The G Protein-Coupled Estrogen Receptor Agonist G-1 Inhibits Nuclear Estrogen Receptor Activity and Stimulates Novel Phosphoproteomic Signatures

Toxicol Sci. 2016 Jun;151(2):434-46. doi: 10.1093/toxsci/kfw057. Epub 2016 Mar 29.


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.

Keywords: G-1; GPER; estrogen; estrogen receptor; phosphoproteomic; xenoestrogens.

MeSH terms

  • Antineoplastic Agents, Hormonal / pharmacology*
  • Benzhydryl Compounds / pharmacology
  • Breast Neoplasms / drug therapy*
  • Breast Neoplasms / genetics
  • Breast Neoplasms / metabolism
  • Breast Neoplasms / pathology
  • Cell Proliferation / drug effects
  • Chromatography, High Pressure Liquid
  • Cyclopentanes / pharmacology
  • Dose-Response Relationship, Drug
  • Estradiol / pharmacology
  • Genistein / pharmacology
  • Humans
  • MCF-7 Cells
  • Nitriles / pharmacology
  • Phenols / pharmacology
  • Phosphoproteins / metabolism*
  • Propionates / pharmacology
  • Proteomics / methods
  • Pyrazoles / pharmacology
  • Quinolines / pharmacology
  • Receptors, Estrogen / antagonists & inhibitors*
  • Receptors, Estrogen / metabolism
  • Receptors, G-Protein-Coupled / agonists*
  • Receptors, G-Protein-Coupled / metabolism
  • Signal Transduction / drug effects
  • Tandem Mass Spectrometry
  • Time Factors


  • 1-(4-(6-bromobenzo(1,3)dioxol-5-yl)-3a,4,5,9b-tetrahydro-3H-cyclopenta(c)quinolin-8-yl)ethanone
  • Antineoplastic Agents, Hormonal
  • Benzhydryl Compounds
  • Cyclopentanes
  • GPER1 protein, human
  • Nitriles
  • Phenols
  • Phosphoproteins
  • Propionates
  • Pyrazoles
  • Quinolines
  • Receptors, Estrogen
  • Receptors, G-Protein-Coupled
  • diarylpropionitrile
  • 4,4',4''-(4-propyl-((1)H)-pyrazole-1,3,5-triyl) tris-phenol
  • Estradiol
  • Genistein
  • bisphenol A