title: The G Protein-Coupled Estrogen Receptor Agonist G-1 Inhibits Nuclear Estrogen Receptor Activity and Stimulates Novel Phosphoproteomic Signatures.
abstract: 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.
authors.affiliation: *Department of Physiological Sciences, Center for Environmental and Human Toxicology, University of Florida, Gainesville, Florida 32611;
authors.forename: L Cody
authors.initials: LC
authors.lastname: Smith
authors.affiliation: Department of Civil and Environmental Engineering, Nicholas School of the Environment, Duke University, Durham, North Carolina 27708.
authors.forename: Kimberly J
authors.initials: KJ
authors.lastname: Ralston-Hooper
authors.affiliation: Department of Civil and Environmental Engineering, Nicholas School of the Environment, Duke University, Durham, North Carolina 27708.
authors.forename: P Lee
authors.initials: PL
authors.lastname: Ferguson
authors.affiliation: Department of Environmental and Global Health, Center for Environmental and Human Toxicology, University of Florida, Gainesville, Florida 32611; sabo@phhp.ufl.edu.
authors.forename: Tara
authors.initials: T
authors.lastname: Sabo-Attwood
mesh_terms: D018931:Antineoplastic Agents, Hormonal / Q000494:pharmacology*; D001559:Benzhydryl Compounds / Q000494:pharmacology; D001943:Breast Neoplasms / Q000188:drug therapy* / Q000235:genetics / Q000378:metabolism / Q000473:pathology; D049109:Cell Proliferation / Q000187:drug effects; D002851:Chromatography, High Pressure Liquid; D003517:Cyclopentanes / Q000494:pharmacology; D004305:Dose-Response Relationship, Drug; D004958:Estradiol / Q000494:pharmacology; D019833:Genistein / Q000494:pharmacology; D006801:Humans; D061986:MCF-7 Cells; D009570:Nitriles / Q000494:pharmacology; D010636:Phenols / Q000494:pharmacology; D010750:Phosphoproteins / Q000378:metabolism*; D011422:Propionates / Q000494:pharmacology; D040901:Proteomics / Q000379:methods; D011720:Pyrazoles / Q000494:pharmacology; D011804:Quinolines / Q000494:pharmacology; D011960:Receptors, Estrogen / Q000037:antagonists & inhibitors* / Q000378:metabolism; D043562:Receptors, G-Protein-Coupled / Q000819:agonists* / Q000378:metabolism; D015398:Signal Transduction / Q000187:drug effects; D053719:Tandem Mass Spectrometry; D013997:Time Factors; D000098826:Bisphenol A Compounds
chemical_list: D018931:Antineoplastic Agents, Hormonal; D001559:Benzhydryl Compounds; D003517:Cyclopentanes; D004958:Estradiol; D019833:Genistein; D009570:Nitriles; D010636:Phenols; D010750:Phosphoproteins; D011422:Propionates; D011720:Pyrazoles; D011804:Quinolines; D011960:Receptors, Estrogen; D043562:Receptors, G-Protein-Coupled; C517943:1-(4-(6-bromobenzo(1,3)dioxol-5-yl)-3a,4,5,9b-tetrahydro-3H-cyclopenta(c)quinolin-8-yl)ethanone; C486182:2,3-bis(4-hydroxyphenyl)-propionitrile; C109584:GPER1 protein, human; C486184:4,4',4''-(4-propyl-((1)H)-pyrazole-1,3,5-triyl) tris-phenol; C006780:bisphenol A; D000098826:Bisphenol A Compounds
keywords: G-1; GPER; estrogen; estrogen receptor; phosphoproteomic; xenoestrogens