Antigen-mediated cross-linking of IgE bound to its receptor, FcRI, stimulates degranulation, phospholipid metabolism, and cytokine production in mast cells and basophils to initiate inflammatory and allergic responses. Previous studies suggested that spatial organization of the clustered receptors affects the assembly of the transmembrane signaling complexes. To investigate systematically the structural constraints in signal initiation, we utilized rigid double-stranded DNA scaffolds to synthesize ligands with tunable lengths. We characterized a series of symmetric trivalent DNA ligands with rigid spacing between 2,4-dinitrophenyl (DNP) haptenic groups in the range of 5-15 nm. These ligands all bind to anti-DNP IgE on RBL mast cells with similar avidity, and they all cross-link IgE-FcRI complexes effectively. We observe length-dependent stimulation of tyrosine phosphorylation of FcRI beta and gamma subunits and the adaptor protein LAT: the shortest ligand is approximately 5-10-fold more potent than the longest. Stimulated Ca2+ mobilization and degranulation also exhibits kinetics and magnitudes that differ as a function of ligand length. In contrast, tyrosine phosphorylation of phospholipase Cgamma1 and consequent Ca2+ release from intracellular stores do not show this dependence on ligand length. Our results with these rigid, DNA-based ligands provide direct support for receptor transphosphorylation as a key step in amplified signaling leading to degranulation, and they further reveal branching of pathways in signaling events.

The asymmetrical structure of bent immunoglobulin E (IgE) bound to its high-affinity receptor, Fc epsilon RI, suggests a possible role for this configuration in the regulation of signaling mediated by cross-linking of Fc epsilon RI on the surface of mast cells and basophils. Indeed, the presence of bound IgE strongly influences the capacity of cross-linked Fc epsilon RI dimers to trigger mast cell degranulation, implicating orientational constraints by bound IgE. Bivalent ligands that cross-link by binding to bivalent IgE can form linear and cyclic chains of IgE/Fc epsilon RI complexes, and these exhibit only limited capacity to stimulate downstream signaling and degranulation, whereas structurally analogous trivalent ligands, which can form branched networks of cross-linked IgE/Fc epsilon RI complexes, are more effective at cell activation. Long bivalent ligands with flexible spacers can form intramolecular cross-links with IgE, and these stable 1:1 complexes are very potent inhibitors of mast cell degranulation stimulated by multivalent antigen. In contrast, trivalent ligands with rigid double-stranded DNA spacers effectively stimulate degranulation responses in a length-dependent manner, providing direct evidence for receptor transphosphorylation as a key step in the mechanism of signaling by Fc epsilon RI. Thus, studies with chemically defined oligovalent ligands show important features of IgE receptor cross-linking that regulate signaling, leading to mast cell activation.

We use electrospray ionization mass spectrometry to quantify >100 phospholipid (PL) components in detergent-resistant membrane (DRM) domains that are related to ordered membrane compartments commonly known as lipid rafts. We previously compared PL compositions of DRMs with plasma membrane vesicles and whole cell lipid extracts from RBL mast cells, and we made the initial observation that antigen stimulation of IgE receptors (FcεRI) causes a significant change in the PL composition of DRMs (Fridriksson, et al., Biochemistry 38: 8056, 1999). We now characterize the signaling requirements and time course for this change, which is manifested as an increase in the recovery of polyunsaturated PL in DRM, particularly in phosphatidylinositol species. We find that this change is largely independent of tyrosine phosphorylation, stimulated by engagement of FcεRI, and can be activated by Ca 2+ ionophore independent of antigen stimulation. Unexpectedly, we found that inhibitors of actin polymerization (cytochalasin D, latrunculin A) cause a similar, but more rapid, change in the PL composition of DRMs in the absence of FcεRI activation, indicating that perturbations in the actin cytoskeleton affect the organization of plasma membrane domains. Consistent with this interpretation, a membrane-permeable stabilizer of F-actin, jasplakinolide, prevents antigen-stimulated changes in DRM PL composition. These results are confirmed by a detailed analysis of multiple experiments, showing that receptor and cytochalasin D-stimulated changes in DRM lipid composition follow first-order kinetics. Analysis in terms of the number of double bonds in the fatty acid chains is valid for total PL of the major head groups and for head groups individually. In this manner we show that, on average, concentrations of saturated or monounsaturated PL decrease in the DRM, whereas concentrations of PL with two or more double bonds (polyunsaturated PL) increase due to cytoskeletal perturbation. We find that these changes are independent of fatty acid chain length. Our mass spectrometric analyses provide a detailed accounting of receptor-activated alterations in the plasma membrane that are regulated by the actin cytoskeleton.

Two series of functional polymers, a,v-bi [2,4-dinitrophenyl][poly(ethylene oxide)-b-poly(2-methoxystyrene)-b-poly(ethylene oxide)] (DNP-PEO-P2MS-PEO-DNP) and a,v-bi[2,4-dinitrophenyl caproic][poly(ethylene oxide)-b-poly(2-methoxystyrene)-b-poly(ethylene oxide)] (CDNP-PEO-P2MS-PEO-CDNP), were synthesized by anionic living polymerization. The polymers were characterized by FT-IR, 1 H-NMR and Gel Permeation Chromatography (GPC). The molecular weight distributions for the lower molecular weight functional polymers were slightly broad (1.3 -1.5). However, the molecular weight distributions for higher molecular weight polymers were narrower (1.1 -1.2). Differential scanning calorimetry (DSC) studies showed thermal transitions indicative of the presence of microphases in the polymer solid state. The polymers were white powders and soluble in tetrahydrofuran. The binding affinity of DNP-PEO-P2MS-PEO-DNP ligands towards anti DNP IgE was determined by titrations with fluorescently labeled FITC-IgE. A water soluble CDNP-PEO-P2MS-PEO-CDNP/DMEG (dimethoxyethylene glycol) complex binds and achieves steady state binding with solution IgE within a few seconds. This strongly suggests that CDNP functional polymers with improved water solubility have potential in therapeutics. Higher molecular weight (water insoluble) CDNP-PEO-P2MS-PEO-CDNP polymers were electrosprayed as fibers (500 nm) on silicon surface. Fluorescence spectroscopy clearly showed that RBL mast cells were interacting with the fibers suggesting that the cell-surface receptors were clustered along the fiber surface. These observations suggest that the functional polymers hold promise for developing an antibody detection device.

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