Journal
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA
Volume 103, Issue 43, Pages 15883-15888Publisher
NATL ACAD SCIENCES
DOI: 10.1073/pnas.0607385103
Keywords
complementation; optical imaging; split reporters
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Funding
- NCI NIH HHS [R24 CA92865, R24 CA092865, P50 CA114747, R01 CA082214, R01 CA82214] Funding Source: Medline
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Strategies for high-throughput analysis of interactions between various hormones and drugs with the estrogen receptor (ER) are crucial for accelerating the understanding of ER biology and pharmacology. Through careful analyses of the crystal structures of the human ER (hER) ligand-binding domain (hER-LBD) in complex with different ligands, we hypothesized that the hER-LBD intramolecular folding pattern could be used to distinguish ER agonists from selective ER modulators and pure antiestrogens. We therefore constructed and validated intramolecular folding sensors encoding various hER-LBD fusion proteins that could lead to split Renilla/firefly luciferase reporter complementation in the presence of the appropriate ligands. A mutant hER-LBD with low affinity for circulating estradiol was also identified for imaging in living subjects. Cells stably expressing the intramolecular folding sensors expressing wild-type and mutant hER-LBD were used for imaging ligand-induced intramolecular folding in living mice. This is the first hER-LBD intramolecular folding sensor suited for high-throughput quantitative analysis of interactions between hER with hormones and drugs using cell lysates, intact cells, and molecular imaging of small living subjects. The strategies developed can also be extended to study and image other important protein intramolecular folding systems.
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