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Updated: Aug 16, 2026

Detecting the Ligand-binding Domain Dimerization Activity of Estrogen Receptor Alpha Using the Mammalian Two-Hybrid Assay
09:07

Detecting the Ligand-binding Domain Dimerization Activity of Estrogen Receptor Alpha Using the Mammalian Two-Hybrid Assay

Published on: December 19, 2018

Rapid estrogen-induced phosphorylation of the SRC-3 coactivator occurs in an extranuclear complex containing estrogen

Fuzhong F Zheng1, Ray-Chang Wu, Carolyn L Smith

  • 1Molecular and Cellular Biology, One Baylor Plaza, Houston, TX 77030, USA.

Molecular and Cellular Biology
|September 2, 2005
PubMed

Insights

Estradiol (E2) rapidly triggers SRC-3 phosphorylation via direct interaction with estrogen receptors (ERalpha/ERbeta), even in the cytoplasm. This early, nongenomic action precedes SRC-3

Area of Science:

  • Molecular Biology
  • Endocrinology
  • Cell Signaling

Background:

  • SRC-3 is a key transcriptional coregulator for estrogen receptors (ER).
  • SRC-3 phosphorylation is induced by various signals, involving multiple kinase pathways.
  • Six SRC-3 phosphorylation sites have been identified.

Purpose of the Study:

  • To investigate the mechanisms of estradiol (E2)-induced SRC-3 phosphorylation.
  • To determine the role of estrogen receptor (ER) subtypes and domains in SRC-3 phosphorylation.
  • To explore the cellular localization and timing of E2-induced SRC-3 phosphorylation.

Main Methods:

  • Utilized phosphospecific antibodies to detect SRC-3 phosphorylation at six sites.
  • Employed site-directed mutagenesis of ERalpha and SRC-3 domains.
  • Assessed SRC-3 phosphorylation in response to E2 with various ERalpha mutants.

Main Results:

  • E2-induced SRC-3 phosphorylation requires activated ERalpha or ERbeta.
  • Maximal phosphorylation induction needs ERalpha's activation function 1 and ligand-binding domains.
  • Direct interaction between ERalpha and SRC-3 is essential for phosphorylation, which can occur in the cytoplasm.

Conclusions:

  • E2-induced SRC-3 phosphorylation is dependent on direct ERalpha-SRC-3 interaction.
  • This phosphorylation event represents an early, nongenomic ER action.
  • The findings support the established genomic roles of estrogen and coactivators.

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