Data_Sheet_3_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.xlsx

Activation of the high-affinity receptor for IgE (FcεRI) 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, FcγRIIB, 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 FcεRI and FcγRIIB, which is not dependent on IgG. Upon supra-optimal FcεRI cross-linking, FcγRIIB phosphorylation is executed by LYN and protected from dephosphorylation by SHIP1. Analysis of FcγRIIB-deficient bone marrow-derived mast cells revealed an ambiguous phenotype upon FcεRI cross-linking. Absence of FcγRIIB 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/FcγRIIB/SHIP1 signalosome in the context of FcεRI activation, particularly at supra-optimal Ag concentrations. The fact that SHIP1 tyrosine phosphorylation/activation not only depends on FcγRIIB, highlights the necessity for its tight backup control.

IgE高亲和力受体（FcεRI）的激活呈现出钟形剂量反应曲线。在超适宜刺激下，肥大细胞的效应反应受到抑制性分子如含SH2结构域的肌醇-5′-磷酸酶SHIP1和SRC家族激酶LYN的调节。为了进一步识别参与负性调控信号复合体的分子，我们筛选出与SHIP1相同酪氨酸磷酸化模式的蛋白质，即在接受超适宜抗原（Ag）刺激时酪氨酸磷酸化程度最高。低亲和力IgG受体FcγRIIB在超适宜条件下表现出最强的磷酸化。这种磷酸化是FcεRI和FcγRIIB被动、Ag/IgE依赖性及渐进性共定位的结果，这种共定位与IgG无关。在超适宜FcεRI交联作用下，FcγRIIB磷酸化由LYN执行，并由SHIP1保护免受去磷酸化。FcγRIIB缺陷型骨髓来源肥大细胞的分析揭示了FcεRI交联作用下的表型模糊性。FcγRIIB的缺失显著降低了SHIP1磷酸化水平，并导致Ca2+动员增加。然而，脱颗粒和IL-6的产生仅发生轻微改变。总体而言，我们的数据确立了FcεRI激活背景下LYN/FcγRIIB/SHIP1信号复合体的存在，尤其是在超适宜Ag浓度条件下。SHIP1酪氨酸磷酸化/激活不仅依赖于FcγRIIB的事实，突显了其紧密辅助控制的必要性。