Bivalent ligands with rigid double-stranded DNA spacers reveal structural constraints on signaling by Fc epsilon RI.
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.
Paar
Jodi M
JM
Department of Chemistry and Chemical Biology, Baker Laboratory, Cornell University, Ithaca, NY 14853, USA.
Harris
Nancie T
NT
Holowka
David
D
Baird
Barbara
B
D019297:2,4-Dinitrophenol / Q000737:chemistry / Q000276:immunology / Q000378:metabolism; D000818:Animals; D018033:Antibodies, Bispecific / Q000737:chemistry / Q000378:metabolism / Q000494:pharmacology; D000911:Antibodies, Monoclonal / Q000737:chemistry / Q000378:metabolism / Q000494:pharmacology; D001665:Binding Sites / Q000276:immunology; D015550:Cell Degranulation / Q000276:immunology; D003432:Cross-Linking Reagents / Q000737:chemistry / Q000378:metabolism; D004247:DNA / Q000138:chemical synthesis / Q000378:metabolism* / Q000502:physiology; D021901:DNA, Intergenic / Q000138:chemical synthesis* / Q000378:metabolism* / Q000502:physiology; D015536:Down-Regulation / Q000276:immunology; D006241:Haptens / Q000737:chemistry / Q000378:metabolism / Q000502:physiology; D007073:Immunoglobulin E / Q000737:chemistry / Q000378:metabolism / Q000502:physiology; D008024:Ligands; D008407:Mast Cells / Q000276:immunology / Q000378:metabolism; D051381:Rats; D017455:Receptors, IgE / Q000037:antagonists & inhibitors / Q000502:physiology*; D015398:Signal Transduction / Q000276:immunology*; D012996:Solutions; D013329:Structure-Activity Relationship; D014407:Tumor Cells, Cultured
D018033:Antibodies, Bispecific; D000911:Antibodies, Monoclonal; D003432:Cross-Linking Reagents; D021901:DNA, Intergenic; D006241:Haptens; D008024:Ligands; D017455:Receptors, IgE; D012996:Solutions; D007073:Immunoglobulin E; D004247:DNA; D019297:2,4-Dinitrophenol