IgG-Independent Co-aggregation of FcεRI and FcγRIIB Results in LYN- and SHIP1-Dependent Tyrosine Phosphorylation of FcγRIIB in Murine Bone Marrow-Derived Mast Cells

Activation of the high-affinity receptor for IgE (Fc epsilon RI) follows a bell-shaped dose-response curve. Upon supra-optimal stimulation, mast cell effector responses are down-regulated by inhibitory molecules like the SH2-containing inositol-5 '-phosphatase SHIP1 and the SRC-family-kinase LYN. To identify further molecules involved in a negative regulatory signalosome, we screened for proteins showing the same pattern of tyrosine phosphorylation as SHIP1, which is tyrosine-phosphorylated strongest upon supra-optimal antigen (Ag) stimulation. The low-affinity IgG receptor, Fc gamma RIIB, was found to be most strongly phosphorylated under supra-optimal conditions. This phosphorylation is the consequence of passive, Ag/IgE-dependent and progressive co-localization of Fc epsilon RI and Fc gamma RIIB, which is not dependent on IgG. Upon supra-optimal Fc epsilon RI cross-linking, Fc gamma RIIB phosphorylation is executed by LYN and protected from dephosphorylation by SHIP1. Analysis of Fc gamma RIIB-deficient bone marrow-derived mast cells revealed an ambiguous phenotype upon Fc epsilon RI cross-linking. Absence of Fc gamma RIIB significantly diminished the level of SHIP1 phosphorylation and resulted in augmented Ca2+ mobilization. Though, degranulation and IL-6 production were only weakly altered. Altogether our data establish the LYN/Fc gamma RIIB/SHIP1 signalosome in the context of Fc epsilon RI activation, particularly at supra-optimal Ag concentrations. The fact that SHIP1 tyrosine phosphorylation/activation not only depends on Fc gamma RIIB, highlights the necessity for its tight backup control.