Separation of a cholesterol-enriched microdomain involved in T-cell signal transduction
Separation of a cholesterol-enriched microdomain involved in T-cell signal transduction
复制标题
DOI：
10.1111/j.1742-4658.2005.04938.x
复制
发表时间：
2005-11-01
期刊：
影响因子：
5.4
通讯作者：
Ohno-Iwashita, Y
中科院分区：
文献类型：
--
作者：
Shimada, Y;Inomata, M;Ohno-Iwashita, Y
我们从Jurkat T细胞的所谓脂筏组分中分离出富含胆固醇的膜亚群，利用其与胆固醇结合探针BC θ的选择性结合。BC θ结合的膜亚群具有比筏级分中BC θ未结合的群体（约0.3）高得多的胆固醇/磷脂（C/P）摩尔比（约1.0）。它不仅含有筏标记物GM 1和flotillin，而且还含有一些T细胞受体（TCR）信号分子，包括Lck、Fyn和LAT。此外，Csk和PAG，TCR信号级联的抑制分子，也包含在BC 0结合的膜中。另一方面，CD 3 β、CD 3 ζ和Zap 70定位于未结合BC θ的膜中，在非刺激条件下与其他TCR信号分子分离。然而，在刺激TCR时，CD 3 β、CD 3 ζ和Zap 70的部分被募集到BC 0结合的膜。用于脂筏制备的Triton X-100浓度既不影响C/P比，也不影响BC θ结合膜的蛋白质组成。这些结果表明，我们的方法可用于分离T细胞的特定富含胆固醇的膜结构域，其可能是控制TCR信号级联的核心结构域。
We isolated a cholesterol-enriched membrane subpopulation from the so-called lipid raft fractions of Jurkat T-cells by taking advantage of its selective binding to a cholesterol-binding probe, BC theta. The BC theta-bound membrane subpopulation has a much higher cholesterol/phospholipid (C/P) molar ratio (approximate to 1.0) than the BC theta-unbound population in raft fractions (approximate to 0.3). It contains not only the raft markers GM1 and flotillin, but also some T-cell receptor (TCR) signalling molecules, including Lck, Fyn and LAT. In addition, Csk and PAG, inhibitory molecules of the TCR signalling cascade, are also contained in the BC theta-bound membranes. On the other hand, CD3 epsilon, CD3 zeta and Zap70 are localized in the BC theta-unbound membranes, segregated from other TCR signalling molecules under nonstimulated conditions. However, upon stimulation of TCR, portions of CD3 epsilon, CD3 zeta and Zap70 are recruited to the BC theta-bound membranes. The Triton X-100 concentration used for lipid raft preparation affects neither the C/P ratio nor protein composition of the BC theta-bound membranes. These results show that our method is useful for isolating a particular cholesterol-rich membrane domain of T-cells, which could be a core domain controlling the TCR signalling cascade.