Fc Epsilon RI Alpha Research Articles

Systemic anaphylaxis in the mouse can be mediated largely through IgG1 and Fc gammaRIII. Assessment of the cardiopulmonary changes, mast cell degranulation, and death associated with active or IgE- or IgG1-dependent passive anaphylaxis.

We attempted to elicit active anaphylaxis to ovalbumin, or passive IgE- or IgG1-dependent anaphylaxis, in mice lacking either the Fc epsilonRI alpha chain or the FcR gamma chain common to Fc epsilonRI and Fc gammaRI/III, or in mice lacking mast cells (KitW/ KitW-v mice), and compared the responses to those in the corresponding wild-type mice. We found that the FcR gamma chain is required for the death, as well as for most of the pathophysiological changes, associated with active anaphylaxis or IgE- or IgG1-dependent passive anaphylaxis. Moreover, some of the physiological changes associated with either active, or IgG1-dependent passive, anaphylactic responses were significantly greater in Fc epsilonRI alpha chain -/- mice than in the corresponding normal mice. Finally, while both KitW/KitW-v and congenic +/+ mice exhibited fatal active anaphylaxis, mast cell-deficient mice exhibited weaker physiological responses than the corresponding wild-type mice in both active and IgG1-dependent passive systemic anaphylaxis. Our findings strongly suggest that while IgE antibodies and Fc epsilonRI may influence the intensity and/or kinetics of some of the pathophysiological changes associated with active anaphylaxis in the mouse, the mortality associated with this response can be mediated largely by IgG1 antibodies and Fc gammaRIII.

GM-CSF downregulates expression of tryptase, Fc epsilon RI and histamine in HMC-1 mast cells.

Fibroblast-derived growth factors like SCF are able to upregulate the expression of mast cell characteristics in human multilineage hematopoietic progenitor cells. Other factors, like GM-CSF, have been reported to inhibit this process, probably by the competitive recruitment of cells not belonging to the mast cell lineage. In this study, we investigated the influence of GM-CSF on immature mast cells of the HMC-1 cell line which already show low-level expression of mast cell tryptase, histamine and Fc epsilonRI alpha. Culture of HMC-1 cells with mast-cell-conditioning medium, containing fibroblast supernatants, upregulated tryptase activity, histamine contents and expression of Fc epsilonRI alpha. However, addition of GM-CSF (10 ng/ml) markedly downregulated these mast cell markers, without affecting proliferation and viability of cells. Thus, GM-CSF may provide an inhibitory signal during mast cell differentiation and probably even downregulates mast cell characteristics in more differentiated cells.

Interaction of human IgE with soluble forms of IgE high affinity receptors.

Interaction of human IgE with its high affinity receptor (Fc epsilon RI) on mast cells and basophils is an important step for initiating IgE mediated immune responses. To characterize the IgE and Fc epsilon RI interaction, we investigated this interaction in terms of stoichiometry and binding affinity in solution. The binding of IgE and IgE Fc epsilon RI alpha chain, the extracellular portion of IgE high affinity receptor (sFc epsilon RI alpha) was compared with the binding of IgE and IgE immunoadhesin (Fc epsilon RI alpha-IgG). The interaction was characterized by analytical ultracentrifugation, size exclusion chromatography, light scattering and ELISA. We show that the sFc epsilon RI alpha is only able to bind to one IgE, while the immunoadhesin can bind to two IgE. The interaction between IgE and Fc epsilon RI is very strong. Both forms of soluble receptors have similar intrinsic binding affinity with IgE. Both soluble receptors (Fc epsilon RI alpha-IgG and sFc epsilon RI alpha) can block the binding of IgE to its high affinity receptors on cell surface. The Fc epsilon RI alpha-IgG is a better IgE binding protein than sFc epsilon RI alpha at physiological relevant conditions. A humanized anti-IgE monoclonal antibody, rhuMAb E25 that also can block the binding of IgE to its high affinity receptors appears to bind to IgE at slightly different regions or in a different manner as the soluble forms of IgE receptors.

Peripheral blood dendritic cells express Fc epsilon RI as a complex composed of Fc epsilon RI alpha- and Fc epsilon RI gamma-chains and can use this receptor for IgE-mediated allergen presentation.

Originally limited to basophils and mast cells, the spectrum of high affinity IgE receptor (Fc epsilon RI-bearing cells has expanded recently to include Langerhans cells, dermal dendritic cells (DC), monocytes, and eosinophils. As a result of studies on the distribution, structure, and function of Fc epsilon RI on APCs, we discovered a minor nonbasophil, nonmonocyte PBMC population that can bind IgE via Fc epsilon RI. This receptor occurs on the surface of these cells as a multimeric structure containing Fc epsilon RI alpha- and Fc epsilon RI gamma-chains but, unlike its counterpart on basophils, lacking Fc epsilon RI beta. Further experiments revealed that these Fc epsilon RI alpha gamma-expressing cells closely resemble peripheral blood DC by immunophenotype (HLA-DRhigh, HLA-DQhhigh; CD4+, CD11a+, CD32+, CD33+, B7/2 (CD86)+; CD11blow, CD14low, CD40low, CD54low, CD64low) and cell morphology. These features allowed us to isolate Fc epsilon RI-expressing DC from the peripheral blood and to investigate their immunostimulatory properties. We found Fc epsilon RI-positive DC to be efficient stimulators of both primary (allogeneic MLR) and Fc epsilon RI/IgE-dependent, secondary T cell responses at low cell numbers. Thus, Fc epsilon RI-expressing DC may not only amplify established type I allergic immune reactions but, unlike Fc epsilon RI-positive semiprofessional APCs, may be able to prime naive T cells to common and/or cryptic epitopes of IgE-reactive Ags.

Dermal Mast Cell Activation by Autoantibodies Against the High Affinity IgE Receptor in Chronic Urticaria

Previous studies identified autoantibodies against the IgE high affinity receptor alpha-chain, Fc epsilon RI alpha, in sera of selected patients with severe chronic idiopathic urticaria. We have now determined the incidence of anti-Fc epsilon RI alpha autoantibodies in a group of 163 patients. Intradermal injection of autologous serum caused skin reactions indicative of mast cell degranulation in 98 (60%) patients. Based on histamine release from IgE-sensitized and nonsensitized basophil leukocytes of healthy donors, we detected anti-Fc epsilon RI alpha autoantibodies in sera from 38 (23%) urticaria patients and evidence for anti-IgE antibodies in a further nine patients. The sera that released histamine from basophils induced histamine release (4-34%, n = 12) from mast cells in incubated skin slices. Protein-G affinity chromatography of sera demonstrated that mast cell histamine release was IgG-mediated. Preincubation of sera or the IgG fraction with a recombinant alpha-chain of Fc epsilon RI inhibited histamine release from mast cells and basophils. Further studies with the mouse anti-human Fc epsilon RI alpha antibody 29C6 showed that mast cells and basophils were similarly sensitive to IgG-mediated direct cross-linking of Fc epsilon RI, with 0.01-1.0 micrograms/ml 29C6 evoking histamine release in each case. These studies demonstrate that circulating levels of anti-Fc epsilon RI alpha autoantibodies mediate histamine release from skin mast cells in vitro and, taken together with in vivo evidence of mast cell degranulation following intradermal injection of autologous serum, support the concept that anti-Fc epsilon RI alpha autoantibodies are relevant to the pathogenesis of severe chronic urticaria in about 25% of patients.

Expression of high-affinity IgE receptor (Fc epsilon RI) on human alveolar macrophages from atopic and non-atopic patients.

The expression of high-affinity IgE receptor (Fc epsilon RI) on alveolar macrophages was examined in order to determine the association of alveolar macrophages with IgE-dependent inflammation. Immunostaining showed that a 15-1 monoclonal antibody against Fc epsilon RI alpha chain reacted with alveolar macrophages from both atopic and non-atopic patients by immunostaining. In addition, preincubation of 15-1 monoclonal antibody specifically blocked monomeric IgE binding to alveolar macrophages. Reverse transcription polymerase chain reaction showed that CD14+ alveolar macrophages expressed Fc epsilon RI alpha, beta and gamma chain mRNA. These results show that alveolar macrophages express Fc epsilon RI and may participate in IgE-dependent inflammation mediated by Fc epsilon RI.

Transgenic mice expressing the human high-affinity immunoglobulin (Ig) E receptor alpha chain respond to human IgE in mast cell degranulation and in allergic reactions.

The high-affinity receptor for immunoglobulin (Ig) E (Fc epsilon RI) on mast cells and basophils plays a key role in IgE-mediated allergies. Fc epsilon RI is composed of one alpha, one beta, and two gamma chains, which are all required for cell surface expression of Fc epsilon RI, but only the alpha chain is involved in the binding to IgE. Fc epsilon RI-IgE interaction is highly species specific, and rodent Fc epsilon RI does not bind human IgE. To obtain a "humanized" animal model that responds to human IgE in allergic reactions, transgenic mice expressing the human Fc epsilon RI alpha chain were generated. The human Fc epsilon RI alpha chain gene with a 1.3-kb promoter region as a transgene was found to be sufficient for mast cell-specific transcription. Cell surface expression of the human Fc epsilon RI alpha chain was indicated by the specific binding of human IgE to mast cells from transgenic mice in flow cytometric analyses. Expression of the transgenic Fc epsilon RI on bone marrow-derived mast cells was 4.7 x 10(4)/cell, and the human IgE-binding affinity was Kd = 6.4 nM in receptor-binding studies using 125I-IgE. The transgenic human Fc epsilon RI alpha chain was complexed with the mouse beta and gamma chains in immunoprecipitation studies. Cross-linking of the transgenic Fc epsilon RI with human IgE and antigens led to mast cell activation as indicated by enhanced tyrosine phosphorylation of the Fc epsilon RI beta and gamma chains and other cellular proteins. Mast cell degranulation in transgenic mice could be triggered by human IgE and antigens, as demonstrated by beta-hexosaminidase release in vitro and passive cutaneous anaphylaxis in vivo. The results demonstrate that the human Fc epsilon RI alpha chain alone not only confers the specificity in human IgE binding, but also can reconstitute a functional receptor by coupling with the mouse beta and gamma chains to trigger mast cell activation and degranulation in a whole animal system. These transgenic mice "humanized" in IgE-mediated allergies may be valuable for development of therapeutic agents that target the binding of IgE to its receptor.

Multiple Regions of Human FcγRII (CD32) Contribute to the Binding of IgG

The low affinity receptor for IgG, Fc gamma RII (CD32), has a wide distribution on hematopoietic cells where it is responsible for a diverse range of cellular responses crucial for immune regulation and resistance to infection. Fc gamma RII is a member of the immunoglobulin superfamily, containing an extracellular region of two Ig-like domains. The IgG binding site of human Fc gamma RII has been localized to an 8-amino acid segment of the second extracellular domain, Asn154-Ser161. In this study, evidence is presented to suggest that domain 1 and two additional regions of domain 2 also contribute to the binding of IgG by Fc gamma RII. Chimeric receptors generated by exchanging the extracellular domains and segments of domain 2 between Fc gamma RII and the structurally related Fc epsilon RI alpha chain were used to demonstrate that substitution of domain 1 in its entirety or the domain 2 regions encompassing residues Ser109-Val116 and Ser130-Thr135 resulted in a loss of the ability of these receptors to bind hIgG1 in dimeric form. Site-directed mutagenesis performed on individual residues within and flanking the Ser109-Val116 and Ser130-Thr135 domain 2 segments indicated that substitution of Lys113, Pro114, Leu115, Val116, Phe129, and His131 profoundly decreased the binding of hIgG1, whereas substitution of Asp133 and Pro134 increased binding. These findings suggest that not only is domain 1 contributing to the affinity of IgG binding by Fc gamma RII but, importantly, that the domain 2 regions Ser109-Val116 and Phe129-Thr135 also play key roles in the binding of hIgG1. The location of these binding regions on a molecular model of the entire extracellular region of Fc gamma RII indicates that they comprise loops that are juxtaposed in domain 2 at the interface with domain 1, with the putative crucial binding residues forming a hydrophobic pocket surrounded by a wall of predominantly aromatic and basic residues.

Structure of the gene for the human myeloid IgA Fc receptor (CD89).

A better understanding of IgA's role in immunity requires insight in IgAR complexity. We have now isolated, characterized, and sequenced the gene encoding the prototypic human myeloid IgA FcR (CD89). The gene consists of five exons and spans approximately 12 kilobase pairs. The leader peptide is encoded by two exons, the second of which is 36 bp long and specifies the predicted peptidase cleavage site. A similar, but shorter (21 bp) mini-exon has been found in the FcR for IgG (Fc gamma R) genes, and the FcR for IgE (Fc epsilon RI alpha) gene (human and rodent). The third and fourth exons code for two homologous Ig-like domains. The final exon encodes a short extracellular region, a hydrophobic transmembrane region, and a short cytoplasmic tail. The sequence of the 5'-flanking region was determined, and one major and several minor transcription initiation sites were mapped by primer extension studies. A putative TATA box was located at an appropriate location relative to the start site. Southern blot analyses of genomic DNA confirm the restriction map generated from cloned DNA. These data define the Fc alpha R gene as a distantly related member of the IgR gene family.

Mast cells lacking the high affinity immunoglobulin E receptor are deficient in Fc epsilon RI gamma messenger RNA.

A population of cells that express mast cell markers, including the membrane protein p161, but that lack expression of the high affinity IgE receptor, Fc epsilon RI, can be routinely grown from bone marrow. Ionomycin, but not IgE immune complexes, causes these cells to release serotonin and to express IL-3 and IL-13 mRNA, consistent with their being FC epsilon RI-deficient mast cells. These p161+/Fc epsilon RI- mast cells expressed normal amounts of Fc epsilon RI alpha and beta chain mRNA, but extremely low levels of Fc epsilon RI gamma chain mRNA. In addition, this novel mast cell population expressed CD3 zeta chain mRNA, which p161+/Fc epsilon RI+ mast cells did not. CD3 zeta stable transfectants of Abelson-murine leukemia virus-transformed p161+/Fc epsilon RI+ mast cells continued to express Fc epsilon RI. This strongly suggests that the failure of p161+/Fc epsilon RI- mast cells to express IgE receptors was not caused by the presence of CD3 zeta chain. Transfection of human Fc epsilon RI gamma cDNA into p161+/Fc epsilon RI- mast cells rescued IgE binding. These stable transfectants released serotonin in response to cross-linkage of Fc epsilon RI, demonstrating that the molecular defect of p161+/Fc epsilon RI- mast cells is indeed the loss of Fc epsilon RI gamma expression.

Quantitation of tryptase, chymase, Fc epsilon RI alpha, and Fc epsilon RI gamma mRNAs in human mast cells and basophils by competitive reverse transcription-polymerase chain reaction.

Competitive reverse transcription-PCR assays developed for human tryptase, chymase, Fc epsilon RI alpha, and Fc epsilon RI gamma mRNA molecules were applied to the HMC-1 leukemic mast cell line, the KU812 leukemic basophil cell line, mast cells dispersed from lung and skin, and peripheral blood basophils. Relative amounts of alpha-tryptase and beta-tryptase mRNA were determined by analysis of BseAI digests of PCR products. Tryptase expression was highest in tissue-derived mast cells, lowest in basophils and KU812 cells, and intermediate in HMC-1 cells. beta-Tryptase mRNA predominated in HMC-1 and KU812 cells; mixtures of alpha- and beta-tryptase were found in tissue mast cells; and alpha-tryptase predominated in basophils. Chymase mRNA was more abundant in skin-derived (nearly all of the MCTC type) than lung-derived (variable amounts of MCTC and MCT cells) mast cells. Small amounts of chymase mRNA were detected in HMC-1 cells; none was found in basophils, in KU812 cells, or in the one preparation of 100% MCT cells derived from lung. Comparable amounts of Fc epsilon RI alpha and Fc epsilon RI gamma mRNA molecules were measured in basophils and tissue-derived mast cells, lesser amounts were detected in KU812 cells, and almost none was detected in HMC-1 cells. Thus, steady state levels of the granule and membrane resident molecules examined in our study are transcriptionally regulated in mast cells and basophils.

Hydrodynamic studies of a complex between the Fc fragment of human IgE and a soluble fragment of the Fc epsilon RI alpha chain.

The interaction between immunoglobulin E (IgE) and its high-affinity receptor Fc epsilon RI is central to allergic disease. The binding site for Fc epsilon RI lies in the third constant region domain of the epsilon heavy chain of IgE (C epsilon 3). Identical epitopes on the two C epsilon 3 domains in the IgE-Fc are predicted to be on opposite sides of the structure, and therefore each could bind independently to a receptor molecule. Titrations, however, reveal that the IgE-Fc forms an equimolar complex with a soluble fragment of the Fc epsilon RI alpha chain (sFc epsilon RI alpha), and the molecular weight of the complex, as determined by sedimentation equilibrium, confirms this stoichiometry. The measured sedimentation coefficients of the two ligands are in good agreement with computed values for a compact IgE-Fc and an elongated sFc epsilon RI alpha structure. The calculated sedimentation coefficients for possible models of a 1:1 complex lead to exclusion of all highly extended geometries for the complex. Possible explanations for the paradoxical stoichiometry of the IgE-Fc/sFc epsilon RI alpha complex, in terms of the curved shape of IgE, a conformational change in IgE when the receptor binds, and steric interference between two molecules of Fc epsilon RI binding to identical sites, are discussed.

Functional characterization of the signal transduction events mediated by Fc epsilon RI alpha and gamma chimeric receptors.

Chimeric receptors containing the Fc epsilon RI alpha and gamma subunit domains were constructed, stably transfected into RBL-2H3 cells, and characterized for the biochemical events which are elicited upon receptor aggregation. Chimeric receptors containing the extracellular (EC) domain of the human Fc epsilon RI alpha subunit, or the EC domain of the p55 subunit of the interleukin-2 receptor were fused to the human Fc epsilon RI gamma subunit transmembrane and cytoplasmic (CT) domains or only the CT domain. The chimeras generated included alpha/gamma/gamma, I/gamma/gamma, alpha/I/gamma or I/I/gamma. The results indicate that both the Fc epsilon RI alpha EC domain and the Fc epsilon RI alpha CT domain are essential for signalling.

Role of glycosylation sites in the IgE Fc molecule.

The Fc region of immunoglobulin E (IgE) comprising the C epsilon 3 and C epsilon 4 domains (residues 329-547) is sufficient for binding to the high-affinity IgE Fc receptor (Fc epsilon RI alpha). Three potential N-linked glycosylation sites are present within the C epsilon 3 domain. To determine the effect of the glycosylation sites on IgE Fc synthesis and on Fc epsilon RI alpha binding, site-directed mutagenesis was performed. Mutant IgE Fc constructs were expressed in COS cells and analyzed for protein synthesis and secretion, and Fc epsilon RI alpha binding activity. We find that only N371 and N394 are glycosylated, and that the residues surrounding the glycosylation site at N394 are required for Fc epsilon RI alpha binding activity.

Identification of the IgG binding site of the human low affinity receptor for IgG Fc gamma RII. Enhancement and ablation of binding by site-directed mutagenesis.

Fc receptor-antibody interactions are key mechanisms through which antibody effector functions are mediated. The low affinity receptor for IgG, Fc gamma RII, is expressed on most hematopoietic cells, and through the binding of immune complexes mediates a large spectrum of biological responses vital for resistance to infection and the regulation of immunity. In this study the key residues of human Fc gamma RII involved in the interaction with IgG1 have been identified. Chimeric receptors composed of extracellular regions of Fc gamma RII and the Fc epsilon RI alpha chain have been used to localize the IgG1 binding site of Fc gamma RII to an 8-residue stretch in the second extracellular domain, Asn154 to Ser161. Site-directed mutagenesis of this region revealed that substitution of Ile155 or Gly156 with alanine ablated the binding of human and mouse IgG1, whereas replacement of Leu159, Phe160, or Ser161 with alanine enhanced binding. Molecular modeling has been used to generate a putative 3-dimensional model structure of the second extracellular domain of Fc gamma RII, suggesting that the binding site lies in an exposed loop region at the interface of domains 1 and 2.

High-level expression of the truncated alpha chain of human high-affinity receptor for IgE as a soluble form by baculovirus-infected insect cells. Biochemical characterization of the recombinant product.

The binding subunit of human high-affinity receptor for IgE (Fc epsilon RI alpha) was efficiently expressed as a truncated form in insect cells. The soluble (s)Fc epsilon RI alpha purified from culture medium by affinity chromatography with an anti-(alpha chain) mAb was nearly homogeneous and had an IgE-binding activity. The amino acid composition and the revealed N-terminal amino acid sequence of sFc epsilon RI alpha suggested that it was properly processed in insect cells. The apparent molecular mass (35 kDa) of purified sFc epsilon RI alpha was smaller than that of sFc epsilon RI alpha produced by CHO transfectants. The reduction of the apparent molecular mass after N-glycanase treatment showed the recombinant product was N-glycosylated. Peptide mapping of native and deglycosylated sFc epsilon RI alpha indicated that three Asn residues (Asn21, Asn42 and Asn166) should be almost fully glycosylated, and that two Asn residues (Asn74 and Asn135) were partially glycosylated.

Characterization of the gene for the human high affinity IgE receptor (Fc epsilon RI) alpha-chain.

The Fc epsilon RI couples the mast cell-surface binding of IgE and Ag to a complex series of intracellular events culminating in cell activation and degranulation. The alpha-chain of Fc epsilon RI constitutes the Ig-binding subunit of this heterotetrameric receptor, and is itself a member of the Ig gene superfamily. We have isolated a human genomic DNA clone containing the entire Fc epsilon RI alpha gene, and completely sequenced a region from 1257 bp 5' of the transcription start site, to 513 bp 3' of the last exon of the gene. As with the previously characterized rat and mouse genes, human Fc epsilon RI alpha consists of five exons and four introns, and spans 5889 bp of genomic DNA. The splice donor and acceptor sites deduced by comparison with the cDNA sequence corresponded exactly to the locations found in analogous rodent genes. By mapping the 5' end of Fc epsilon RI alpha transcripts we found three major transcription initiation sites 24, 27, and 29 bp upstream of the ATG translation initiation codon. As well, several longer minor transcripts were seen, with a maximum of 60 nt of 5'-untranslated sequence. About 650 bp of DNA upstream of the ATG translation initiation codon were compared among human, rat, and mouse Fc epsilon RI alpha sequences in search of common motifs that might mediate conserved regulatory interactions with DNA binding proteins. A 172-bp region of the human Fc epsilon RI alpha 5'-flanking sequence was highly conserved in both rodent species. Further studies will be required to determine whether these or other sequences are involved in Fc epsilon RI alpha gene regulation.

Identification of sites on the human Fc epsilon RI alpha subunit which are involved in binding human and rat IgE.

The high affinity IgE Fc receptor (Fc epsilon RI), found on mast cells and basophils, is a tetrameric receptor complex. The extracellular portion of the Fc epsilon RI alpha subunit consists of two immunoglobulin-like domains and binds IgE in the absence of the other subunits. To localize the high affinity IgE binding site within the Fc epsilon RI alpha subunit, we generated a series of chimeric receptor constructs where one of the two immunoglobulin-like domains was either deleted or substituted with those from the human Fc gamma RIIIA alpha or the rat Fc epsilon RI alpha subunit. The chimeric receptors were monitored for their capacity to bind human and rat IgE, and their reactivity with different antireceptor antibodies. Domain I substitutions maintained high affinity human IgE binding. Domain II substitutions resulted in a total loss of both human and rat IgE binding. Single-domain alpha subunits could not bind IgE, suggesting that both extracellular domains are required for proper protein folding or IgE binding. To further localize the IgE binding sites, homolog-scanning mutagenesis was performed. At least three independent regions of domain II encompassing residues 118-129, 136-150, and 148-162 were required for IgE binding. Our results suggest that domain II of the human Fc epsilon RI alpha confers most of the important contributions to the binding of the human IgE Fc molecule, whereas domain I of the rat Fc epsilon RI alpha makes important contributions to the binding of rat IgE.

Immobilization of Fc epsilon receptors by wheat germ agglutinin. Receptor dynamics in IgE-mediated signal transduction.

Transmembrane signaling initiated by the receptor with high affinity for Fc stem of IgE(Fc epsilon RI) requires the diffusion-dependent cross-linkage and persistent aggregation of the Fc epsilon RI. Disruption or prevention of receptor cross-links at any time during the secretory response quickly terminates secretion. We found that in the rat basophilic leukemia mast cell line, addition of wheat germ agglutinin, a lectin that binds to the Fc epsilon RI alpha subunit, caused a precipitous decline in the lateral diffusional and electrokinetic mobilities of the Fc epsilon RI. Both the unoccupied Fc epsilon RI and IgE-Fc epsilon RI complexes became immobilized, as determined from in situ electromigration and postelectric field relaxation. Immobilization of the Fc epsilon RI by wheat germ agglutinin was accompanied by a ligand-reversible association of 125I-IgE-Fc epsilon RI complexes with the Triton X-100-insoluble cytoskeletal fraction. Wheat germ agglutinin rapidly inhibited Fc epsilon RI-mediated signal transduction and secretion, whether cross-linkage was initiated by multivalent antigen, covalent IgE oligomers, anti-IgE, or anti-Fc epsilon RI antibody. Inhibition of signaling and secretion occurred on simultaneous addition of wheat germ agglutinin and antigen, and also when wheat germ agglutinin was added at increasing times after induction of Fc epsilon RI cross-linkage. Wheat germ agglutinin neither reduced the affinity of anti-DNP IgE for haptenic DNP-lysine nor reversed the binding of IgE to the Fc epsilon RI. Although wheat germ agglutinin caused internalization of the Fc epsilon RI, the onset of inhibition preceded and its extent exceeded that of internalization. Wheat germ agglutinin did not interfere with the secretory apparatus, as indicated by its lack of inhibition of secretion elicited by calcium ionophores. These findings suggest that inhibition of signal transduction is secondary to an initial event linked to immobilization of the Fc epsilon RI. Implications of these results are discussed with respect to the dynamics of Fc epsilon RI aggregation on rat basophilic leukemia cells.

Effects of subunit mutation on the localization to coated pits and internalization of cross-linked IgE-receptor complexes.

IgE receptors of mast cells, Fc epsilon RI, localize to coated pits and internalize after cross-linking. We investigated whether any one of the receptor's four distinctive cytoplasmic domains regulates these phenomena. COS cells, which lack Fc epsilon RI entirely, and P815 mouse mastocytoma cells that lack the alpha and beta subunits of the tetrameric Fc epsilon RI (alpha beta gamma 2), were transfected with wild-type, incomplete, or variant Fc epsilon RI. IgE-receptor complexes were observed by electron microscopy. Before cross-linking with anti-IgE gold particles, receptors were not preferentially localized to coated pits, which occupy approximately 1% of the cell surface. After cross-linking, up to 10 to 20% of the wild-type and most other receptor variants were in coated pits in transfected P815 cells at any one time. beta-less variants localized normally but, surprisingly, receptors containing a variant beta subunit showed reduced localization. "Receptors" consisting simply of the lipid-anchored ectodomains of the human alpha subunit failed to localize to coated pits. In general, cross-linked receptors that localized to coated pits were progressively internalized, whereas receptors that failed to accumulate in coated pits were not. We conclude that no single cytoplasmic domain of the Fc epsilon RI uniquely controls its ligand-induced localization to coated pits and internalization.