detecting-ligand-binding-domain-dimerization-activity-estrogen

Detecting the Ligand-binding Domain Dimerization Activity of Estrogen Receptor Alpha Using the Mammalian Two-Hybrid Assay

Estrogen receptor alpha (ER&#945;) is an estrogenic ligand-dependent transcription regulator. The sequence of ER&#945; protein is highly conserved among ...

National Institutes of Health (NIH)

Estrogen receptor α (ERα) is a major transducer of estrogen-mediated physiological signals. ERα is a member of the nuclear receptor superfamily, which encompasses ligand-dependent transcription factors. The C terminus of nuclear receptors, termed the F domain, is the least homologous region among the members of this family. The ERα F domain possesses 45 amino acids; however, its function remains unclear. We noticed that the homology of the F domains between mouse and human ERαs is remarkably lower (75.6% similarity) than that between the entire proteins (94.7% similarity). To assess the functionality of the ERα F domains, here we generated chimeric ERα expression constructs with mouse-human-exchanged F domains. Using cell-based  assays, we analyzed the transcriptional coactivator interaction and ligand-binding domain dimerization activities of these mouse-human F domain-swapped ERαs. We found that the transcriptional activity of the mouse WT ERα is more potent than that of the human WT ERα in the human hepatoma cell line HepG2. 4-Hydroxytamoxifen (4OHT)-mediated transcriptional activity of mouse-human F domain-swapped ERαs was the inverse of the WT ERα activities but not estradiol-mediated transcriptional activities. Further experiments with constructs containing deletion or point mutations of a predicted β-strand region within the F domain suggested that this region governs the species-specific 4OHT-mediated transcriptional activity of ERα. We conclude that the ERα F domain has a species-specific function in 4OHT-mediated receptor transactivation and that mouse-human F domain-swapped ERα mutants enable key insights into ERα F domain structure and function.

The F domain of estrogen receptor α is involved in species-specific, tamoxifen-mediated transactivation.

Studies using the estrogen receptor alpha (ERα) knock-out (αERKO) mice have demonstrated that ERα plays a crucial role in various estrogen-mediated metabolic regulations. ERα is a ligand dependent transcription regulator and its activity is regulated by estrogenic compounds. ERα consists of two transcriptional activation domains, AF-1 and AF-2. The activities of these domains are regulated through different mechanisms; however, the specific physiological role in metabolic regulation by these domains is still unclear.

Yukitomo Arao

Reproductive Developmental Biology Laboratory,
National Institute of Environmental Health Sciences,
Reproductive Developmental Biology Laboratory, National Institute of Environmental Health Sciences

National Institutes of Health (NIH)

Detecting the Ligand-binding Domain Dimerization Activity of Estrogen Receptor Alpha Using the Mammalian Two-Hybrid Assay

Yukitomo Arao

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National Institutes of Health (NIH)

Detecting the Ligand-binding Domain Dimerization Activity of Estrogen Receptor Alpha Using the Mammalian Two-Hybrid Assay

Reproductive Developmental Biology Laboratory,
National Institute of Environmental Health Sciences,
Reproductive Developmental Biology Laboratory, National Institute of Environmental Health Sciences