Membrane-bound IgE receptor complexes fused with rat basophilic leukemia cells mediate degranulation

The high affinity receptor for IgE on rat basophilic leukemia (RBL) cells mediates antigen-triggered cellular degranulation. Polyethylene glycol-induced membrane fusion methods were used to introduce exogenous IgE receptors into living RBL cells, and these were tested for normal activities. In cell-cell fusion experiments, RBL cells with fluorescein-labeled rat IgE bound to receptors and containing [5-1,2-3H(N)]hydroxytryptamine binoxalate ([3H]5HT) in their secretory granules were fused to cells with receptors occupied by rhodamine-labeled anti-dinitrophenyl mouse IgE. The fused cells showed a uniform surface distribution of both types of IgE, which could be patched independently by anti-IgE or dinitrophenylated bovine gamma globulin (DNP16BGG). [3H]5HT release could be triggered specifically by DNP16BGG. In vesicle-cell fusion experiments, plasma membrane vesicles, with receptors occupied by fluorescein- and 125I-labeled anti-DNP mouse IgE, were fused to RBL cells containing [3H]5HT. The cells showed substantial associated fluorescein fluorescence and 125I counts, and [3H]5HT release could be triggered specifically by DNP16BGG. These experiments indicate that IgE receptors can be dissociated from their natural cellular interactions and retain the ability to reassociate with another cell's components to deliver the transmembrane signal for degranulation.     

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.     

Interaction of antigen-specific T cell factors with unique "receptors" on the surface of mast cells: demonstration in vitro by an indirect rosetting technique

Picryl (trinitrophenyl) chloride (PCL) contact sensitization of mice induces T cells that release an antigen-binding T cell factor (PCLF) that plays an important role in the initiation of contact sensitivity responses, in part via activation of mast cells. The current study employs an in vitro indirect rosette assay to demonstrate that PCLF can interact with the mast cell surface. Sheep red blood cells (SRBC) were hapten conjugated with trinitrophenyl (TNP), dinitrophenyl (DNP), or oxazolone (OX). When TNP-conjugated SRBC were coated with PCLF, monoclonal anti-DNP IgE, or anti-DNP IgG1, they produced 40 to 50% rosettes with purified normal mouse peritoneal mast cells. Analogous antigen-binding factors, from lymphoid cells of OX and dinitrofluorobenzene contact-sensitized mice, gave similar mast cell rosetting levels with OX-SRBC and DNP-SRBC, respectively. PCLF demonstrated a high degree of hapten specificity in that it formed rosettes with TNP-SRBC but not with DNP-SRBC, unlike IgE and IgG1, or DNPF, which formed rosettes with either SRBC type. Similarly, soluble TNP-BSA could inhibit PCLF rosette-forming capacity, but soluble DNP-BSA could not. In addition to mouse mast cells, PCLF formed rosettes with rat basophil leukemia cells, mouse peritoneal exudate macrophages, mouse alveolar macrophages, and J 774 cultured mouse macrophages; it did not form rosettes with rat mast cells, rat alveolar macrophages, or mouse spleen cells. Thus, PCLF-formed rosettes were antigen specific, relatively species specific, and mast cell/macrophage specific. PCLF-mediated rosette-forming activity could be detected in the presence of nanogram quantities of PCLF. More than 10 times greater IgE was needed to produce IgE-mediated rosettes. Reduction and alkylation eliminated the rosetting activity of IgE, but the rosetting activity of PCLF was not affected. PCLF, but not IgE rosette-forming activity, could be removed by and eluted from affinity columns linked with a monoclonal antibody specific for T cell-derived antigen-binding factors, whereas PCLF rosetting activity was not retained by an anti-immunoglobulin affinity column. Preincubation of mast cells with rat myeloma IgE or mouse monoclonal IgE of various specificities blocked IgE rosettes but not PCLF-induced rosettes. Other immunoglobulin isotypes likewise did not block PCLF rosettes. However, PCLF rosettes could be blocked by preincubation of mast cells with OX factor (OXF),and OXF-mediated rosettes could be blocked similarly by PCLF. These results suggest that the antigen-binding T cell factor PCLF interacts with a unique receptor on the surface of mouse mast cells.     

Study of the transit of an integral membrane protein from secretory granules through the plasma membrane of secreting rat basophilic leukemia cells using a specific monoclonal antibody

The monoclonal antibody 5G10 reacted specifically with an 80-kD integral membrane protein in rat basophilic leukemia (RBL) cells. Immunofluorescence microscopy studies of RBL cells, fixed and permeabilized, revealed that the 80-kD protein was located in the membrane of cytoplasmic vesicles. The vesicles were identified as secretory granules by their content in immunoreactive serotonin. Expression of the 5G10 antigen on the surface of unstimulated RBL cells was low. However, RBL cells stimulated to secrete with anti-dinitrophenyl IgE followed by dinitrophenyl-bovine serum albumin or with the Ca2+ ionophore A-23187 displayed an increased expression of the antigen on their surface. Surface exposure of the 5G10 antigen was maximal at 5 min after stimulation of secretion. Removal of dinitrophenyl-bovine serum albumin from the incubation medium resulted in internalization of 50% of the antigen within 10 min.     

Rotational motion of monomeric and dimeric immunoglobulin E-receptor complexes.

Erythrosin 5'-thiosemicarbazide labeled immunoglobulin E (IgE) was used to monitor the rotational dynamics of monomeric and dimeric Fc epsilon RI receptors for IgE on rat basophilic leukemia (RBL) basophilic leukemia (RBL) cells using time-resolved phosphorescence anisotropy. Receptors were studied both on living RBL cells and on membrane vesicles derived from RBL cell plasma membrane. The un-cross-linked IgE-receptor complexes on cells and vesicles exhibit rotational correlation times that are consistent with those expected for freely rotating monomers, but a small fraction of these complexes on cells may be rotationally immobile. A comparison of the initial phosphorescence anisotropy values for erythrosin-labeled IgE-receptor complexes on cells and vesicles reveals a fast component of rotational motion that is greater on the vesicles and may be due to a site of segmental flexibility in the receptor itself. Dimers of IgE-receptor complexes formed with anti-IgE monoclonal antibodies appear to be largely immobile on cells, but they are mobile on vesicles with a 2-fold larger rotational correlation time than the monomeric complexes. The results suggest that dimeric IgE-receptor complexes undergo interactions with other membrane components on intact cells that do not occur on the membrane vesicles. The possible significance of these interactions to receptor function is discussed.     

Triggering of histamine release from rat mast cells by divalent antibodies against IgE-receptors.

Antibodies against receptor molecules for IgE on rat basophilic leukemic (RBL) cells were prepared by immunization of a rabbit with immune precipitates composed of IgE-receptor complexes and anti-IgE. Antibodies against cell surface components were specifically purified by using RBL cells and rendered specific for mast cells by appropriate absorption. The major antibodies in the final preparation (anti-RBL) were directed against receptor molecules. It was found that the F(ab')2 fragments of anti-RBL induced histamine release from rat mast cells and caused immediate skin reactions in normal rats. These reactions by anti-RBL or its F(ab')2 fragments were inhibited if the receptors on mast cells had been saturated with IgE. The Fab' fragments of anti-RBL could bind with receptors on RBL cells and blocked passive sensitization of mast cells with IgE antibodies, but failed to induce skin reactions and histamine release from normal mast cells. Sensitization of normal rat skin with the Fab' fragment followed by an i.v. injection of anti-rabbit IgG induced skin reactions. The results indicated that bridging of receptor molecules by divalent anti-receptor antibody triggered mast cells for histamine release.     

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.     

Effect of Nippostrongylus brasiliensis infection on anti-hapten IgE antibody response in the mouse: I. Induction of carrier specific helper cells

Inoculation of infective larvae of Nippostrongylus brasiliensis into A/J, BALB/c, and SJL mice primed intraperitoneally (ip) 3 weeks before infection with 1 μg of dinitrophenylated keyhole limpet hemocyanin (DNP-KLH) mixed with 1 mg Al(OH)₃ induced a carrier effect on anti-DNP IgE and IgG₁ antibody responses when the experimental mice were secondarily immunized with an ip injection of 1 μg of DNP-coupled N. brasiliensis extract (DNP-Nb) plus alum 2 weeks after infection. The magnitude of the hapten specific antibody response did not correlate rigidly with the number of larvae in the inoculum. Thus, a dose of 100 larvae was as effective in inducing the carrier effect as a dose of 800 larvae. Kinetic studies in A/J and BALB/c mice revealed that the anti-DNP IgE antibody response reached a maximum titer 7 days after the secondary immunization. These studies also showed that the enhanced IgE antibody response persisted for more than 40 days, while the response in all control groups terminated prior to that time. Using the adoptive transfer system, it was demonstrated that lymphoid cells obtained from the spleens or the mesenteric lymph nodes of infected mice cooperated with DNP-KLH primed cells to produce hapten specific IgE and IgG, antibodies when the challenge was made with DNP-Nb but not when it was made with 1 μg DNP-ovalbumin, clearly indicating carrier specificity. The helper activity of the cells obtained from infected mice was completely abolished or greatly reduced by the in vitro treatment with anti-θ serum and complement. The helper cells with maximum activity were present as early as 14 days after inoculation. The level of helper activity gradually decreased after 14 days. The results indicate that N. brasiliensis infection is effective in inducing carrier specific helper cells of thymic origin (T cells) in anti-DNP antibody responses. These results confirm those obtained by other investigators and add the new observation that N. brasiliensis infection elicits special helper T cells which induce an enhancement as well as a prolongation of anti-DNP IgE antibody response.     

The effect of receptor density on the forward rate constant for binding of ligands to cell surface receptors.

For monovalent ligands interacting with cell surface receptors we have directly observed the functional dependence of the forward rate constant on the number of receptors per cell (N). The experimental system we studied consisted of monovalent ligand, 2,4-dinitrophenyl (DNP)-aminocaproyl-L-tyrosine (DCT), binding to bivalent, monoclonal anti-DNP immunoglobulin E (IgE) anchored to its high affinity receptor on rat basophilic leukemia (RBL) cells. To measure the fractional occupation of antibody combining sites by DNP we employed a recently developed fluorescence technique (Erickson, J., Kane, B. Goldstein, D. Holowka, and B. Baird, 1986, Mol. Immunol., 72:769-781). Our results are well fitted by the equation (Berg and Purcell, 1977, Biophys. J., 20:193-219) konc = 4 pi DaN kappa on/[4 pi Da + N kappa on] where konc is the forward rate constant for binding to the cell, D is the diffusion constant of the ligand, a is the radius of the cell, and kappa on is the intrinsic forward rate constant describing a single IgE combining site-DNP interaction. If D is fixed at 10(-5) cm2/s, the best fit of accumulated data predicts an average cell radius of approximately 4 microns and kappa on of approximately 1.8 x 10(-13) cm3/s [1.1 x 10(8)(M . s)-1]; both in excellent agreement with RBL cell size and the single-site forward rate constant for the binding of DCT to IgE in solution, respectively. We believe this is the first report of experimental evidence that directly illustrates the effect of surface density in determining the rates of binding for small molecules to membrane receptors.     

Large-scale co-aggregation of fluorescent lipid probes with cell surface proteins

Large scale aggregation of fluorescein-labeled immunoglobulin E (IgE) receptor complexes on the surface of RBL cells results in the co- aggregation of a large fraction of the lipophilic fluorescent probe 3,3'-dihexadecylindocarbocyanine (diI) that labels the plasma membranes much more uniformly in the absence of receptor aggregation. Most of the diI molecules that are localized in patches of aggregated receptors have lost their lateral mobility as determined by fluorescence photobleaching recovery. The diI outside of patches is mobile, and its mobility is similar to that in control cells without receptor aggregates. It is unlikely that the co-aggregation of diI with IgE receptors is due to specific interactions between these components, as two other lipophilic probes of different structures are also observed to redistribute with aggregated IgE receptors, and aggregation of two other cell surface antigens also results in the coredistribution of diI at the RBL cell surface. Quantitative analysis of CCD images of labeled cells reveals some differences in the spatial distributions of co- aggregated diI and IgE receptors. The results indicate that cross- linking of specific cell surface antigens causes a substantial change in the organization of the plasma membrane by redistributing pre- existing membrane domains or causing their formation.     

Inhibition of anti-DNP antibody formation by high doses of DNP-polyacrylamide molecules; effects of hapten density and hapten valence

High-dose inhibition of anti-DNP antibody formation by a series of DNP-polyacrylamide molecules of varying hapten density and hapten valence was measured. It was found that a molecule's inhibitory ability correlated directly with its hapten density, but not with its hapten valence nor with its own ability at optimal dose to stimulate an anti-DNP response.     

Induction of serotonin release from mast cells by lymphocyte activators is dependent upon implantation of lymphocyte plasma membrane components.

Plasma membranes (PM) isolated from mouse splenic lymphocytes were successfully fused to rat basophilic leukemia (RBL) cells using intact Sendai virus virions (SV). A two-step procedure was used in which SV were first fused with the PM to create PM + SV vesicles; the vesicles formed were then incubated with the RBL cells. Insertion of lymphocyte PM into the RBL cell's membrane endows a high rate of serotonin secretion upon stimulation of the implanted RBL cells with antimouse Ig antibodies or with concanavalin-A. The results of the present work clearly suggest that activation signals can be delivered via implantation of foreign membrane preparation containing specific receptors, thus rendering the target cells susceptible to stimulation by specific reagents.     

Contact hypersensitivity reactions to dinitrofluorobenzene mediated by monoclonal IgE anti-DNP antibodies

Several studies have suggested a possible role for IgE antibodies in the pathogenesis of cutaneous hypersensitivity reactions that reach maximum intensity 24 to 48 hr after antigen challenge. The recent availability of murine monoclonal IgE anti-hapten antibodies has made possible the direct examination of the range of cutaneous inflammatory reactions that can be mediated by such antibodies. We have examined the effects of passively sensitizing BALB/c mice with monoclonal IgE anti-dinitrophenyl (DNP) antibody 48 hr before antigen challenge. Inflammatory responses were assessed by measuring ear swelling in mice challenged on the ears with the reactive hapten 2,4-dinitrofluorobenzene (DNFB). Compared with unsensitized controls, the ears of mice passively sensitized with IgE anti-DNP displayed a biphasic pattern of ear swelling after DNFB challenge. An early, transient response (present within 15 to 30 min of challenge and returning to control levels within 4 to 9 hr) was followed by a second, more persistent increase in ear swelling that peaked 24 to 48 hr after challenge. This biphasic pattern of ear swelling seen in IgE-sensitized mice was temporally indistinguishable from that observed in mice conventionally sensitized for allergic contact dermatitis reactions by epicutaneous application of DNFB 5 days before DNFB ear challenge. Antigen specificity of the IgE-mediated contact hypersensitivity reactions was demonstrated by the failure of mice passively sensitized with IgE anti-DNP to display early or delayed ear swelling greater than unsensitized controls when challenged with either of two noncross-reacting haptens, fluorescein isothiocyanate or oxazolone. Mice passively sensitized with a monoclonal IgA anti-DNP antibody (MOPC 315) 48 hr before DNFB challenge failed to display early or delayed ear swelling greater than unsensitized controls. Heat inactivation of the IgE anti-DNP ascitic fluid at 56 degrees C for 30 min completely abolished its capacity to passively sensitize mice for contact hypersensitivity reactions after DNFB challenge. These results document the existence of an antigen-specific, IgE-mediated, delayed-in-time cutaneous hypersensitivity response that can be elicited by epicutaneous challenge (contract) with a reactive hapten.     

Characterization of the target cell receptor for IgE. II. Polyacrylamide gel analysis of the surface IgE receptor from normal rat mast cells and from rat basophilic leukemia cells.

Purified rat peritoneal mast cells (RMC) and cultured rat basophilic leukemia (RBL) cells were surface labeled with 125I by using lactoperoxidase, incubated with unlabeled rat monoclonal IgE and subjected to solubilization by treatment with Nonidet P-40 (NP-40). With both cell types significant amounts of radioiodinated material could be specifically precipitated by a "sandwich" system consisting of rabbit anti-rat epsilon-chain and goat anti-rabbit Ig. The precipitates were dissociated with sodium dodecyl sulfate (SDS) and urea and subsequently analyzed by SDS-polyacrylamide gel electrophoresis. With RMC three radioactive bands were seen. One corresponded to IgE present on the RMC at the time of isolation. A small band migrating in the region of light chain was seen with both sepcific (anti-IgE) and control precipitates. It showed no demonstrable relationship to IgE. The major radioactive band corresponded to a m.w. of 62,000. This band was dependent upon the presence of IgE and was not found when non-IgE binding control cells were used. With RBL cells, only the IgE-dependent 62,000 dalton peak was present. Saturation of the IgE receptor sites of the RMC or RBL cells before lactoperoxidase labeling almost totally eliminated this radioactive band, indicating that cell-bound IgE rendered this membrane component inaccessible to the radiolabel. These results strongly suggest that this cellular component is identical, at least in part, with the target cell surface receptor for reaginic antibody. The data also further support the hypothesis that the neoplastic RBL cells have a normal surface receptor for IgE.     

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.     

The physical state of membrane lipids modulates the activation of the first component of complement.

Activation of the first component of human complement (C1) by bilayer-embedded nitroxide spin label lipid haptens and specific rabbit antinitroxide antibody has been measured. The nitroxide spin label hapten was contained in host bilayers of either dimyristoyl phosphatidylcholine or dipalmitoyl phosphatidylcholine in the form of both liposomes and vesicles. At a temperature of 32 degrees C, which is intermediate between the hydrocarbon chain-melting temperatures of the two phospholipids, activation of C1 in such vesicles and liposomes is more efficient in the fluid membrane. Studies of C1 activation in binary mixtures of cholesterol and dipalmitoyl phosphatidylcholine indicate that the activation of C1 is not limited by the lateral diffusion of the lipid haptens in these membranes.     

Membranes of sorting organelles display lateral heterogeneity in receptor distribution

This study describes the distribution of an intrinsic membrane protein, the asialoglycoprotein receptor (ASGP-R) in the trans-Golgi reticulum and compartment of uncoupling receptor and ligand (CURL) of rat liver cells. Using quantitative immunogold electron microscopy and membrane length measurements, we showed lateral nonhomogeneity of receptors in the membranes of trans-Golgi reticulum and CURL, in particular in the membranes of secretory vesicles (identified by their content of albumin and very low density lipoprotein particles) and of CURL vesicles (endosomes), including multivesicular bodies. The characteristic tubulovesicular morphology of both sorting organelles defines the transition of receptor-rich tubular membrane and the receptor-poor limiting membrane of the attached vesicles. There was a direct relationship between the size of the secretory and CURL vesicles and the density of ASGP-Rs in their membranes. Receptor density in the smallest vesicles was similar to that found in adjacent continuous tubules. The larger the vesicles, the less receptor was detectable in their membranes. We propose that the receptor molecules are excluded from the vesicle membranes by dynamic lateral redistribution. Nonrandom receptor distribution in the CURL vesicle membranes was present even at the multivesicular body stage. These observations strongly suggest the existence of barriers to ASGP-R diffusion at the junctions of tubules and vesicles. In addition, our observations suggest that ASGP-Rs are transported to the plasma membrane via a mechanism other than the normal secretory pathway.     

Membrane domains of intestinal epithelial cells: distribution of Na+,K+- ATPase and the membrane skeleton in adult rat intestine during fetal development and after epithelial isolation

The organization of the basolateral membrane domain of highly polarized intestinal absorptive cells was studied in adult rat intestinal mucosa, during development of polarity in fetal intestine, and in isolated epithelial sheets. Semi-thin frozen sections of these tissues were stained with a monoclonal antibody (mAb 4C4) directed against Na+,K+-ATPase, and with other reagents to visualize distributions of the membrane skeleton (fodrin), an epithelial cell adhesion molecule (uvomorulin), an apical membrane enzyme (aminopeptidase), and filamentous actin. In intact adult epithelium, Na+,K+-ATPase, membrane-associated fodrin, and uvomorulin were concentrated in the lateral, but not basal, subdomain. In the stratified epithelium of fetal intestine, both fodrin and uvomorulin were localized in areas of cell-cell contact at 16 and 17 d gestation, a stage when Na+,K+-ATPase was not yet expressed. These molecules were excluded from apical domains and from cell surfaces in contact with basal lamina. When Na+,K+-ATPase appeared at 18-19 d, it was codistributed with fodrin. Detachment of epithelial sheets from adult intestinal mucosa did not disrupt intercellular junctions or lateral cell contacts, but cytoplasmic blebs appeared at basal cell surfaces, and a diffuse pool of fodrin and actin accumulated in them. At the same time, Na+,K+-ATPase moved into the basal membrane subdomain, and extensive endocytosis of basolateral membrane, including Na+,K+-ATPase, occurred. Endocytosis of uvomorulin was not detected and no fodrin was associated with endocytic vesicles. Uvomorulin, along with some membrane-associated fodrin and some Na+,K+-ATPase, remained in the lateral membrane as long as intercellular contacts were maintained. Thus, in this polarized epithelium, interaction of lateral cell-cell adhesion molecules as well as basal cell-substrate interactions are required for maintaining the stability of the lateral membrane skeleton and the position of resident membrane proteins concentrated in the lateral membrane domain.     

Binding mechanisms of PEGylated ligands reveal multiple effects of the PEG scaffold

A series of synthetic ligands consisting of poly(ethylene glycol) (PEG), capped on one or both ends with the hapten 2,4-dinitrophenyl (DNP), were previously shown to be potent inhibitors of cellular activation in RBL mast cells stimulated by a multivalent antigen [Baird, E. J., Holowka, D., Coates, G. W., and Baird, B. (2003) Biochemistry 42, 12739-12748]. In this study, we systematically investigated the effect of increasing length of the PEG scaffold on the binding of these monovalent and bivalent ligands to anti-DNP IgE in solution. Our analysis reveals evidence for an energetically favorable interaction between two monovalent ligands bound to the same receptor, when the PEG molecular mass exceeds approximately 5 kDa. Additionally, for ligands with much higher molecular masses (>10 kDa PEG), the binding of a single ligand apparently leads to a steric exclusion of the second binding site by the bulky PEG scaffold. These results are further corroborated by data from an alternate fluorescence-based assay that we developed to quantify the capacity of these ligands to displace a small hapten bound to IgE. This new assay monitors the displacement of a small, receptor-bound hapten by a competitive monovalent ligand and thus quantifies the competitive inhibition offered by a monovalent ligand. We also show that, for bivalent ligands, inhibitory capacity is correlated with the capacity to form effective intramolecular cross-links with IgE.