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

Abstract

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.

Signaling assemblies formed in mast cells activated via Fcϵ receptor I dimers

“…4. study. Although the likelihood of this possibility seems to be strengthened by data showing that the aggregated H10 mAb induced activation comparable in extent to multivalent Ag [9], our own unpublished data render this explanation unlikely because the removal of aggregates by ultracentrifugation did not alter the properties of the mAb. Furthermore, confocal microscopy revealed an almost homogeneous distribution of the H10 mAb on cells incubated with the Ab for 5 min before fixation and staining (not shown).…”

“…These complexes are relatively stable and are not rapidly internalized, as suggested by the absence of an increased amount of clathrin-coated pits in the vicinity of Fc 4 RI dimers/oligomers (see also [9]). How these small complexes are internalized remains to be solved.…”

“…Because these osmiophilic membranes accumulated several other signaling molecules, including Syk, PLC + 2, PI3K, Gab2 and Grb2, Wilson et al suggested that they represent the primary signaling domains that function as sites of signaling from cross-linked Fc 4 RI to downstream responses [2,8]. This hypothesis was supported by data indicating that Fc 4 RI dimers that did not localize in osmiophilic patches were capable of inducing tyrosine phosphorylation of Fc 4 RI subunits, but not Syk activation and other more distant signaling events [9]. Interestingly, full activation response could be restored after induced aggregation of the Fc 4 RI dimers.…”

The SCHOOL of nature: I. Transmembrane signaling

“…Multichain Fc receptors, such as FcεRI, FcαRI, FcγRI and FcγRIII have been known to initiate cell signaling following interactions with multivalent ligands that induce their clustering. 35,62,63,132,140,163,[169][170][171][172][173][238][239][240][241] FcεRI aggregates as small as dimers have been reported to be capable of providing an effective activation signal to cause mediator secretion. 163 Using a set of chemically well-defined ligands of valences 1-3, the magnitude of the cellular response has been demonstrated to dramatically increase as the valency of a ligand raises from two to three.…”

“…Clearly, the strength of the ligand determines not only duration of the ligand-MIRR contact but also lifetime of an individual receptor in the engaged MIRR thus suggesting the importance of the orientational restraint in ligand-specific FceRI dimers/oligomers for generating competent activation signal. 163,164,171,178,179,261 Further, in the IgA receptor, FcαRI, a positively charged arginine residue within the TM domain of ligand recognition α chain promotes association with the signaling FcRγ chain. 153 Studies of signaling through mutants of the FcαRI have shown that a vertical relocation of this TM positive charge does not have any significant effect on proximal and distal receptor functions, whereas a lateral transfer of the positive charge completely abrogates these functions.…”