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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
An intramolecular folding sensor for imaging estrogen receptor-ligand interactions
Ramasamy Paulmurugan1, Sanjiv S Gambhir
1Department of Radiology, Stanford University School of Medicine, James H. Clark Center, 318 Campus Drive, East Wing, First Floor, Stanford, CA 94305-5427, USA. paulmur8@stanford.edu
Summary
Researchers developed novel estrogen receptor (ER) intramolecular folding sensors for high-throughput drug screening. These sensors enable precise analysis of hormone and drug interactions with the ER in various biological contexts.
Area of Science:
- Biochemistry
- Molecular Biology
- Pharmacology
Background:
- Understanding estrogen receptor (ER) interactions with hormones and drugs is vital for ER biology and pharmacology.
- Crystal structure analysis of the human ER ligand-binding domain (hER-LBD) suggested ligand-specific folding patterns.
Purpose of the Study:
- To develop and validate intramolecular folding sensors for high-throughput analysis of hER-ligand interactions.
- To identify a mutant hER-LBD for in vivo imaging applications.
Main Methods:
- Construction and validation of intramolecular folding sensors utilizing split Renilla/firefly luciferase reporter complementation.
- Engineering of a low-affinity estradiol mutant hER-LBD.
- Stable cell line generation for sensor expression and imaging in living mice.
Main Results:
- Developed functional intramolecular folding sensors capable of distinguishing ER agonists, modulators, and antiestrogens.
- Identified a mutant hER-LBD suitable for in vivo imaging.
- Demonstrated successful imaging of ligand-induced intramolecular folding in living mice.
Conclusions:
- The developed hER-LBD intramolecular folding sensors enable high-throughput quantitative analysis of ER interactions.
- This technology is applicable to cell lysates, intact cells, and molecular imaging in living subjects.
- The strategy can be extended to study other protein intramolecular folding systems.
