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1.
Biochemistry ; 43(35): 11352-60, 2004 Sep 07.
Article in English | MEDLINE | ID: mdl-15366945

ABSTRACT

Aggregation of FcepsilonRI, the high-affinity cell-surface receptor for IgE antibody, is required for degranulation of basophils and mast cells, but not all receptor aggregates elicit this cellular response. The stereochemical constraints on clusters of FcepsilonRI that are able to signal cellular responses, such as degranulation, have yet to be fully defined. To improve our understanding of the properties of FcepsilonRI aggregates that influence receptor signaling, we have studied the interaction of 23G3, a rat IgG(1)(kappa) IgE-specific monoclonal antibody, with IgE-FcepsilonRI complexes on rat mucosal-type mast cells (RBL-2H3 line). We find that 23G3 is a potent secretagogue. This property and the structural features of 23G3 (two symmetrically arrayed IgE-specific binding sites) make 23G3 a potentially valuable reagent for investigating the relationship between FcepsilonRI clustering and FcepsilonRI-mediated signaling events. To develop a mathematical model of 23G3-induced aggregation of FcepsilonRI, we used fluorimetry and flow cytometry to quantitatively monitor equilibrium binding of FITC-labeled 23G3 intact Ab and its Fab' fragment to cell-surface IgE. The results indicate that IgE bound to FcepsilonRI expresses two epitopes for 23G3 binding; that 23G3 binds IgE resident on the cell surface with negative cooperativity; and that 23G3 appears to induce mostly but not exclusively noncyclic dimeric aggregates of FcepsilonRI. There is no simple relationship between receptor aggregation at equilibrium and the degranulation response. Further studies are needed to establish how 23G3-induced aggregation of IgE-FcepsilonRI correlates with cellular responses.


Subject(s)
Antibodies, Monoclonal/metabolism , Antibody Specificity , Cell Degranulation , Immunoglobulin E/metabolism , Receptors, Antigen, B-Cell/metabolism , Receptors, IgE/metabolism , Animals , Binding Sites , Cell Degranulation/immunology , Cell Line, Tumor , Dimerization , Epitopes/biosynthesis , Epitopes/immunology , Immunoglobulin E/biosynthesis , Ligands , Models, Immunological , Protein Binding , Rats , Receptor Aggregation , Receptors, IgE/physiology
2.
J Immunol ; 169(2): 856-64, 2002 Jul 15.
Article in English | MEDLINE | ID: mdl-12097389

ABSTRACT

Degranulation of mast cells and basophils during the allergic response is initiated by Ag-induced cross-linking of cell surface IgE-Fc epsilon RI receptor complexes. To investigate how separation distances between cross-linked receptors affect the competency of signal transduction, we synthesized and characterized bivalent dinitrophenyl (DNP)-modified dsDNA oligomers with rigid spacing lengths of approximately 40-100 A. All of these bivalent ligands effectively bind and cross-link anti-DNP IgE with similar affinities in the nanomolar range. The 13-mer (dsDNA length of 44 A), 15-mer (51 A), and flexible 30-mer ligands stimulate similar amounts of cellular degranulation, about one-third of that with multivalent Ag, whereas the 20-mer (68 A) ligand is less effective and the rigid 30-mer (102 A) ligand is ineffective. Surprisingly, all stimulate tyrosine phosphorylation of Fc epsilon RI beta, Syk, and linker for activation of T cells to similar extents as multivalent Ag at optimal ligand concentrations. The magnitudes of Ca(2+) responses stimulated by these bivalent DNP-dsDNA ligands are small, implicating activation of Ca(2+) mobilization by stimulated tyrosine phosphorylation as a limiting process. The results indicate that structural constraints on cross-linked IgE-Fc epsilon RI complexes imposed by these rigid DNP-dsDNA ligands prevent robust activation of signaling immediately downstream of early tyrosine phosphorylation events. To account for these results, we propose that activation of a key downstream target is limited by the spacing between cross-linked, phosphorylated receptors and their associated components.


Subject(s)
DNA, Intergenic/chemical synthesis , DNA, Intergenic/metabolism , DNA/metabolism , Receptors, IgE/physiology , Signal Transduction/immunology , 2,4-Dinitrophenol/chemistry , 2,4-Dinitrophenol/immunology , 2,4-Dinitrophenol/metabolism , Animals , Antibodies, Bispecific/chemistry , Antibodies, Bispecific/metabolism , Antibodies, Bispecific/pharmacology , Antibodies, Monoclonal/chemistry , Antibodies, Monoclonal/metabolism , Antibodies, Monoclonal/pharmacology , Binding Sites/immunology , Cell Degranulation/immunology , Cross-Linking Reagents/chemistry , Cross-Linking Reagents/metabolism , DNA/chemical synthesis , DNA/physiology , DNA, Intergenic/physiology , Down-Regulation/immunology , Haptens/chemistry , Haptens/metabolism , Haptens/physiology , Immunoglobulin E/chemistry , Immunoglobulin E/metabolism , Immunoglobulin E/physiology , Ligands , Mast Cells/immunology , Mast Cells/metabolism , Rats , Receptors, IgE/antagonists & inhibitors , Solutions , Structure-Activity Relationship , Tumor Cells, Cultured
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