Overcoming the signaling defect of Lyn-sequestering, signal-curtailing Fc epsilon RI dimers: Aggregated dimers can dissociate from Lyn and form signaling complexes with Syk

Clustering the tetrameric (alpha beta gamma (2)) IgE receptor, Fe epsilon R I, on basophils and mast cells activates the Src-family tyrosine kinase, Ly n, which phosphorylates Fc epsilon RI beta and gamma subunit tyrosines, cre ating binding sites for the recruitment and activation of Syk. We reported previously that Fc epsilon RI dimers formed by a particular anti-Fe epsilon RI alpha mAb (H10) initiate signaling through Lyn activation and Fc epsilo n ERI subunit phosphorylation, but cause only modest activation of Syk and little Ca2+ mobilization and secretion. Curtailed signaling was linked to t he formation of unusual, detergent-resistant complexes between Lyn and phos phorylated receptor subunits. Here, we show that H10-Fc epsilon RI multimer s, induced by adding F(ab ')(2) of goat anti-mouse IgG to H10-treated cells , support strong Ca2+ mobilization and secretion. Accompanying the recovery of signaling, H10-Fc epsilon RI multimers do not form stable complexes wit h Lyn and do support the phosphorylation of Syk and phospholipase C gamma2. Immunogold electron microscopy showed that H10-Fc epsilon RI dimers coloca lize preferentially with Lyn and are rarely within the osmiophilic "signali ng domains" that accumulate Fc epsilon RI and Syk in Ag-treated cells. In c ontrast, H10-Fc epsilon RI multimers frequently colocalize with Syk within osmiophilic patches. In sucrose gradient centrifugation analyses of deterge nt-extracted cells, H10-treated cells show a more complete redistribution o f Fc epsilon RI beta from heavy (detergent-soluble) to light (Lyn-enriched, detergent-resistant) fractions than cells activated with Fc epsilon RI mul timers. We hypothesize that restraints imposed by the particular orientatio n of H10-Fc epsilon RI dimers traps them in signal-initiating Lyn microdoma ins, and that converting the dimers to multimers permits receptors to disso ciate from Lyn and redistribute to separate membrane domains that support S yk-dependent signal propagation.