Disulfide-linked dimerization of the FcRγ chain is required for positive and negative regulation of mast cell activation via FcεRI
Disulfide-linked dimerization of the FcRγ chain is required for positive and negative regulation of mast cell activation via FcεRI
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FcRγ 链的二硫键连接二聚化是通过 FcεRI 肥大细胞激活的正向和负向调节所必需的
DOI：
10.1016/j.alit.2017.04.010
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发表时间：
2017
影响因子：
6.8
通讯作者：
Okayama Y
中科院分区：
文献类型：
--
作者：
Nunomura S;Ra C;Terui T;Okayama Y
肥大细胞（MC）表达高亲和力IgE受体（FcεRI），由IgE结合Fcε RI链和两个信号亚基组成：FcRb链和FcRg链的二硫键连接的同源二聚体。FcRg链同源二聚体是啮齿类动物和人类MC中的信号转导子，是FcεRI的关键组分。先前使用具有FcRg链胞内结构域的嵌合抗原受体的研究表明，需要FcRg链同源二聚化以在MC中诱导足够的抗原依赖性细胞应答。1，2这一发现表明，二硫键介导的FcRg链同源二聚化可能通过含有FcεRIa和FcRb链的FcεRI复合物在MC活化中发挥重要作用。然而，与完整的FcεRI不同，这种嵌合受体不能组装FcεRIa和FcRb链。因此，对Fcε RI介导的细胞应答中FcRg链之间二硫键的生物学意义了解甚少。在本研究中，我们制备了在FcRg跨膜（TM）区的半胱氨酸7残基（C7 S）处具有或不具有丝氨酸取代的鼠骨髓来源的MC（BMMC），并使用逆转录病毒基因转移将FcRg TM构建体引入FcRg β/β MC（图1A）。野生型和突变的FcRg BMMC分别命名为abgWT和abgC 7S。在abgC 7 S BMMC中，在存在或不存在还原剂的情况下，FcRg链的二硫键连接的二聚化被完全消除（图1 B）。然而，在存在或不存在IgE的情况下，abgWT和abgC 7S BMMC之间的细胞表面Fcε RIa表达相当（图1C、D）。这些数据表明，在存在或不存在IgE的情况下，FcRg链之间的二硫键对BMMC中FcεRI的细胞表面表达的调节没有贡献。尽管abgC 7S BMMC中FcRg链蛋白的表达水平显著降低（图2A），但与FcRb蛋白共沉淀的FcRg蛋白的量在使用温和去污剂毛地黄皂苷溶解的abgWT和abgC 7S细胞裂解物中相当。此外，通过将C7 S突变引入FcRg TM区，BMMC中FcεRI增强诱导的受体内化降低（数据未显示）。总之，这些结果提高了abgC 7S BMMC在FcεRI交联后表现出改变的细胞应答的可能性。最后，我们检查了FcRg链TM结构域中的C7 S突变是否影响Fcε RI触发的脱粒反应和细胞因子产生。由于通过FcεRI的MC活化导致钟形细胞应答曲线，这取决于刺激强度，因此使用次优（2 ng/ml）、最优（20 ng/ml）和超优（200 ng/ml）抗原浓度刺激3、4 IgE致敏的abgWT和abgC 7S BMMC。如图2 C和D所示，将C7 S突变引入FcRg链TM结构域导致Fcε RI依赖性脱粒和细胞因子产生的各种改变。在次优抗原刺激后，abgC 7S BMMC显示出比abgWT BMMC更低水平的脱粒和细胞因子产生。然而，当用高剂量的抗原刺激时，在abgC 7S中的脱粒应答和细胞因子产生显著大于在abgWT BMMC中。这些发现表明，在MC中，FcRg通过Cys 7残基的二聚化有助于控制最佳脱粒和细胞因子产生，这取决于FcεRI刺激的强度。据我们所知，这份报告是第一份解决FcRg链之间的二硫键在调节Fcε RI依赖性MC活化中的生物学作用的报告。
Mast cells (MCs) express the high-affinity IgE receptor (FcεRI) as a tetramer consisting of the IgE-binding FcεRIachain and two signaling subunits: the FcRb chain and a disulfide-linked homodimer of FcRg chains. The FcRg chain homodimer, a signal transducer in rodent and human MCs, is a critical component of FcεRI. Previous studies using chimeric antigen receptors with the FcRg chain intracellular domain have suggested that FcRg chain homodimerization is required to induce sufficient antigen-dependent cellular responses in MCs. 1, 2 Such a finding suggests that disulfide-mediated homodimerization of FcRgchains may play an important role in MC activation via the FcεRI complex containing FcεRIa and FcRb chains. Unlike intact FcεRI, however, such chimeric receptors cannot assemble FcεRIa and FcRb chains. Thus, the biological significance of the disulfide bond between FcRg chains in FcεRI-mediated cellular responses is poorly understood. In this study, we prepared murine bone marrow-derived MCs (BMMCs) with or without a serine substitution at the cysteine7 residue (C7S) of the FcRg transmembrane (TM) region and used retroviral gene transfer to introduce the FcRg TM constructs into FcRgÀ/À MCs (Fig. 1 A). Wild-type and mutated FcRg BMMCs were named abgWT and abgC7S, respectively. In abgC7S BMMCs, disulfide-linked dimerization of the FcRg chain was completely abolished in the presence or absence of a reducing agent (Fig. 1 B). However, cell surface FcεRIaexpression was comparable between abgWT and abgC7S BMMCs in the presence or absence of IgE (Fig. 1 C, D). These data suggest that the disulfide bond between FcRg chains does not contribute to the regulation of cell surface expression of FcεRI in BMMCs in the presence or absence of IgE. Although the expression level of FcRg chain proteins in abgC7S BMMCs was markedly reduced (Fig. 2 A), the amount of FcRg protein co-precipitating with the FcRb protein was comparable in abgWT and abgC7S cell lysates solubilized using the mild detergent digitonin. Moreover, FcεRI engagement-induced receptor internalization was decreased in BMMCs by introduction of the C7S mutation into the FcRg TM region (data not shown). Collectively, these results raise the possibility that abgC7S BMMCs exhibit altered cellular responses following FcεRI cross-linking. Finally, we examined whether the C7S mutation in the FcRg chain TM domain affects FcεRI-triggered degranulation responses and cytokine production. Because MC activation through FcεRI leads to bellshaped cellular response curves depending on the strength of the stimulation, 3, 4 IgE-sensitized abgWT and abgC7S BMMCs were stimulated with sub-optimal (2 ng/ml), optimal (20 ng/ml), and supraoptimal (200 ng/ml) antigen concentrations. As shown in Figure 2 C and D, introduction of the C7S mutation into the FcRg chain TM domain resulted in various alterations in FcεRI-dependent degranulation and cytokine production. Upon sub-optimal antigen stimulation, abgC7S BMMCs showed lower levels of degranulation and cytokine production than abgWT BMMCs. However, when stimulated with a high dose of antigen, degranulation responses and cytokine production were significantly greater in abgC7S than in abgWT BMMCs. These findings suggest that in MCs, FcRg dimerization through the Cys7 residue contributes to controlling optimal degranulation and cytokine production, depending on the strength of FcεRI stimulation. To the best of our knowledge, this report is the first to address the biological actions of the disulfide bond between FcRg chains in the regulation of FcεRI-dependent MC activation …