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Detecting the Ligand-binding Domain Dimerization Activity of Estrogen Receptor Alpha Using the Mammalian Two-Hybrid Assay
Published on: December 19, 2018
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Transactivation Function-2 of Estrogen Receptor α Contains Transactivation Function-1-regulating Element.
Yukitomo Arao1, Laurel A Coons2, William J Zuercher3
1From the Receptor Biology Section, Reproductive and Developmental Biology Laboratory, NIEHS, National Institutes of Health, Research Triangle Park, North Carolina 27709.
The Journal of Biological Chemistry
|June 2, 2015
Summary
Ligand-dependent estrogen receptor alpha (ERα) functions involve complex interactions between AF-1 and AF-2 domains. This study reveals AF-2 normally suppresses AF-1, a mechanism disrupted by mutations, leading to unexpected antagonist activity.
Area of Science:
- Molecular Endocrinology
- Receptor Biology
- Pharmacology
Background:
- Estrogen receptor alpha (ERα) possesses ligand-dependent (AF-2) and ligand-independent (AF-1) transactivation functions.
- The cooperative regulation of AF-1 and AF-2 by ligands remains incompletely understood.
Purpose of the Study:
- To investigate the role of AF-1 in the estrogenic activities of various compounds.
- To elucidate the regulatory relationship between ERα's AF-1 and AF-2 domains.
Main Methods:
- Utilized AF-1-truncated and AF-2-mutated ERα mutants, including flexible and static region mutants of AF-2.
- Employed a novel hybrid reporter assay to analyze the transcription activity of physically separated AF-1 and AF-2 domains.
Main Results:
- AF-2 mutants, unresponsive to agonists, were surprisingly activated by antagonists and selective estrogen receptor modulators (SERMs), with this activity originating from AF-1.
- Demonstrated that AF-2 normally suppresses AF-1 activity.
- Physically separated AF-1 and AF-2 showed that AF-1 was not suppressed by AF-2, and SERMs did not activate the separated AF-2 mutant, unlike the intact protein.
Conclusions:
- Mutation of AF-2 disrupts its suppressive function on AF-1, leading to antagonist reversal.
- SERM activity relies on ligand-induced conformational changes in full-length ERα, enabling AF-1 activation.
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