Quantitative measurement of estrogen-induced ERK 1 and 2 activation via multiple membrane-initiated signaling pathways

Steroids. 2004 Mar;69(3):181-92. doi: 10.1016/j.steroids.2003.12.003.


Estradiol (E2) and other steroids have recently been shown to initiate various intracellular signaling cascades from the plasma membrane, including those stimulating mitogen-activated protein kinases (MAPKs), and particularly extracellular-regulated kinases (ERKs). In this study we demonstrated the ability of E2 to activate ERKs in the GH3/B6/F10 pituitary tumor cell line, originally selected for its enhanced expression of membrane estrogen receptor-alpha (mERalpha). We compared E2 to its cell-impermeable analog (E2 conjugated to peroxidase, E2-P), and to the synthetic estrogen diethylstilbestrol (DES). Time-dependent ERK activation was quantified with a novel fixed cell-based immunoassay developed to efficiently determine activation by multiple compounds over multiple parameters. Both E2 and DES produced bimodal responses, but with distinctly different time courses of enzyme phosphorylation (activation) and inactivation; E2-P induced a monophasic ERK activation. E2 also phosphorylated ERKs in concentration-dependent manner with two concentration optima (10(-14) and 10(-8)M). Inhibitors were employed to determine pathway (ER, EGFR, membrane organization, PI3 kinase, Src kinase, Ca2+) involvement and timing of pathway activations; all affected ERK activation as early as 3-6 min, suggesting simultaneous, not sequential, activation. Therefore, E2 and other estrogenic compounds can produce rapid ERK phosphorylations via nongenomic pathways, using more than one pathway for signal generation.

Publication types

  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Animals
  • Antifungal Agents / pharmacology
  • Cell Line, Tumor
  • Cell Membrane / drug effects*
  • Cell Membrane / metabolism
  • Chelating Agents / pharmacology
  • Diethylstilbestrol / pharmacology
  • Egtazic Acid / analogs & derivatives*
  • Egtazic Acid / pharmacology
  • Enzyme Activation / drug effects
  • Enzyme-Linked Immunosorbent Assay
  • Epidermal Growth Factor / pharmacology
  • Estrogen Antagonists / pharmacology
  • Estrogens / chemistry
  • Estrogens / pharmacology*
  • Flavonoids / pharmacology
  • MAP Kinase Signaling System / drug effects*
  • Mitogen-Activated Protein Kinase 1 / metabolism*
  • Mitogen-Activated Protein Kinase 3 / metabolism*
  • Nystatin / pharmacology
  • Peroxidase / chemistry
  • Peroxidase / pharmacology
  • Phosphorylation / drug effects
  • Pyrimidines / pharmacology
  • Rats


  • AG 1879
  • Antifungal Agents
  • Chelating Agents
  • Estrogen Antagonists
  • Estrogens
  • Flavonoids
  • Pyrimidines
  • 1,2-bis(2-aminophenoxy)ethane N,N,N',N'-tetraacetic acid acetoxymethyl ester
  • Nystatin
  • Egtazic Acid
  • Epidermal Growth Factor
  • Diethylstilbestrol
  • Peroxidase
  • Mitogen-Activated Protein Kinase 1
  • Mitogen-Activated Protein Kinase 3
  • 2-(2-amino-3-methoxyphenyl)-4H-1-benzopyran-4-one