The G Protein-Coupled Estrogen Receptor Agonist G-1 Inhibits Nuclear Estrogen Receptor Activity and Stimulates Novel Phosphoproteomic Signatures.
The G Protein-Coupled Estrogen Receptor Agonist G-1 Inhibits Nuclear Estrogen Receptor Activity and Stimulates Novel Phosphoproteomic Signatures.
复制标题
DOI：
10.1093/toxsci/kfw057
复制
发表时间：
2016-06
期刊：
影响因子：
--
通讯作者：
L. Cody Smith;Kimberly J. Ralston-Hooper;P. Lee Ferguson;T. Sabo-Attwood
中科院分区：
文献类型：
--
作者：
L. Cody Smith;Kimberly J. Ralston-Hooper;P. Lee Ferguson;T. Sabo-Attwood
雌激素通过核（ESR 1和ESR 2）和膜结合雌激素受体（G蛋白偶联雌激素受体，GPER）发挥细胞效应;然而，目前尚不清楚它们是否独立作用或参与串扰影响激素反应。为了研究每种受体在乳腺癌细胞（MCF-7）增殖、转录激活和蛋白磷酸化中的作用，我们采用了ESR 1丙基吡唑三醇（PPT）、ESR 2二芳基丙腈（DPN）和GPER（G-1）的选择性激动剂，并确定了异种雌激素双酚A（BPA）和染料木素对这些效应的影响。正如预期的那样，17β-雌二醇（E2）、PPT、DPN、BPA和染料木黄酮均增强了ERE驱动的报告基因的增殖和激活，而G-1没有显著影响。然而，在大于500 nM的剂量下，G-1显著降低E2-、PPT-、DPN-、BPA-和染料木黄酮诱导的增殖和ERE活化，表明G-1介导的抑制不是ESR同种型特异性的。由于膜受体启动磷酸化事件的级联反应，我们对暴露于E2或G-1的细胞进行了全局磷酸化蛋白质组学分析，以通过下游蛋白磷酸化靶标鉴定受体串扰的潜在靶标。在鉴定的211种磷酸化蛋白中，分别有40种和13种磷酸化蛋白被E2和G-1特异性修饰。子网络富集分析显示，与E2相比，G-1特异性富集了与细胞周期相关的几个过程。此外，还存在许多新鉴定的被G-1特异性磷酸化的蛋白质。这些磷酸化网络突出了可能调节G-1抑制作用的特定蛋白质，并提示了E2和外源性雌激素驱动的核受体活性干扰的新作用。
Estrogen exerts cellular effects through both nuclear (ESR1 and ESR2) and membrane-bound estrogen receptors (G-protein coupled estrogen receptor, GPER); however, it is unclear if they act independently or engage in crosstalk to influence hormonal responses. To investigate each receptor's role in proliferation, transcriptional activation, and protein phosphorylation in breast cancer cells (MCF-7), we employed selective agonists for ESR1 propyl-pyrazole-triol (PPT), ESR2 diarylpropionitrile (DPN), and GPER (G-1) and also determined the impact of xenoestrogens bisphenol-A (BPA) and genistein on these effects. As anticipated, 17β-estradiol (E2), PPT, DPN, BPA, and genistein each enhanced proliferation and activation of an ERE-driven reporter gene whereas G-1 had no significant impact. However, G-1 significantly reduced E2-, PPT-, DPN-, BPA-, and genistein-induced proliferation and ERE activation at doses greater than 500 nM indicating that G-1 mediated inhibition is not ESR isotype specific. As membrane receptors initiate cascades of phosphorylation events, we performed a global phosphoproteomic analysis on cells exposed to E2 or G-1 to identify potential targets of receptor crosstalk via downstream protein phosphorylation targets. Of the 211 phosphorylated proteins identified, 40 and 13 phosphoproteins were specifically modified by E2 and G-1, respectively. Subnetwork enrichment analysis revealed several processes related to cell cycle were specifically enriched by G-1 compared with E2. Further there existed a number of newly identified proteins that were specifically phosphorylated by G-1. These phosphorylation networks highlight specific proteins that may modulate the inhibitory effects of G-1 and suggest a novel role for interference with nuclear receptor activity driven by E2 and xenoestrogens.