Overcoming the Signaling Defect of Lyn-Sequestering, Signal-Curtailing FcεRI Dimers: Aggregated Dimers Can Dissociate from Lyn and Form Signaling Complexes with Syk

Clustering the tetrameric (αβγ2) IgE receptor, FcεRI, on basophils and mast cells activates the Src-family tyrosine kinase, Lyn, which phosphorylates FcεRI β and γ subunit tyrosines, creating binding sites for the recruitment and activation of Syk. We reported previously that FcεRI dimers formed by a particular anti-FcεRI α mAb (H10) initiate signaling through Lyn activation and FcεRI subunit phosphorylation, but cause only modest activation of Syk and little Ca2+ mobilization and secretion. Curtailed signaling was linked to the formation of unusual, detergent-resistant complexes between Lyn and phosphorylated receptor subunits. Here, we show that H10-FcεRI multimers, 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-FcεRI multimers do not form stable complexes with Lyn and do support the phosphorylation of Syk and phospholipase Cγ2. Immunogold electron microscopy showed that H10-FcεRI dimers colocalize preferentially with Lyn and are rarely within the osmiophilic “signaling domains” that accumulate FcεRI and Syk in Ag-treated cells. In contrast, H10-FcεRI multimers frequently colocalize with Syk within osmiophilic patches. In sucrose gradient centrifugation analyses of detergent-extracted cells, H10-treated cells show a more complete redistribution of FcεRI β from heavy (detergent-soluble) to light (Lyn-enriched, detergent-resistant) fractions than cells activated with FcεRI multimers. We hypothesize that restraints imposed by the particular orientation of H10-FcεRI dimers traps them in signal-initiating Lyn microdomains, and that converting the dimers to multimers permits receptors to dissociate from Lyn and redistribute to separate membrane domains that support Syk-dependent signal propagation.