Estrogen- and xenoestrogen-induced ERK signaling in pituitary tumor cells involves estrogen receptor-α interactions with G protein-αi and caveolin I.
Estrogen- and xenoestrogen-induced ERK signaling in pituitary tumor cells involves estrogen receptor-α interactions with G protein-αi and caveolin I.
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DOI：
10.1016/j.steroids.2011.12.025
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发表时间：
2012-04
期刊：
影响因子：
2.7
通讯作者：
Guptarak J
中科院分区：
文献类型：
--
作者：
Watson CS;Jeng YJ;Hu G;Wozniak A;Bulayeva N;Guptarak J
多种生理雌激素(雌二醇、雌三醇和雌酮)以及异种雌激素化合物(包括烷基酚和双酚A)可以通过质膜雌激素受体α(merα)的配基启动的非基因组信号起作用。我们研究了异三聚体G蛋白参与导致细胞外调节激酶激活的大鼠垂体前叶肿瘤细胞系GH3/B6/F10，这些细胞表达大量的Merα，少量表达Merβ和GPR30。微阵列、免疫印迹和定量免疫分析相结合，证明了这些细胞中表达所有α，β和γG蛋白类别的成员。选择性抑制剂的使用表明，G-ERKI亚型是下游α信号的主要启动子。使用针对Gα蛋白I和S亚型的GTP结合型抗体，我们发现异种雌激素(双酚A、壬基酚)在15-30秒内激活GαI；随后检测的所有烷基酚在5分钟内抑制激活。与GTPαS不可逆的累积结合增强了GγI对所有雌激素的激活作用，而GαS既不被雌激素激活，也不被雌激素灭活。ERα和GαI共定位于胞核外，可共同被免疫捕获。通过表位邻近连接实验证实ERα与GαI和小窝蛋白I的相互作用。ERα/β拮抗剂(ICI182780)和腔泡结构的选择性干扰剂(制霉菌素)可阻断雌激素诱导的ERK激活。异种雌激素和生理性雌激素一样，可以激活下游的激酶信号，涉及ERα与GαI和小窝蛋白I的选择性相互作用，但具有一些不同的特征，这可以解释它们的干扰作用。
Multiple physiologic estrogens (estradiol, estriol, and estrone), as well as xenoestrogenic compounds (including alkylphenols and bisphenol A), can act via nongenomic signaling initiated by liganding of the plasma membrane estrogen receptor-α (mERα). We examined heterotrimeric G protein involvement leading to extracellular-regulated kinase (ERK) activation in GH3/B6/F10 rat anterior pituitary tumor cells that express abundant mERα, and smaller amounts of mERβ and GPR30. A combination of microarrays, immunoblots, and quantitative immunoassays demonstrated the expression of members of all α, β, and γ G protein classes in these cells. Use of selective inhibitors showed that the Gαi subtype was the primary initiator of downstream ERK signaling. Using antibodies against the GTP-bound form of Gα protein subtypes i and s, we showed that xenoestrogens (bisphenol A, nonylphenol) activated Gαi at 15-30 sec; all alkylphenols examined subsequently suppressed activation by 5 min. GTP-activation of Gαi for all estrogens was enhanced by irreversible cumulative binding to GTPγS. In contrast, Gαs was neither activated nor deactivated by these treatments with estrogens. ERα and Gαi co-localized outside nuclei and could be immuno-captured together. Interactions of ERα with Gαi and caveolin I were demonstrated by epitope proximity ligation assays. An ERα/β antagonist (ICI182780) and a selective disruptor of caveolar structures (nystatin) blocked estrogen-induced ERK activation. Xenoestrogens, like physiologic estrogens, can evoke downstream kinase signaling involving selective interactions of ERα with Gαi and caveolin I, but with some different characteristics, which could explain their disruptive actions.