Stauffer T P, Meyer T
Department of Cell Biology, Duke University Medical Center, Durham, North Carolina 27710, USA.
J Cell Biol. 1997 Dec 15;139(6):1447-54. doi: 10.1083/jcb.139.6.1447.
Several receptor-mediated signal transduction pathways, including EGF and IgE receptor pathways, have been proposed to be spatially restricted to plasma membrane microdomains. However, the experimental evidence for signaling events in these microdomains is largely based on biochemical fractionation and immunocytochemical studies and only little is known about their spatial dynamics in living cells. Here we constructed green fluorescent protein-tagged SH2 domains to investigate where and when IgE receptor (FcepsilonRI)-mediated tyrosine phosphorylation occurs in living tumor mast cells. Strikingly, within minutes after antigen addition, tandem SH2 domains from Syk or PLC-gamma1 translocated from a uniform cytosolic distribution to punctuate plasma membrane microdomains. Colocalization experiments showed that the microdomains where tyrosine phosphorylation occurred were indistinguishable from those stained by cholera toxin B, a marker for glycosphingolipids. Competitive binding studies with coelectroporated unlabeled Syk, PLC-gamma1, and other SH2 domains selectively suppressed the induction of IgE receptor-mediated calcium signals as well as the binding of the fluorescent SH2 domains. This supports the hypothesis that PLC-gamma1 and Syk SH2 domains selectively bind to Syk and IgE receptors, respectively. Unlike the predicted prelocalization of EGF receptors to caveolae microdomains, fluorescently labeled IgE receptors were found to be uniformly distributed in the plasma membrane of unstimulated cells and only transiently translocated to glycosphingolipid rich microdomains after antigen addition. Thus, these in vivo studies support a plasma membrane signaling mechanism by which IgE receptors transiently associate with microdomains and induce the spatially restricted activation of Syk and PLC-gamma1.
包括表皮生长因子（EGF）和免疫球蛋白E（IgE）受体途径在内的几种受体介导的信号转导途径，被认为在空间上局限于质膜微结构域。然而，这些微结构域中信号事件的实验证据主要基于生化分级分离和免疫细胞化学研究，对于它们在活细胞中的空间动态了解甚少。在此，我们构建了绿色荧光蛋白标记的Src同源2（SH2）结构域，以研究IgE受体（FcepsilonRI）介导的酪氨酸磷酸化在活的肿瘤肥大细胞中的发生位置和时间。令人惊讶的是，在添加抗原后的几分钟内，来自脾酪氨酸激酶（Syk）或磷脂酶C-γ1（PLC-γ1）的串联SH2结构域从均匀的胞质分布转位至点状的质膜微结构域。共定位实验表明，发生酪氨酸磷酸化的微结构域与霍乱毒素B染色的微结构域无法区分，霍乱毒素B是糖鞘脂的标志物。与共电穿孔的未标记Syk、PLC-γ1和其他SH2结构域的竞争性结合研究选择性地抑制了IgE受体介导的钙信号诱导以及荧光SH2结构域的结合。这支持了以下假设，即PLC-γ1和Syk的SH2结构域分别选择性地结合Syk和IgE受体。与表皮生长因子受体定位于小窝微结构域的预测不同，荧光标记的IgE受体在未刺激细胞的质膜中均匀分布，仅在添加抗原后短暂转位至富含糖鞘脂的微结构域。因此，这些体内研究支持了一种质膜信号传导机制，通过该机制IgE受体与微结构域短暂结合并诱导Syk和PLC-γ1在空间上受限的激活。
