EGF-dependent translocation of green fluorescent protein-tagged PLC-gamma1 to the plasma membrane and endosomes.
EGF-dependent translocation of green fluorescent protein-tagged PLC-gamma1 to the plasma membrane and endosomes.
复制标题
绿色荧光蛋白标记的 PLC-gamma1 依赖于 EGF 易位至质膜和内体。
DOI：
10.1006/excr.2001.5241
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发表时间：
2001
期刊：
影响因子：
--
通讯作者：
Carpenter,G
中科院分区：
文献类型：
--
作者：
Wang,XJ;Liao,HJ;Chattopadhyay,A;Carpenter,G
使用在人表皮样癌 A-431 细胞中表达的绿色荧光蛋白标记的 PLC-γ1 (PLC-γ1–GFP) 研究了磷脂酶 C-γ1 (PLC-γ1) 的生长因子依赖性易位。在缺乏生长因子的情况下，PLC-γ1-GFP 存在于 A-431 细胞的整个细胞质中。用表皮生长因子 (EGF) 处理细胞，使 PLC-γ1-GFP 以不均匀的方式非常快速地重新分布到质膜上。这种向质膜的易位对磷脂酰肌醇3-激酶抑制剂不敏感并且与细胞粘附无关。然而，易位被解聚肌动蛋白细胞骨架的试剂破坏。后来添加 EGF 后，PLC-γ1-GFP 似乎与细胞内囊泡相关。德克萨斯红结合 EGF 刺激 A-431 细胞超过 10 分钟，导致细胞内出现点状 PLC-γ1-GFP 分布，与德克萨斯红结合 EGF 共定位。这表明 PLC-γ1 在 EGF 处理后可能通过与内化和自磷酸化的 EGF 受体结合而易位至内体。分级研究表明，EGF 诱导的质膜定位 PLC-γ1 集中在小凹微区中。用甲基-β-环糊精破坏小窝可消除EGF 诱导的细胞内Ca2+ 动员，但不会消除缓激肽诱导的细胞内Ca2+ 动员。然而，这种处理仅部分减少了 PLC-γ1 膜易位。
Growth factor-dependent translocation of phospholipase C-γ1 (PLC-γ1) was investigated using a green fluorescent protein-tagged PLC-γ1 (PLC-γ1–GFP) expressed in human epidermoid carcinoma A-431 cells. In the absence of growth factors, PLC-γ1–GFP was present throughout the cytoplasm of A-431 cells. Treatment of the cells with epidermal growth factor (EGF) produced a very rapid redistribution of PLC-γ1–GFP to the plasma membrane in a nonuniform manner. This translocation to the plasma membrane was insensitive to an inhibitor of phosphatidylinositol 3-kinase and was independent of cell adhesion. However, the translocation was disrupted by an agent which depolymerizes the actin cytoskeleton. At later times following the addition of EGF, PLC-γ1–GFP appeared associated with intracellular vesicles. Stimulation of A-431 cells by Texas red-conjugated EGF for more than 10 min resulted in punctate intracellular PLC-γ1–GFP distribution that colocalized with Texas red-conjugated EGF. This suggests that PLC-γ1 is translocated to endosomes after EGF treatment, probably by associating with the internalized and autophosphorylated EGF receptor. Fractionation studies demonstrated that the EGF-induced plasma membrane-localized PLC-γ1 is concentrated in caveolae microdomains. Disruption of caveolae with methyl-β-cyclodextrin resulted in the ablation of EGF-induced, but not bradykinin-induced, mobilization of intracellular Ca2+. This treatment, however, only partially decreased PLC-γ1 membrane translocation.