Tadokoro Satoshi, Kurimoto Toru, Nakanishi Mamoru, Hirashima Naohide
Graduate School of Pharmaceutical Sciences, Nagoya City University, 3-1 Tanabe-dori, Mizuho-ku, Nagoya 467-8603, Japan.
Mol Immunol. 2007 Jul;44(13):3427-33. doi: 10.1016/j.molimm.2007.02.013. Epub 2007 Apr 3.
Recent studies have revealed that SNARE proteins are involved in exocytotic granular content release in mast cells as well as in neurotransmitter release in neural cells. However, the proteins that regulate the structure and activity of SNARE proteins in mast cells are not well understood. Munc18 is one such regulatory protein that plays a crucial role in neurotransmitter release. In this study, we investigated the role of Munc18 and its mechanism for regulating exocytotic release (degranulation) in rat basophilic leukemia cells (RBL-2H3). We obtained RBL-2H3 cells that express a low level of Munc18-2 and found that degranulation was remarkably inhibited in knockdown cells without any change in the expression level of syntaxins or Ca(2+) mobilization. We also observed the behavior of secretory granules in a single cell, and found no significant changes in their number and distribution in Munc18-2 knockdown cells. Using chimera proteins fused with fluorescent proteins, we demonstrated that Munc18-2 interacted with syntaxin-3, but not with syntaxin-4, in vivo. Interestingly, this interaction occurred not only on plasma membrane but also on secretory granules, suggesting that Munc18-2 may regulate granule-granule fusion as well as granule-plasma membrane fusion. These observations suggest that Munc18-2 together with syntaxin-3 regulate degranulation positively during the process of membrane fusion between secretory granules and plasma membrane, rather than during processes that regulate the number or behavior of secretory granules.
最近的研究表明，SNARE蛋白参与肥大细胞中颗粒内容物的胞吐释放以及神经细胞中神经递质的释放。然而，在肥大细胞中调节SNARE蛋白结构和活性的蛋白质尚未得到充分了解。Munc18就是这样一种调节蛋白，它在神经递质释放中起关键作用。在本研究中，我们调查了Munc18在大鼠嗜碱性白血病细胞（RBL-2H3）中的作用及其调节胞吐释放（脱颗粒）的机制。我们获得了表达低水平Munc18-2的RBL-2H3细胞，发现敲低细胞中的脱颗粒受到显著抑制，而Syntaxin的表达水平或Ca(2+)动员没有任何变化。我们还观察了单个细胞中分泌颗粒的行为，发现在Munc18-2敲低细胞中它们的数量和分布没有显著变化。使用与荧光蛋白融合的嵌合蛋白，我们证明Munc18-2在体内与Syntaxin-3相互作用，但不与Syntaxin-4相互作用。有趣的是，这种相互作用不仅发生在质膜上，也发生在分泌颗粒上，这表明Munc18-2可能调节颗粒-颗粒融合以及颗粒-质膜融合。这些观察结果表明，Munc18-2与Syntaxin-3一起在分泌颗粒与质膜之间的膜融合过程中正向调节脱颗粒，而不是在调节分泌颗粒数量或行为的过程中。
