PubMed · 11503442
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M Tkaczyk, K Kalita. 2001. [Estrogen receptor beta--structure, regulation and function].. https://pubmed.ncbi.nlm.nih.gov/11503442/
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This article describes the development and validation of two fluorescent receptor assays for the hRec-estrogen receptor subtypes alpha and beta. As a labelled ligand an autofluorescent phyto-estrogen (coumestrol) has been used. The estrogen receptor (ER) belongs to the nuclear receptor family, a class of soluble DNA binding proteins, mainly present in the cytoplasm of the cell, that act as ligand-activated enhancer factors. It consists of two different forms, expressed as ER-alpha (66 kDa) and ER-beta (59 kDa). The ER-alpha is mainly located in the uterus and the ER-beta can be found in vascular tissue. Detection and identification of compounds having estrogenic effects is of importance in drug discovery programmes within the pharmaceutical industry for their search for ER-subtype selective (ant)agonists which may prove to be of therapeutic value in treating a variety of estrogen-linked pathologies (breast cancer, osteoporosis, cardiovascular disease, type II diabetes and Alzheimer disease). Furthermore, interactions of (xeno-)estrogens with the endogenous hormonal system of the exposed organism can affect embryos, gonads, and reproductive behaviour. The latter can eventually lead to reduced reproduction and deterioration of a population. For that reason, monitoring of (xeno-)estrogens in food products and in the environment, attracts considerable attention by health councils throughout the world. The following characteristics were obtained for the human recombinant (hRec) estrogen receptor-beta assay, which is suitable for ER subtype selective drug-discovery purposes (IC50 values for 17-beta-estradiol and genistein were 5.1 nM and 25 nM, respectively): goodness of fit (R2) was always > 0.98 (x = 0.9933, n = 10). LLOQ of the assay is typically > or = 500 picomolar, whereas the ULOQ of the assay is < or = 20.0 nanomolar. For the hRec-estrogen receptor-alpha assay, which is suitable for monitoring of (xeno-)estrogens (IC50 values for 17-beta-estradiol and genistein were 0.68 nM and 65 nM, respectively) the following characteristics were obtained: goodness of fit (R2) was always > 0.96 (x = 0.9838, n = 10). LLOQ of the assay is typically > or = 200 picomolar, whereas the ULOQ of the assay is < or = 5.0 nanomolar.
Estrogen receptors (ERs) are transcription factors that can be modulated by both estrogen-dependent and growth factor-dependent phosphorylation. A yeast two-hybrid screening identified a serine/threonine protein phosphatase (PP5) as an interactant of ERbeta (1-481), a dominant negative ERbeta mutant. Glutathione S-transferase pull-down assays, mammalian two-hybrid assays, and immunoprecipitation studies showed that PP5 directly binds to both ERalpha and ERbeta via its tetratricopeptide repeat domain. E domains of ERalpha and ERbeta, without containing activation domain core regions in transcription activation function 2, were required for the binding to PP5. In ERalpha-positive breast cancer MCF7 cells, estrogen- and epidermal growth factor-dependent phosphorylation of ERalpha on serine residue 118, a major phosphorylation site of the receptor, was reduced by expressing PP5 but enhanced by PP5 antisense oligonucleotide. Estrogen-induced transcriptional activities of both ERalpha and ERbeta and mRNA expression of estrogen-responsive genes, including pS2, c-myc, and cyclin D1, were suppressed by PP5 but enhanced by PP5 antisense oligonucleotide. A truncated PP5 mutant consisting only of its tetratricopeptide repeat domain acted as a dominant negative PP5 that enhanced serine residue 118 phosphorylation of ERalpha and transactivations by ERalpha and ERbeta. We present the first evidence that PP5 functions as an inhibitory regulator of ER phosphorylation and transcriptional activation in vivo.