Trivalent Ligands with Rigid DNA Spacers Reveal Structural Requirements For IgE Receptor Signaling in RBL Mast Cells

Abstract

Antigen-mediated cross-linking of IgE bound to its receptor, FcϵRI, 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–FcϵRI complexes effectively. We observe length-dependent stimulation of tyrosine phosphorylation of FcϵRI β and γ subunits and the adaptor protein LAT: the shortest ligand is ∼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 Cγ1 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.