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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
Structure, affinity, and availability of estrogen receptor complexes in the cellular environment
Eric M Kofoed1, Martin Guerbadot, Fred Schaufele
1Diabetes Center, University of California, San Francisco, California 94143-0540, USA.
The Journal of Biological Chemistry
|November 21, 2009
Summary
New Förster resonance energy transfer (FRET) methods quantify protein complex structure and biochemistry in living cells. These techniques reveal how estrogen receptor alpha (ER alpha) interacts with cofactors SRC1, SRC2, and SRC3.
Area of Science:
- Cellular biochemistry
- Molecular biology
- Biophysics
Background:
- Understanding protein complex dynamics in vivo is crucial for cellular function.
- Existing methods often lack the precision to quantify interactions within the cellular environment.
Purpose of the Study:
- To develop and validate Förster resonance energy transfer (FRET) methods for measuring biochemical and structural parameters of protein complexes in living cells.
- To investigate the interactions between the human estrogen receptor alpha-isoform (ER alpha) and receptor interacting domains (RIDs) of cofactors SRC1, SRC2, and SRC3.
Main Methods:
- Widely applicable, calibrated FRET methods were employed.
- Measurements were conducted in the nucleus of living cells.
- Quantified structural and biochemical parameters of ER alpha-cofactor interactions.
Main Results:
- ER alpha formed high-affinity complexes with SRC-RIDs, transitioning from dimeric to monomeric states with increasing cofactor concentration.
- Complex affinity decreased from high (low concentration) to lower (high concentration) levels.
- Only a subpopulation of ER alpha was available for complex formation with SRC-RIDs.
Conclusions:
- The developed FRET methods provide unprecedented detail on the biochemistry and structure of intracellular protein complexes.
- These methods serve as a template for studying diverse molecular interactions within the cellular environment.
- Revealed concentration-dependent structural and affinity changes in ER alpha-cofactor interactions.
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