An intramolecular folding sensor for imaging estrogen receptor–ligand interactions

Abstract

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 Renilla͞firefly 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.complementation ͉ optical imaging ͉ split reporters E strogens are responsible for the growth, development, and maintenance of the reproductive, skeletal, neuronal, and immune systems as well as and other systems. The physiological effects of these hormones are mediated by the estrogen receptor (ER), which is a ligand-inducible nuclear transcription factor (1). In the classical pathway of steroid hormone action, 17␤-estradiol (E2), hormones, and a variety of other estrogens bind to the ligandbinding domain (LBD) of ER and lead to its dimerization and subsequent binding to a specific regulatory sequence in the promoters of ER target genes known as the estrogen response elements (2, 3), which then trigger activation or repression of many downstream target genes (4). The deficiency or excess of estrogens can lead to various pathological conditions including osteoporosis and breast carcinomas (5), making ER a major cellular therapeutic target.Elegant crystallographic studies with ER-LBD have shown that conformation of helix 12 (H12) is critical in responses observed with various ER ligands (4, 6, 7). The conformation of H12 behaves as a ''molecular switch'' that either prevents or enhances ER from binding to an array of coactivator proteins, which then activates transcription of many downstream estrogen-regulated genes responsible for cell growth. Given the critical role of H12 in ER signaling, we reasoned that it may be feasible to develop an intramolecular ER folding sensor with specific split reporter complementation patterns to study ligand pharmacology based directly on the conformational changes of H12 in response to different ligands (Fig. 1).We used a spli...

An Improved Bioluminescence Resonance Energy Transfer Strategy for Imaging Intracellular Events in Single Cells and Living Subjects

“…Several adaptations of bioluminescence imaging have already been devised by our lab and others to detect protein functions and protein interactions in small living animals, such as the inducible luciferase yeast two-hybrid system (15,16), luciferase complementation (17)(18)(19)(20)(21), and, more recently, bioluminescence resonance energy transfer (BRET; refs. 22,23).…”

A multi‐parameter imaging assay identifies different stages of ligand‐induced androgen receptor activation

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In Vitro and In Vivo Effects of Tamoxifen against Larval Stage Echinococcus granulosus

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