the high affinity receptor for immunoglobulin E, is not worse than standard method of determining, using monoclonal antibodies to IgE. Number of eosinophils in peripheral blood correlates with the amount of IgE+-cells. In children with eosinophilia IgE+ -cell number is higher than in children with normal eosinophils level. There is a higher correlation IgE+-cell number with the eosinophils number in case of eosinophilia. In 14% of cases the other IgE+-cells (except eosinophils) were dominant.

The high-affinity immunoglobulin E receptor as pharmacological target

The high-affinity receptor for immunoglobulin E is expressed mainly on mast cells and basophils, but also on neutrophils, eosinophils, platelets, monocytes, Langerhans and dendritic cells, airway smooth muscle cells and some nerve cells. Its main function is, upon its engagement by IgE and specific antigen, to trigger a powerful defense against invading pathogens and a rapid neutralization of dangerous toxic substances introduced in the body. This powerful response could be wielded against tumors. But, when control over this receptor is lost, its unchecked activation can induce an array of diseases, some of which can lead to death. In this review we will summarize the pharmacological approaches and strategies that are currently used, or under study, to harness or wield activation of this receptor for therapeutic purposes.

Molecular editing of cellular responses by the high-affinity receptor for IgE.

Cellular responses elicited by cell surface receptors differ according to stimulus strength. We investigated how the high-affinity receptor for immunoglobulin E (IgE) modulates the response of mast cells to a high- or low-affinity stimulus. Both high- and low-affinity stimuli elicited similar receptor phosphorylation; however, differences were observed in receptor cluster size, mobility, distribution, and the cells' effector responses. Low-affinity stimulation increased receptor association with the Src family kinase Fgr and shifted signals from the adapter LAT1 to the related adapter LAT2. LAT1-dependent calcium signals required for mast cell degranulation were dampened, but the role of LAT2 in chemokine production was enhanced, altering immune cell recruitment at the site of inflammation. These findings uncover how receptor discrimination of stimulus strength can be interpreted as distinct in vivo outcomes.

Natural Vanadium-Containing Jeju Groundwater Inhibits Immunoglobulin E-Mediated Anaphylactic Reaction and Suppresses Eicosanoid Generation and Degranulation in Bone Marrow Derived-Mast Cells

The high-affinity receptor for immunoglobulin E (IgE) (FcεRI)-mediated activation of mast cells plays an important role in various allergic diseases. To assess the anti-allergic activity of natural vanadium-containing Jeju groundwater (JW), an in vivo passive cutaneous anaphylaxis (PCA) animal model and in vitro mouse bone marrow-derived mast cells (BMMCs) was used. JW inhibited cyclooxygenase-2 (COX-2)-dependent prostaglandin D(2) (PGD(2)) generation in a dose-dependent manner, with a concomitant reduction of COX-2 protein expression in IgE-induced BMMCs. In addition, JW inhibited 5-lipoxygenase (5-LOX)-dependent generation of leukotriene C(4) (LTC(4)) as well as degranulation in a dose-dependent manner. These results demonstrate that JW has dual COX-2/5-LOX inhibitory activity. In addition, vanadium pentoxide (V(2)O(5)), which is the major vanadium component of JW, also inhibited PGD(2) and LTC(4) generation as well as degranulation in IgE-induced BMMCs. Furthermore, oral administration of JW dose-dependently inhibited mast cell-dependent passive anaphylactic reaction in IgE-sensitized mice. Taken together, these results suggest that JW may be useful in regulating mast cell-mediated allergic response through the suppression of eicosanoid generation and degranulation in mast cells.

Association of polymorphisms in the promoter region of FCER1A gene with atopic dermatitis, chronic uticaria, asthma, and serum immunoglobulin E levels in a Han Chinese population

The high-affinity receptor for immunoglobulin E (IgE) plays a central role in allergy diseases. Previous studies have reported the association of variants in the proximal promoter of FCER1A with IgE levels as well as allergy disorders. Another promoter gene polymorphism that is located upstream of exon 1 has not been investigated. We investigated the association of variants in the promoter located upstream of FCER1A exon 1 with serum IgE levels and allergy diseases in a Han Chinese population. A total of 97 patients with atopic dermatitis (AD), 123 patients with chronic urticaria (CU), 286 children with asthma, and control groups were screened for polymorphisms in the promoter region located upstream of FCER1A exon 1 by the polymerase chain reaction-ligation detection reaction method. Total serum IgE levels were tested in groups. The rare allele A of the rs2427837 A/G polymorphism was significantly different in the AD group compared with the controls. No association with the polymorphism was observed in the CU group. In asthmatic patients, IgE levels were higher in the mutation genotypes GA of rs2427837 and TC of rs2251746 compared with normal genotype individuals. The minor allele of rs2427837 and rs2251746 in FCER1A is a genetic risk factor of high IgE levels.

The lectin ArtinM binds to mast cells inducing cell activation and mediator release

Mast cells are inflammatory cells that respond to signals of innate and adaptive immunity with immediate and delayed release of mediators. ArtinM, a lectin from Artocarpus integrifolia with immunomodulatory activities, is able to induce mast cell activation, but the mechanisms remain unknown. This study sought to further investigate the effects of the lectin on mast cells. We showed that ArtinM binds to mast cells, possibly to the high affinity receptor for immunoglobulin E (IgE) – FcεRI – and/or to IgE bound to FcεRI. Binding of the lectin resulted in protein tyrosine phosphorylation and release of the pre- and newly-formed mediators, β-hexosaminidase and LTB4 by mast cells, activities that were potentiated in the presence of IgE. ArtinM also induced the activation of the transcription factors NFκB and NFAT, resulting in expression of some of their target genes such as IL-4 and TNF-α. In view of the established significance of mast cells in many immunological and inflammatory reactions, a better understanding of the mechanisms involved in mast cell activation by ArtinM is crucial to the pharmacological application of the lectin.

Sequestration of phosphoinositides by mutated MARCKS effector domain inhibits stimulated Ca2+mobilization and degranulation in mast cells

Protein kinase C β (PKCβ) participates in antigen-stimulated mast cell degranulation mediated by the high-affinity receptor for immunoglobulin E, FcεRI, but the molecular basis is unclear. We investigated the hypothesis that the polybasic effector domain (ED) of the abundant intracellular substrate for protein kinase C known as myristoylated alanine-rich protein kinase C substrate (MARCKS) sequesters phosphoinositides at the inner leaflet of the plasma membrane until MARCKS dissociates after phosphorylation by activated PKC. Real-time fluorescence imaging confirms synchronization between stimulated oscillations of intracellular Ca(2+) concentrations and oscillatory association of PKCβ-enhanced green fluorescent protein with the plasma membrane. Similarly, MARCKS-ED tagged with monomeric red fluorescent protein undergoes antigen-stimulated oscillatory dissociation and rebinding to the plasma membrane with a time course that is synchronized with reversible plasma membrane association of PKCβ. We find that MARCKS-ED dissociation is prevented by mutation of four serine residues that are potential sites of phosphorylation by PKC. Cells expressing this mutated MARCKS-ED SA4 show delayed onset of antigen-stimulated Ca(2+) mobilization and substantial inhibition of granule exocytosis. Stimulation of degranulation by thapsigargin, which bypasses inositol 1,4,5-trisphosphate production, is also substantially reduced in the presence of MARCKS-ED SA4, but store-operated Ca(2+) entry is not inhibited. These results show the capacity of MARCKS-ED to regulate granule exocytosis in a PKC-dependent manner, consistent with regulated sequestration of phosphoinositides that mediate granule fusion at the plasma membrane.

Common variants in FCER1A influence total serum IgE levels from cord blood up to six years of life

In a recent genome wide scan, a functional promoter variant (rs2251746) in the gene encoding the alpha chain of the high affinity receptor for immunoglobulin E (IgE) (FCER1A) was identified as major determinant of serum IgE levels. The aim of this study was to investigate the role of rs2251746 on total IgE levels measured at different stages of life from birth (cord blood) up to the age of 6 and to evaluate its interaction with the environmental influences in two German birth cohorts. Data from two German birth cohorts were analysed (n = 1043 for the LISA cohort and n = 1842 for the GINI cohort). In the studies, total serum IgE was measured from cord blood, and blood samples taken at the age of 2/3 and 6 years. In a subgroup of the LISA study, house dust samples were collected at age of 3 months and the amount of endotoxin was determined. Random effect models were used to analyse the longitudinal health outcomes. In the two cohorts, the heterozygote and the rare homozygote of rs2251746 was consistently associated with lower total IgE levels from birth up to the age of 6 years with an allele-dose effect (P < 0.02 for blood samples taken at each time point in both cohorts). No interaction between the two FCER1A encoding gene and environmental exposures including endotoxin, worm infestation and day care centre attendance during early childhood were observed. Common variants in FCER1A strongly influence basal IgE production independently from environmental stimuli. These effects can be observed already in cord blood pointing to altered gene expression in foetus.

Genetic associations of variants of the high affinity receptor for immunoglobulin E in Wegener’s granulomatosis

Immunoglobulin E (IgE) and the high affinity IgE receptor (FcepsilonRI) have been suggested to contribute to the pathogenesis of autoimmune disorders. Their role in Wegener's granulomatosis (WG) are, however, poorly recognized. We sought a genetic association between laboratory markers for the disease, i.e. anti-proteinase 3 antibodies (anti-PR3), anti-myeloperoxidase antibodies, anti-cyclic citrullinated peptide antibodies, C-reactive protein (CRP), C3c and C4 complement components, and total serum IgE levels in WG subjects with common genetic variants of FcepsilonRI subunits. Anti-PR3 and CRP and serum IgE levels showed significant associations, while complement components tended to be associated, with -18483A > C and/or -344C > T FCER1A (FcepsilonRI alpha-subunit gene) polymorphisms. Moreover, a correlation between -109T > C FCER1B (FcepsilonRI beta-subunit gene) genotypes and serum IgE was observed. Both WG specific auto-antibodies and other blood inflammatory markers displayed correlations with serum total IgE levels and genetic variants of the high affinity receptor for this immunoglobulin. This observation suggests a functional relantionship of FcepsilonRI in the regulation of autoimmune response observed in WG.

Characterization of dendritic cell phenotype in allergic conjunctiva: increased expression of FcɛRI, the high-affinity receptor for immunoglobulin E

Dendritic cells (DCs) express the high-affinity receptor for IgE (Fc(epsilon)RI) on their surface, which may enhance their ability to capture and internalize antigens for presentation to T-lymphocytes. The aim of this study was to determine if expression of Fc(epsilon)RI(+) DCs is increased in the conjunctivae of vernal keratoconjunctivitis (VKC) patients compared with those of normal controls. Conjunctival biopsies were obtained from non-atopic and VKC patients. Double immunohistochemical staining was carried out using antibodies against Fc(epsilon)RI and the CD1a antigen, a DC marker. The double-positive cells were counted in five representative fields of view for each conjunctival sample. Fc(epsilon)RI(+) CD1a(+) cells were present in significantly higher numbers in VKC conjunctivae compared with normal controls (mean cell count of 21.3 in VKC vs5.0 in controls, P<0.005). In normal patients the Fc(epsilon)RI-expressing DCs tended to be confined to the epithelial layer or the superficial substantia propria, but in the VKC samples these Fc(epsilon)RI(+) cells were mainly concentrated in the deeper substantia propria. Fc(epsilon)RI(+) DC numbers are elevated in the conjunctivae of VKC patients, a finding consistent with the results of other studies focusing on atopic conditions. Elevated expression of Fc(epsilon)RI on DCs would facilitate antigen presentation and enhance T-cell priming, thereby contributing to ocular symptoms.

Detecting Anti-FcεRI Autoantibodies in Patients with Asthma by Flow Cytometry

Autoimmune diseases have been implicated in the development of intrinsic asthma, however little data are available on the role of autoimmunity in the pathogenesis of asthma. The purpose of this study was to investigate circulating autoantibodies against the high-affinity receptor for immunoglobulin E, FcepsilonRI, in patients with asthma. Seventy-eight patients with asthma and 32 healthy control subjects were included. All individuals were tested using a triple-staining flow cytometry-based basophil activation test (BAT) for the potential presence of autoantibodies against FcepsilonRI. Of the 78 asthma patients, 29 (37.2%) had a positive BAT result, indicating that their serum was able to activate basophils, compared with only four (12.5%) of the control group, a statistically significant between-group difference. These data suggest that some asthma patients have aberrant anti-FcepsilonRI autoantibodies, which implies that autoimmunity may be one factor involved in the pathogenesis of intrinsic asthma.

Diacylglycerol kinases in immune cell function and self‐tolerance

Both diacylglycerol (DAG) and phosphatidic acid (PA) are important second messengers involved in signal transduction from many immune cell receptors and can be generated and metabolized through multiple mechanisms. Recent studies indicate that diacylglycerol kinases (DGKs), the enzymes that catalyze phosphorylation of DAG to produce PA, play critical roles in regulating the functions of multiple immune cell lineages. In T cells, two DGK isoforms, alpha and zeta, inhibit DAG-mediated signaling following T-cell receptor engagement and prevent T-cell hyperactivation. DGK alpha and zeta synergistically promote T-cell anergy and are critical for T-cell tolerance. In mast cells, DGKzeta plays differential roles in their activation by promoting degranulation but attenuating cytokine production following engagement of the high affinity receptor for immunoglobulin E. In dendritic cells and macrophages, DGKzeta positively regulates Toll-like receptor-induced proinflammatory cytokine production through its product PA and is critical for host defense against Toxoplasma gondii infection. These studies demonstrate pivotal roles of DGKs in regulating immune cell function by acting both as signal terminator and initiator.

Suppressive effect of Elf-1 on FcεRI α-chain expression in primary mast cells

The high-affinity receptor for immunoglobulin E (IgE), FcepsilonRI, is specifically expressed in mast cells and basophils and plays a key role in IgE-mediated allergic reactions. The transcription factor Elf-1 has been previously identified to bind to the promoter of the human FcepsilonRI alpha-chain, which is essential for the function and expression of FcepsilonRI. In the present study, Elf-1 siRNA was conducted to evaluate the effects of Elf-1 on FcepsilonRI alpha-chain expression in the primary mouse mast cells, bone marrow-derived mast cells (BMMC). Introduction of Elf-1 siRNA effectively reduced expression levels of Elf-1 mRNA and protein in BMMC. Transient reporter assay showed that the knockdown of Elf-1 by siRNA resulted in increased FcepsilonRI alpha-chain promoter activity, while overexpression of Elf-1 suppressed alpha-chain promoter activity in BMMC. Elf-1 siRNA-treated BMMC exhibited marked upregulation of FcepsilonRI alpha-chain transcription, whereas beta-chain mRNA was not affected by Elf-1 siRNA. Chromatin immunoprecipitation assay showed that the amount of transcription factor PU.1, recognizing the cis-element close to the Elf-1-site on the FcepsilonRI alpha-chain promoter, was significantly increased by introduction of Elf-1 siRNA. These results indicate that Elf-1 negatively regulates FcepsilonRI alpha-chain expression by suppressing PU.1-mediated transcription of the alpha-chain in BMMC.

The leukocyte receptor CD84 inhibits FcɛRI-mediated signaling through homophilic interaction in transfected RBL-2H3 cells

Signaling through the high-affinity receptor for immunoglobulin E (FcɛRI) results in the coordinated activation of tyrosine kinases, thus leading to calcium mobilization, degranulation, and leukotriene and cytokine synthesis. Here, we show that CD84, a member of the CD150 family of leukocyte receptors, inhibits FcɛRI-mediated mast cell degranulation in CD84-transfected rat basophilic leukaemia-2H3 mast cell line cells (RBL-2H3) through homophilic interaction. There was no reduction in overall protein phosphorylation following IgE triggering in CD84 RBL-2H3 cells. Indeed, phosphorylation of Dok-1 and c-Cbl increased in CD84 RBL-2H3, suggesting that inhibition is mediated by these molecules. MAP kinase phosphorylation (ERK1/2, JNK and p38) and cytokine synthesis were impaired in CD84 RBL-2H3. This inhibitory mechanism was independent of SAP and SHP-2 recruitment. Interestingly, CD84 mutants in tyrosines (Y279F and ΔY324) reversed this inhibitory profile. These data suggest that CD84 may play a role in modulating FcɛRI-mediated signaling in mast cells. Thus, CD84 could play a protective role against undesired allergic and inflammatory responses.

Role of the extracellular domain of FcɛRIα in intracellular processing and surface expression of the high affinity receptor for IgE FcɛRI

The high affinity receptor for immunoglobulin E, FcɛRI, is a critical component of IgE-mediated allergic reactions. It is expressed as a tetramer (αβγ 2) made of an IgE-binding α chain and a signaling module formed by the β chain and a dimer of γ chains. It is expressed in humans and rodents on basophils and mast cells at a high level, and, upon activation, it induces the liberation of allergy mediators. In humans a trimeric form lacking the β chain also exists (αγ 2). This trimeric form is expressed on antigen presenting cells where it acts to facilitate antigen presentation via IgE. Both the expression and the signaling capacity of the trimer are lower than those of the tetramer. The differences between human (tetrameric and trimeric) and murine (tetrameric only) expression is explained in part by the fact that mouse α cannot be expressed at the cell surface in the absence of β, while human α can. Here we demonstrate that the capacity of human α to be expressed at the cell surface in the absence of β is encoded entirely in its extracellular domain. These findings show that the extracellular domain of the type I transmembrane protein FcɛRI α plays a role in FcɛRI intracellular processing and expression at the cell surface.

Comparative analysis of nasal and oral mucosa dendritic cells

Mucosal dendritic cells (DC) play a crucial role in tolerance induction as seen in mucosal immunotherapy of atopic diseases. Nevertheless little is known about the phenotypical differences of oral and nasal mucosal DC (nmDC). Recently, we could show that oral mucosal myeloid CD1a(+) DC (omDC) differ from their skin counterparts especially by the expression of high affinity receptor for immunoglobulin E (IgE; FcepsilonRI). However, expression pattern of FcepsilonRI and phenotypical characteristics of CD1a(+) nmDC have not been elucidated in detailed yet. We performed detailed phenotypical comparison of nmDC and omDC of atopic and nonatopic individuals. As reported for omDC, FcepsilonRI on nmDC of atopic donors was elevated and mostly occupied by IgE while FcepsilonRI was present only in low amounts on nmDC of nonatopic donors. Nevertheless, the highest FcepsilonRI expression has been observed on omDC. Furthermore, significant amounts of costimulatory molecules CD40, CD80 and CD86 could be detected on nmDC that expressed more CD80 compared with omDC. Moreover, nmDC displayed less major histocompatability complex (MHC) class I and II molecules than omDC. In addition, nmDC expressed more C-type lectins CD205, CD206 as well as myeloid marker CD11b while omDC displayed increased expression of CD207 and lipopolysaccharide (LPS) receptor CD14. Together these data imply that nmDC phenotypical differ from omDC which might result in diverse functional properties and might be of relevance for selecting routes for immunotherapy of atopic diseases. Moreover these data provide a basis for further studies investigating immunological mechanisms underlying mucosal immunotherapy.

Costimulation of mast cells by 4-1BB, a member of the tumor necrosis factor receptor superfamily, with the high-affinity IgE receptor

Mast cells are the major effector-cell type for immediate hypersensitivity and other forms of allergic reactions. Expression of 4-1BB, a member of the tumor necrosis factor receptor superfamily, is induced at mRNA and protein levels on stimulation through the high-affinity receptor for immunoglobulin E (IgE; FcepsilonRI). In this study, we present evidence that agonistic anti-4-1BB antibodies can enhance FcepsilonRI-induced cytokine production and secretion. Consistent with this, 4-1BB-deficient mast cells exhibit reduced degranulation and cytokine production on FcepsilonRI stimulation. Analysis of 4-1BB ligand (4-1BBL)-deficient cells supported this notion. As a potential mechanism for these defects, we identified a defect in Ca2+ flux induced by FcepsilonRI stimulation. The defective Ca2+ flux could be accounted for by the reduced activity of Lyn/Btk/phospholipase C-gamma2 pathway and constitutive interactions between 4-1BB and Lyn. Therefore, FcepsilonRI-inducible 4-1BB plays a costimulatory function together with FcepsilonRI stimulation.

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), FcɛRI, 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-FcɛRI 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 FcɛRI 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 FcɛRI and a chimeric EGFP that contains only the membrane-targeting sequence from Lyn. Our results reveal real-time interactions between Lyn and cross-linked FcɛRI 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.