Lateral electromigration and diffusion of Fc epsilon receptors on rat basophilic leukemia cells: effects of IgE binding

We have used in situ electromigration and post-field relaxation (Poo, M.-m., 1981, Annu. Rev. Biophys. Bioeng., 10:245-276) to assess the effect of immunoglobulin E (IgE) binding on the lateral mobility of IgE- Fc receptors in the plasmalemma of rat basophilic leukemia (RBL) cells. Bound IgE sharply increased the receptor's electrokinetic mobility, whereas removal of cell surface neuraminic acids cut it to near zero. In contrast, we found only a small difference between the lateral diffusion coefficients (D) of vacant and IgE-occupied Fc receptors (D: 4 vs. 3 X 10(-10) cm2/s at 24 degrees C). This is true for monomeric rat IgE; with mouse IgE, the difference in apparent diffusion rates was slightly greater (D: 4.5 vs. 2.3 X 10(-10) cm2/s at 24 degrees C). This range of D values is close to that found in previous photobleaching studies of the IgE-Fc epsilon receptor complex in RBL cells and rat mast cells. Moreover, enzymatic depletion of cell coat components did not measurably alter the diffusion rate of IgE-occupied receptors. Thus, binding of fluorescent macromolecular probes to cell surface proteins need not severely impede lateral diffusion of the probed species. If the glycocalyx of RBL cells does limit lateral diffusion of the Fc epsilon receptor, it must act primarily on the receptor itself, rather than on receptor-bound IgE.     

Rotational dynamics of the Fc receptor for immunoglobulin E on histamine-releasing rat basophilic leukemia cells

The rotational diffusion of immunoglobulin E (IgE) bound to its specific Fc receptor on the surface of living rat basophilic leukemia cells was determined from time-resolved phosphorescence emission and anisotropy measurements. The IgE-receptor complexes are mobile throughout the range of temperatures of 5-38 degrees C. The residual anisotropy does not reach zero, indicating that the rotational diffusion is hindered. The values of rotational correlation times for each temperature are consistent with dispersed receptors rotating freely in the cell membrane and rule out any significant aggregation of occupied receptors before cross-linking by antigen or anti-IgE antibodies. The rotational correlation times decrease with increasing temperature from 65 microseconds at 5.5 degrees C to 23 microseconds at 38 degrees C. However, the degree of orientational constraint experienced by the probe is unchanged. Thus, the temperature dependence can be attributed primarily to a change in the effective viscosity of the cellular plasma membrane. The phosphorescence depolarization technique is very sensitive (our probe concentrations were 10-100 nM) and thus generally applicable to studies of surface receptors and antigens on living cells.     

Binding of monoclonal antibody AA4 to gangliosides on rat basophilic leukemia cells produces changes similar to those seen with Fc epsilon receptor activation.

The mAb AA4 binds to novel derivatives of the ganglioside Gd1b on rat basophilic leukemia (RBL-2H3) cells. Some of the gangliosides are located close to the high affinity IgE receptor (Fc epsilon RI), and binding of mAb AA4 inhibits Fc epsilon RI-mediated histamine release. In the present study, mAb AA4 was found to bind exclusively to mast cells in all rat tissues examined. In vitro, within 1 min of mAb AA4 binding, the cells underwent striking morphologic changes. They lost their normal spindle shaped appearance, increased their ruffling, and spread over the surface of the culture dish. These changes were accompanied by a redistribution of the cytoskeletal elements, actin, tubulin, and vimentin, but only the actin was associated with the membrane ruffles. Binding of mAb AA4 also induces a rise in intracellular calcium, stimulates phosphatidyl inositol breakdown, and activates PKC. However, the extent of these changes was less than that observed when the cells were stimulated with antigen or antibody directed against the Fc epsilon RI. None of these changes associated with mAb AA4 binding were seen when the cells were exposed to nonspecific IgG, IgE, or four other anti-cell surface antibodies, nor were the changes induced by binding mAb AA4 at 4 degrees C or in the absence of extracellular calcium. Although mAb AA4 does not stimulate histamine release, it enhances the effect of the calcium ionophore A23187 mediated release. The morphological and biochemical effects produced by mAb AA4 are similar to those seen following activation of the cell through the IgE receptor. Therefore, the surface gangliosides which bind mAb AA4 may function in modulating secretory events.     

The interaction of murine IgG subclass proteins with human monocyte Fc receptors

The use of murine monoclonal antibodies in the immunotherapy of human disease has prompted interest in the interactions of murine IgG with Fc receptors (FcR) expressed on human effector cells. We examined the heterocytophilic interactions between monomeric murine IgG subclass proteins and the FcR expressed on human monocytic cells (peripheral blood monocytes and interferon (IFN)-gamma-induced U937 cells). All four murine IgG2a antibodies and both murine IgG3 antibodies that were tested bound to human monocyte FcR with high affinity (10(8) to 10(9) M-1). By contrast, the affinities of four murine IgG1 and four IgG2b monomers were 100-fold to 1000-fold lower than the affinity of the human IgG1-FcR interaction. A 68,000 to 72,000 dalton protein was isolated by affinity chromatography from blood monocytes and from IFN-gamma-induced U937 cells on murine IgG2a, IgG3, and human IgG immunoadsorbents. In binding assays with IFN-stimulated U937 cells, murine IgG2a and IgG3 antibodies showed complete cross-blocking with a human IgG1 myeloma protein, indicating that murine and human IgG interact with the same population of Fc-binding proteins. No evidence for heterogeneity of cross-reactive FcR was observed. The ability of murine IgG2a and IgG3 monomers to compete with human IgG1 monomers for binding to human monocyte FcR suggests the potential usefulness of antibodies of these isotypes in the immunotherapy of diseases in which monocyte- or macrophage-mediated, antibody-dependent cellular cytotoxicity may play a role in the modification or remission of disease.     

Aglycosylation of human IgG1 and IgG3 monoclonal antibodies can eliminate recognition by human cells expressing Fc gamma RI and/or Fc gamma RII receptors.

Aglycosylated human IgG1 and IgG3 monoclonal anti-D (Rh) and human IgG1 and IgG3 chimaeric anti-5-iodo-4-hydroxy-3-nitrophenacetyl (anti-NIP) monoclonal antibodies produced in the presence of tunicamycin have been compared with the native glycosylated proteins with respect to recognition by human Fc gamma RI and/or Fc gamma RII receptors on U937, Daudi or K562 cells. Human red cells sensitized with glycosylated IgG3 form rosettes via Fc gamma RI with 60% of U937 cells. Inhibition of rosette formation required greater than 35-fold concentrated more aglycosylated than glycosylated human monoclonal anti-D (Rh) antibody. Unlabelled polyclonal human IgG and glycosylated monoclonal IgG1 and anti-D (Rh) antibody inhibited the binding of 125I-labelled monomeric human IgG binding by U937 Fc gamma RI at concentrations greater than 50-fold lower than the aglycosylated monoclonal IgG1 anti-D (Rh) (K50 approximately 3 x 10(-9) M and approximately 6 x 10(-7) M respectively). Similar results were obtained using glycosylated and aglycosylated monoclonal human IgG1 or IgG3 chimaeric anti-NIP antibody-sensitized red cells rosetting with Fc gamma RI-/Fc gamma RII+ Daudi and K562 cells. Rosette formation could be inhibited by the glycosylated form (at greater than 10(-6) M) but not by the aglycosylated form. Haemagglutination analysis using a panel of murine monoclonal antibodies specific for epitopes located on C gamma 2, C gamma 3 or C gamma 2/C gamma 3 interface regions did not demonstrate differences in Fc conformation between the glycosylated or aglycosylated human monoclonal antibodies. These data suggest that the Fc gamma RI and Fc gamma RII sites on human IgG are highly conformation-dependent and that the carbohydrate moiety serves to stabilize the Fc structure rather than interacting directly with Fc receptors.     

The fate of IgE bound to rat basophilic leukemia cells.

The present study investigates the fate of the cell-bound IgE by using a well-characterized rat basophilic leukemia cell line and a purifed IgE myeloma protein. Both histamine-releasing and nonreleasing cell lines were examined. In both cases, no evidence for cell-mediated IgE catabolism could be elicited. Both the dissociated IgE and the receptors remained intact for prolonged periods of time, as demonstrated by binding assays. Internalization and/or recycling of membrane-bound IgE could not be demonstrated by E. M. autoradiography. We found only limited time-dependent changes in accessibility to anti-IgE antibody, trypsin, or elution at low pH (2.9 to 3.1). A biphasic dissociation of cell-bound 125I-IgE during incubation in the presence of excess unlabeled IgE was reproducibly observed; the more slowly dissociated IgE was also less readily dissociated at pH 3.4. These studies lead us to conclude that, in vitro, IgE resides in a functional orientation on the surface of RBL-1 cells, for prolonged periods of time.     

Mast cell membrane antigens and Fc receptors in anaphylaxis: II. Functionally distinct receptors for IgG and for IgE on mouse mast cells

The present work was aimed at analyzing the functional relationships between mouse mast cell receptors for IgG and IgE antibodies. It was based on a study of the inhibition of IgG1-and IgE-induced passive mast cell degranulation produced by various immunoglobulin preparations capable of interfering with Fc receptors. Rat myeloma IgE, a high-affinity ligand for IgE receptors, was used to search for a possible participation of IgE receptors in IgG1-dependent degranulation. Mouse myeloma IgG, which inhibited only weakly IgG1-mediated reactions, had no chance to compete successfully with high-affinity IgE antibodies, but aggregated HGG was found to behave as a high-affinity ligand for IgG receptors. This enabled us to search for a possible participation of IgG receptors in IgE-dependent degranulation. The results show that rat myeloma IgE and aggregated HGG specifically inhibited IgE-induced and IgG1-induced reactions, respectively, but failed to inhibit reactions not requiring free Fc receptors. The conclusion was that receptors for IgG and for IgE are functionally independent on mouse mast cells, and are both expressed on the same cells.     

Analysis of Fc receptors for IgG on guinea pig peritoneal macrophages by the use of a monoclonal antibody to one of the Fc receptors

The variety and properties of Fc receptors (FcR's) for homologous IgG on guinea pig peritoneal macrophages were investigated with the use of a mouse monoclonal antibody, VIA2 IgG1, prepared by fusion of splenic cells of a mouse immunized with guinea pig macrophages with a mouse myeloma cell line. VIA2 IgG1 completely inhibited the formation of macrophage rosettes with IgG1 antibody-sensitized erythrocytes, but not that with IgG2 antibody-sensitized erythrocytes. The Fab' of VIA2 IgG1 also completely inhibited the bindings of both monomeric and ovalbumin-bound IgG1 antibodies to macrophages. On the other hand, the Fab' did not affect the binding of monomeric IgG2 antibody to macrophages, although it partially inhibited that of ovalbumin-bound IgG2 antibody. These results show that at least two distinct types of FcR are present on guinea pig macrophages; one (FcR1,2) binds monomeric IgG1 antibody and also antigen-bound IgG1 and IgG2 antibodies, and the other (FcR2) binds monomeric and antigen-bound IgG2 antibodies alone, and also that VIA2 IgG1 binds specifically to FcR1,2. When FcR1,2 was isolated by affinity chromatography on F(ab')2 of VIA2 IgG1 coupled to Sepharose, it gave a main band with a molecular weight of 55,000 on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, which was indistinguishable from the main band isolated with the IgG1 immune complex. The number of FcR1,2 per macrophage cell was estimated to be 2 X 10(5) by measuring the binding of 125I-Fab' of VIA2 IgG1.     

A monoclonal antibody that inhibits secretion from rat basophilic leukemia cells and binds to a novel membrane component

Rat basophilic leukemia (RBL-2H3) cells, like mast cells and basophils, carry monovalent membrane receptors with high affinity for IgE (Fc epsilon R). Cross-linking of these receptors provides the immunologic stimulus which initiates a series of biochemical events, culminating in secretion of inflammatory mediators. In an attempt to identify membrane components involved in the stimulus-secretion coupling of these cells, hybridomas were produced from splenocytes of mice immunized with intact RBL-2H3 cells. Here we report the production of a mAb (designated G63) that inhibits the Fc epsilon R-mediated secretion from RBL cells. At low degrees of Fc epsilon R aggregation, the mAb G63-induced inhibition may be complete, whereas at the maximum of secretion the inhibition is in the range of 30 to 40%. The relative degree of inhibition of secretion is dependent on the dose of mAb G63. Furthermore, inhibition requires the bivalency of G63, as the Fab fragments are inactive. The number of antigenic epitopes recognized by G63 per RBL-2H3 cell is 1.8 x 10(4) epitopes/cell, as determined by direct binding studies of 125I-labeled Fab fragments of G63. This number is 20 to 30 times smaller than that of Fc epsilon R on the same cells. The membrane component to which G63 binds has been identified by immunoprecipitation as a glycoprotein with an apparent Mr of 58 to 70 kDa. All of these results, and the fact that no competition for binding to RBL cells between mAb G63 and IgE can be resolved, indicate that mAb G63 binds to a membrane component which is distinct from the Fc epsilon R. mAb G63 suppresses the Fc epsilon R-mediated rise in cytoplasmic concentration of free Ca2+ ions, known to be one of the biochemical signals involved in the stimulus-secretion coupling in RBL-2H3 cells. G63 does not affect, however, the degranulation induced by the Ca2+ ionophore A23187. Therefore, mAb G63 probably exerts its inhibitory effect on a step preceding the rise in cytoplasmic free Ca2+. Thus, mAb G63 defines a previously unidentified membrane component that is involved in one of the early steps of the RBL-2H3 activation mediated by their Fc epsilon R.     

The fate of IgE bound to rat basophilic leukemia cells. IV. Functional association between the receptors for IgE

Rat basophilic leukemia cells (RBL-2H3) have receptors for immunoglobulin E (IgE) and immunoglobulin G (IgG). These receptors for IgE mediate the endocytosis of chemically or immunochemically cross-linked IgE but not monomeric IgE. However, unoccupied receptors were endocytosed with cross-linked IgE. To further assess the degree and specificity of the observed coendocytosis, we exposed cells carrying monomeric rat IgE and monomeric mouse IgE anti-DNP to a DNP-protein conjugate. We found that up to 30% of the surface-bound monomeric rat IgE redistributed at 0 to 4 degrees C and was then internalized at 37 degrees C with the immunochemically cross-linked mouse IgE. To assess the specificity of the coendocytosis, we exposed cells carrying monomeric rat IgE to immunochemically cross-linked mouse IgG. We found that the binding, patching, and endocytosis of cross-linked mouse IgG had no effect on the monomerically bound rat IgE. The rate of coendocytosis was the same as the rate of endocytosis (t 1/2 3 to 5 min). The extent of coendocytosis depended on the extent of endocytosis but was relatively insensitive to changes in the ratio between mouse and rat IgE over a broad range. These results indicate that some of the receptors for IgE are associated in a specific fashion.     

Cross-linking of immunoglobulin E-receptor complexes induces their interaction with the cytoskeleton of rat basophilic leukemia cells

Bridging of immunoglobulin E (IgE)-receptor complexes on rat basophilic leukemia cells by polyclonal anti-IgE antibodies induces a detergent-resistant association of these complexes with the cellular cytoskeleton. In dose-response curves the extent of the cytoskeletal association appears to follow the extent of bridging, continuing to increase beyond where stimulated degranulation is maximal. This stable association is maintained after the aggregated IgE-receptor complexes have been internalized by the cell. Multivalent antigen and trimeric IgE cause less extensive receptor cross-linking than anti-IgE and stimulate degranulation; they also induce receptor association with the cytoskeleton that is revealed only after stabilization by addition of a chemical cross-linking reagent. The ability of a membrane impermeant chemical cross-linker to stabilize this association suggests that the receptor-cytoskeletal interaction may be mediated by a transmembrane protein that is exposed at the cell surface. Monomeric and dimeric IgE bound to receptors fail to induce a stable interaction with the cytoskeleton even in the presence of chemical cross-linkers and are poor (dimers) or insignificant (monomers) stimulators of cellular degranulation. These findings are consistent with a possible relationship between receptor attachment to the cytoskeleton, receptor immobilization as measured by fluorescence photobleaching recovery, and the triggering of cellular degranulation.     

A chymotrypsin-type serine protease in rat basophilic leukemia cells: evidence for its immunologic identity with atypical mast cell protease

Activity of a chymotrypsin-type serine protease was found in a subline of rat basophilic leukemia (RBL-2H3) cells. The protease was immunologically cross-reactive with anti-atypical mast cell protease immunoglobulin (Ig) G, and its activity was inhibited in a dose-dependent manner by the antibody. The apparent m.w. of the protease that reacted with the antibody was 25,000, which was identical with that of atypical mast cell protease in rat mucosal mast cells. These results show that the chymotrypsin type serine protease in RBL-2H3 cells is immunologically identical with atypical mast cell protease, which was first purified from rat small intestine. Immunohistochemical studies showed that the protease was located not only in intracytoplasmic granules but also in organelles synthesizing protein, such as cisternae of the rough endoplasmic reticulum, perinuclear spaces, and the Golgi apparatus. However, no immunoreactivity was demonstrated in rat basophils. The activity of the protease increased in the exponential phase of growth of RBL-2H3 cells in which some activity was also detected in the medium, and it decreased in the late stationary phase.     

Characterization of increased K+ permeability associated with the stimulation of receptors for immunoglobulin E on rat basophilic leukemia cells.

Aggregation of immunoglobulin E-receptor complexes on the surface of rat basophilic leukemia cells stimulates an increase in plasma membrane K+ permeability that is monitored as an increase in the rate of efflux of preloaded 86Rb+. A major component of this stimulated 86Rb+ efflux appears to be due to a Ca(2+)-activated K+ channel because it is inhibited by quinidine in parallel with the inhibition of degranulation and membrane potential repolarization, it is blocked by 0.1 mM La3+, and it is dependent on external Ca2+. Depolarization of the plasma membrane by carbonyl cyanide 3-chlorophenylhydrazone inhibits stimulated Ca2+ influx and prevents antigen-induced 86Rb+ efflux, and increased external Ca2+ partially restores 86Rb+ efflux under these conditions. In addition, potentiation of antigen-stimulated Ca2+ influx by pretreatment with cholera toxin increases the initial rate of stimulated 86Rb+ efflux. Another component of antigen-stimulated K+ efflux appears to be mediated by a guanine nucleotide-binding protein because pretreatment of rat basophilic leukemia cells with pertussis toxin decreases the initial rate of antigen-stimulated 86Rb+ efflux to 40% of that for the untreated cells. Stimulated 86Rb+ efflux is also observed when ionomycin is used to increase cytoplasmic Ca2+ and to trigger membrane depolarization. The efflux stimulated by ionomycin is inhibited by quinidine but not by pertussis toxin pretreatment; thus, it appears to occur through the Ca(2+)-activated K+ efflux pathway. It is proposed that these K+ efflux pathways serve to sustain the Ca2+ influx that is necessary for receptor-mediated triggering of cellular degranulation.     

The IgG Fc contains distinct Fc receptor (FcR) binding sites: the leukocyte receptors Fc gamma RI and Fc gamma RIIa bind to a region in the Fc distinct from that recognized by neonatal FcR and protein A

The CH2-CH3 interface of the IgG Fc domain contains the binding sites for a number of Fc receptors including Staphylococcal protein A and the neonatal Fc receptor (FcRn). It has recently been proposed that the CH2-CH3 interface also contains the principal binding site for an isoform of the low affinity IgG Fc receptor II (Fc gamma RIIb). The Fc gamma RI and Fc gamma RII binding sites have previously been mapped to the lower hinge and the adjacent surface of the CH2 domain although contributions of the CH2-CH3 interface to binding have been suggested. This study addresses the question whether the CH2-CH3 interface plays a role in the interaction of IgG with Fc gamma RI and Fc gamma RIIa. We demonstrate that recombinant soluble murine Fc gamma RI and human Fc gamma RIIa did not compete with protein A and FcRn for binding to IgG, and that the CH2-CH3 interface therefore appears not to be involved in Fc gamma RI and Fc gamma RIIa binding. The importance of the lower hinge was confirmed by introducing mutations in the proposed binding site (LL234,235AA) which abrogated binding of recombinant soluble Fc gamma RIIa to human IgG1. We conclude that the lower hinge and the adjacent region of the CH2 domain of IgG Fc is critical for the interaction between Fc gamma RIIa and human IgG, whereas contributions of the CH2-CH3 interface appear to be insignificant.     

Maintenance of Fc receptors in hybrid cell lines obtained by fusing mouse splenic macrophages with mouse myeloma cells.

Mouse splenic macrophages were fused with cells of the mouse myeloma line P3-X63-Ag8 in the presence of inactivated Sendai virus. Two continuously growing hybrid cell lines were established from fusion mixtures. These hybrid cell lines exhibited macrophage-like morphology and continued to express macrophage derived Fc receptor activity even after prolonged culture in vitro.     

Phospholipase C-gamma 1 is translocated to the membrane of rat basophilic leukemia cells in response to aggregation of IgE receptors.

Aggregation of the high affinity receptor for IgE (Fc epsilon RI) on the surface of mast cells results in the rapid hydrolysis of membrane inositol phospholipids by phospholipase C (PLC). Although at least seven isoenzymes of PLC have been characterized in different mammalian cells, the isoenzyme involved in Fc epsilon RI-mediated signal transduction and the mechanism of its activation have not been demonstrated. We now report that PLC-gamma 1 is translocated to the membrane of mast cells after aggregation of Fc epsilon RI. Activation of rat basophilic leukemia cells, a rat mast cell line, with oligomeric IgE resulted in an increase in PLC activity in washed membrane preparations in a cell free assay containing exogenous [3H]phosphatidylinositol (PI). The increase in PLC activity has the same dose-response to oligomeric IgE as receptor mediated hydrolysis of inositol lipids (PI hydrolysis) in intact cells. Analysis by Western blot probed with anti-PLC-gamma 1 antibody revealed that there is a three- to fourfold increase in PLC-gamma 1 in membranes from activated cells. The increase in PLC activity is augmented a further 20% by the addition of orthovanadate to the incubation medium suggesting that a tyrosine phosphatase is involved in the down-regulation of this phenomenon. These findings demonstrate translocation of PLC-gamma 1 to the membrane following activation of a receptor which does not contain intrinsic tyrosine kinase activity. Activation of PLC-gamma 1 by this pathway may account for Fc epsilon RI-mediated PI hydrolysis.     

The Fc receptor for IgG on human natural killer cells: phenotypic, functional, and comparative studies with monoclonal antibodies

We compare five monoclonal antibodies ( B73 .1, 3G8 , Leu- 11a , Leu- 11b , and VEP13 ) that react with natural killer (NK) cells and polymorphonuclear cells (PMN). We show that all of these antibodies are directed against and inhibit the functional properties of the receptor for the Fc portion of IgG (FcR). Modulation of the FcR on NK cells after reaction with immune complexes induces the disappearance of the antigen(s) recognized by each of the five antibodies. Conversely, the antibodies block binding of IgG-sensitized erythrocytes to the NK cells and PMN and inhibit their ability to mediate cytotoxicity against antibody-sensitized tumor target cells. By using two-color immunofluorescence techniques, we characterize directly the lymphocyte population recognized by these antibodies and show that it is a homogeneous subset that does not bear markers of either B or T cells, with the exception of the 33,000 dalton antigen characteristic of suppressor/cytotoxic T cells present in 20 to 50% of the cells, and the 45,000 dalton receptor for sheep erythrocytes present on 80 to 90% of the cells. The phenotype of the cells reacting with the monoclonal antibodies corresponds to that of NK cells. Cross-competition experiments indicate that these antibodies detect at least two distinct epitopes on FcR, one ( B73 .1) preferentially expressed on NK cells and one or more ( 3G8 /Leu- 11a /Leu- 11b / VEP13 ) preferentially expressed on PMN. The lack of reactivity of these antibodies with B cells suggests that human B cells bear a different FcR from that on NK cells and PMN.     

Aggregation of IgE-receptor complexes on rat basophilic leukemia cells does not change the intrinsic affinity but can alter the kinetics of the ligand-IgE interaction.

The aggregation of IgE anchored to high-affinity Fc epsilon receptors on rat basophilic leukemia (RBL) cells by multivalent antigens initiates transmembrane signaling and ultimately cellular degranulation. Previous studies have shown that the rate of dissociation of bivalent and multivalent DNP ligands from RBL cells sensitized with anti-DNP IgE decreases with increasing ligand incubation times. One mechanism proposed for this effect is that when IgE molecules are aggregated, a conformational change occurs that results in an increase in the intrinsic affinity of IgE for antigen. This possibility was tested by measuring the equilibrium constant for the binding of monovalent DNP-lysine to anti-DNP IgE under two conditions, where the cell-bound IgE is dispersed and where it has been aggregated into visible patches on the cell surface using anti-IgE and a secondary antibody. No difference in the equilibrium constant in these two cases was observed. We also measured the rate of dissociation of a monovalent ligand from cell surface IgE under these two conditions. Whereas the affinity for monovalent ligand is not altered by IgE aggregation, we observe that the rate of ligand dissociation from IgE in clusters is slower than the rate of ligand dissociation from unaggregated IgE. These results are discussed in terms of recent theoretical developments concerning effects of receptor density on ligand binding to cell surfaces.     

A cell surface glycoprotein of rat basophilic leukemia cells close to the high affinity IgE receptor (Fc epsilon RI). Similarity to human melanoma differentiation antigen ME491.

A monoclonal antibody (mAb), AD1, was isolated that recognized a cell surface protein on rat basophilic leukemia cells (RBL-2H3). At high concentration, this antibody inhibited IgE-mediated but not calcium ionophore-induced histamine release (49% inhibition at 100 micrograms/ml). The mAb AD1 did not inhibit the binding of IgE or of several antibodies directed to the high affinity IgE receptor (Fc epsilon RI). Likewise, IgE did not inhibit mAb AD1 binding. However, several anti-Fc epsilon RI antibodies did inhibit mAb AD1 binding as intact molecules but not as Fab fragments. Therefore, the sites on the cell surface to which mAb AD1 binds are close to Fc epsilon RI. The mAb AD1 immunoprecipitated a broad, 50-60-kDa band from 125I-surface-labeled RBL-2H3 cells that upon peptide N-glycosidase F treatment was transformed into a sharp 27-kDa band. A similar 27-kDa protein was immunoprecipitated from surface-radiolabeled cells after culture with tunicamycin. Thus, the protein recognized by mAb AD1 is highly glycosylated with predominantly N-linked oligosaccharides. The N-terminal sequence of 43 amino acids was found to be different from any subunit of Fc epsilon RI but nearly identical to that of the human melanoma-associated antigen ME491. Therefore, mAb AD1 binds to a surface glycoprotein on RBL-2H3 cells sterically close to the Fc epsilon RI but distinct from the recognized subunits of the receptor.