Involvement of the ras-like GTPase rab3d in RBL-2H3 mast cell exocytosis following stimulation via high affinity IgE receptors (Fc epsilonRI).
Involvement of the ras-like GTPase rab3d in RBL-2H3 mast cell exocytosis following stimulation via high affinity IgE receptors (Fc epsilonRI).
复制标题
通过高亲和力 IgE 受体 (Fc epsilonRI) 刺激后，ras 样 GTPase rab3d 参与 RBL-2H3 肥大细胞胞吐作用。
DOI：
10.4049/jimmunol.159.6.2815
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发表时间：
1997
影响因子：
4.4
通讯作者：
Ulrich Blank
中科院分区：
文献类型：
--
作者：
Michèle Roa;F. Paumet;J. L. Mao;Bernard David;Ulrich Blank
表达在肥大细胞和嗜碱性粒细胞上的高亲和力IgE受体(Fc EpsilonRI)的聚集是从胞浆颗粒释放炎性介质的有力刺激。依赖于Fc epsilonRI的胞吐作用需要蛋白激酶C的激活和细胞内和细胞外钙的动员。然而，这些事件如何最终调节细胞质颗粒和质膜之间的膜融合步骤仍不清楚。在这项研究中，我们研究了Rab3亚家族的小GTP酶在大鼠嗜碱性白血病细胞(RBL-2H3)刺激后调节胞吐中的作用。逆转录-聚合酶链式反应分析表明，RBL-2H3细胞表达Rab3a和rab3d亚型，其中Rab3d在mRNA水平上占优势。亚细胞分布的研究表明，Rab3a主要在胞浆中表达，而rab3d主要在膜部分表达。为了确定这些蛋白是否在Fc epsilonRI触发的胞吐作用中发挥作用，我们建立了过表达野生型和表达GTP结合突变形式(N135I)的rab3a和rab3d的RBL-2H3转基因细胞。然而，根据β-己糖苷酶释放测试，Rab3a蛋白的表达对脱颗粒没有显著影响，而野生型和突变型Rab3d蛋白的表达都抑制了脱颗粒。对最初的快释率和第二缓释率的计算表明，它们都被抑制了大约两倍，这表明Rab3d干扰了Fc epsilonRI刺激的胞吐作用中的限速步骤。
Aggregation of high affinity IgE receptors (Fc epsilonRI) expressed on mast cells and basophils is a potent stimulus for the release of inflammatory mediators from cytoplasmic granules. Fc epsilonRI-dependent exocytosis requires activation of protein kinase C and mobilization of calcium from intra- and extracellular stores. However, how these events ultimately regulate the membrane fusion step between cytoplasmic granules and the plasma membrane still remains unclear. In this study, we investigated the role of the small GTPases of the rab3 subfamily in the regulated exocytosis following stimulation of rat basophilic leukemia cells (RBL-2H3). Analysis using reverse-transcriptase-based PCR showed that RBL-2H3 cells expressed rab3a and rab3d isoforms, with a predominance of rab3d at the mRNA level. Investigation of the subcellular distribution using isoform-specific Abs demonstrated that the majority of rab3a was expressed in the cytosol, whereas rab3d was found predominantly in the membrane fraction. To determine whether these proteins play a role in Fc epsilonRI-triggered exocytosis, we established RBL-2H3 transfectants that overexpressed wild-type and expressed GTP-binding mutant forms (N135I) of rab3a and rab3d. Whereas expression of rab3a proteins did not significantly affect degranulation as tested by beta-hexosaminidase release, those of both wild-type and mutant rab3d proteins inhibited degranulation. Calculations of the initial fast and of the second slow release rates showed that they are both inhibited about twofold, suggesting that rab3d interferes with a rate-limiting step in Fc epsilonRI-stimulated exocytosis.