Binding of antigen to IgE-receptor complexes on the surface of RBL-2H3 rat basophilic leukemia cells is the first event leading to the release of cellular serotonin, histamine, and other mediators of allergic, asthmatic, and inflammatory responses. We have used dinitrophenol-conjugated bovine serum albumin (DNP-BSA) as well as the fluorescent antigen, DNP-B-phycoerythrin, and the electron-dense antigen, DNP-BSA-gold, to investigate dynamic membrane and cytoskeletal events associated with the release of [3H]serotonin from anti-DNP-IgE-primed RBL-2H3 cells. These multivalent antigens bind rapidly to cell surface IgE-receptor complexes. Their distribution is initially uniform, but within 2 min DNP-BSAgold is found in coated pits and is subsequently internalized. Antigen internalization occurs in the presence and absence of extracellular Ca 2÷. The F-actin content of the detergent-extracted cell matrices analyzed by SDS PAGE decreases during the first 10-30 s of antigen binding and then increases by 1 min to almost double the control levels. A rapid and sustained increase is also observed when total F-actin is quantified by flow cytometry after binding of rhodaminephalloidin. The antigen-stimulated increase in F-actin coincides with (and may cause) the transformation of the cell surface from a finely microvillous to a highly folded or plicated topography. Other early membrane responses include increased cell spreading and a 2-3-fold increase in the uptake of fluorescein-dextran by fluid pinocytosis. The surface and Factin changes show the same dependence on DNP-protein concentration as stimulated [3HI serotonin release; and both the membrane responses and the release of mediators are terminated by the addition of the non-cross-linking monovalent ligand, DNP-lysine. These data indicate that the same antigen-stimulated transduction pathway controls both the membrane/cytoskeletal and secretory events. However, the membrane and actin responses to IgEreceptor cross-linking are independent of extracellular Ca 2+ and are mimicked by phorbol myristate acetate, whereas ligand-dependent mediator release depends on extracellular Ca 2+ and is mimicked by the Ca 2+ ionophore A23187.The release of histamine, serotonin, and other inflammatory mediators from mast cells and basophils is the precipitating event in a variety of acute allergic, asthmatic, and inflammatory reactions (38). This release occurs by the fusion of cytoplasmic granules with each other and with the plasma membrane, leading to the discharge of granule matrix and soluble mediators (13). Current studies of the sequence of biochemical and morphological events leading to degranulation are focused on the rat basophil-like cell line, . In this cell line degranulation is measured by the release of [3H]serotonin or histamine after cross-linking of surface IgE-receptor complexes with multivalent antigen. Biochemical studies have shown that antigen binding stimulates phosphatidylinositol turnover and generates two second messengers, diacylglycerol to activate pro...

In RBL-2H3 rat tumor mast cells, cross- The RBL-2H3 rat tumor mast cell line has proven to be an excellent experimental model for studies of receptormediated cell activation. Antigen-mediated cross-linking of the high-affinity IgE receptor, FceRI, on these cells leads to Ca2+ influx, phosphatidylinositol turnover, release of serotonin and other substances from granules, polymerization of actin, and transformation of the cell surface from a microvillous to a lamellar architecture (reviewed in refs. 1 and 2). These antigen-stimulated responses are inhibited by tyrosine kinase inhibitors and enhanced by tyrosine phosphatase inhibitors, suggesting the involvement of a receptorstimulated protein-tyrosine kinase(s) in FcERI-mediated RBL-2H3 cell activation (3, 4). The discovery that antigen binding stimulates tyrosine phosphorylation of multiple RBL-2H3 cell proteins supported this hypothesis (5-8 9107The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

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In basophils, mast cells, and the RBL-2H3 tumor mast cell line, cross-linking the high-affinity immunoglobulin E receptor (FcER1) stimulates a series of responses, particularly the activation of phospholipase C (PLC), that lead to allergic and other immediate hypersensitivity reactions. The mechanism of activation of PLC, however, is not clear. Here, we show that cross-linking FceR1 on RBL-2H3 cells causes the tyrosine phosphorylation of at least 12 cellular proteins, including PLC'yi (PLC-yl) and the receptor 18 and -y subunits.32P-labeled PLC'yl can be detected by anti-phosphotyrosine antibody as early as 10 s after the addition of antigen. The tyrosine-phosphorylated 33-kDa subunit and 9-to 11-kDa -y subunit of the FcER1 are additionally phosphorylated on serine and theonine residues, respectively, and are found as complexes with other phosphotyrosine-containing proteins in antigen-stimulated cells. Our results indicate a means by which the FceRl may control PLC activity in RBL-2H3 cells and raise the possibility that other receptor-mediated signalling events in mast cells may also be controlled through protein tyrosine phosphorylation.In RBL-2H3 rat basophilic leukemia cells, a model for mucosal mast cells, antigens that cross-link the high-affinity immunoglobulin E (IgE) receptor FceR1 lead to Ca2+ influx, phosphatidylinositol (PI) turnover, the release of serotonin and other substances from granules, the polymerization of actin, and changes in the cell surface from a microvillous to a lamellar architecture (for reviews, see references 32 and 36). On the basis of experiments with cholera toxin in intact cells and with GTPyS in permeabilized cells, previous investigators have suggested that the FccRi-mediated signal transduction pathway in RBL-2H3 cells is controlled through the activation of GTP-binding proteins (2,3,31 (6, 15-19, 22, 24, 26, 34, 35, 41, 43, 45, 46, 48). By analogy with results obtained with lymphoid cells, it has been proposed that FccRi-stimulated protein tyrosine phosphorylation in RBL-2H3 mast cells may also be mediated by a receptor-associated src family kinase (4,6,16). Other studies have shown that the substrates for ligandstimulated tyrosine phosphorylation in T and B cells include receptor subunits and phospholipase C-yl (8, 9, 24, 37, 42).We show here that antigen binding causes the tyrosine phosphorylation of multiple proteins in RBL-2H3 cells,

In RBL-2H3 rat mucosal mast cells, the crosslinking of cell-surface IgE-receptor complexes by multivalent antigens initiates a sequence of responses leading to degranulation. We have developed a family of dinitrophenol (DNF')-conjugated fluorescent antigens to study dynamic membrane events associated with these responses. Lysyl groups on the phycobiliproteins, B-phycoerythrin and Cphycocyanin, were labelled with DNP, yielding fluorescent conjugates that cause the release of [3H]serotonin from anti-DNP-IgEprimed RBL-2H3 cells. The binding of these antigens to IgE-receptor complexes was observed by fluorescence microscopy and quantified by flow cytometry. Incubation with 1 pglml DNP42-B-phycoerythrin stimulates maximum degranulation from IgE-saturated cells. Under these conditions, approximately 26 x 103 molecules of DNP42-B-phycoerythrin are bound per cell at equilibrium. The rate and extent of antigen binding and of antigenstimulated mediator release decrease in parallel as the concentration and DNPprotein ratio of the fluorescent conjugates is reduced. Secretion stops immediately when the nonfluorescent monovalent antigen, DNP-lysine, is added to degranulating cell suspensions.DNP-lysine also displaces surface-bound antigen when added during the first minutes after multivalent antigen. However, the ability of DNP-lysine to displace surface-bound DNP42-B-phycoerythrin from IgE-receptor complexes decreases progressively with time. Treatment with dihydrocytochalasin B and several analogs that prevent antigen-stimulated F-actin assembly enhances secretion and delays the transition of antigen to its DNPlysine-resistant form. Cytochalasin treatment also permits the long-range movement of antigen into surface caps. Based on these data, we propose that secretion is triggered by the act of IgE-receptor crosslinking or by a shortlived excited state of the crosslinked antigenIgE-receptor complex. We propose further that antigen-stimulated F-actin assembly contributes to the transition of antigen-IgEreceptor complexes to a DNP-lysine-resistant form that does not trigger secretion. Two possible mechanisms for the transition to DNPlysine resistance are discussed.Key terms: Transmembrane signalling, ligand-receptor binding kinetics, phycobiliproteins, mast cells, IgE receptorsThe IgE-dependent release of histamine, serotonin, and other inflammatory mediators from mast cells and basophils is the triggering event in a variety of acute allergic, asthmatic, and inflammatory conditions. Although the events leading to release are still imperfectly understood, the development of new tools and techniques has contributed to recent progress. In particular, Siraganian and colleagues (2,26)