Function of estrogen receptor tyrosine 537 in hormone binding, DNA binding, and transactivation.

The human estrogen receptor (hER) is a ligand-activated transcription factor which, like many other members of the nuclear receptor protein family, exhibits a dimerization-dependent transcriptional activation. Several previous reports have provided evidence of the phosphorylation of the hER at tyrosine 537 (Y537). However, the exact function of a putative phosphorylation at this site remains controversial.

The role of phosphorylation in human estrogen receptor function.

We have studied the role of phosphorylation of the human estrogen receptor (hER) at serine 118, which has been previously identified as a site important for transactivation. We have tested this transactivation in yeast and cell-free transcription assays, and have shown that mutation of serine 118 to alanine results in a 30-40% decrease in hER-dependent transcription. Furthermore, we investigated the functional significance of phosphorylation at this site by hormone binding and DNA binding.

Molecular and kinetic basis for the mixed agonist/antagonist activity of estriol.

Estriol acts as a weak estrogen when administered in a single dose into immature or ovariectomized laboratory animals, but produces full estrogenic responses upon chronic administration. However, when estriol is injected together with estradiol it acts as an antiestrogen. We studied the dual agonist/antagonist properties of estriol, using recombinant human estrogen receptor (hER) in ligand-binding assay, cell-free transcription assay, electrophoretic mobility shift assay with cVitII estrogen response element (ERE), and ERE-Sepharose chromatography.

Phosphorylation of serine-167 on the human oestrogen receptor is important for oestrogen response element binding and transcriptional activation.

We have studied the role of phosphorylation of the human oestrogen receptor (hOR; otherwise known as hER) at serine-167, which has been identified previously as the major oestrogen-induced phosphorylation site. We have tested transactivation by the hOR in yeast and cell-free transcription assays, and shown that mutation of serine-167 results in a 70% decrease in hOR-dependent transcription. Furthermore we explored the functional significance of phosphorylation at this site by hormone binding and DNA binding.

Transcriptional activation of the human estrogen receptor by DDT isomers and metabolites in yeast and MCF-7 cells.

In this study, we determined whether the DDT isomers p,p'-DDT [1,1,1,-trichloro-2,2-bis(p-chlorophenyl)ethane], o,p'-DDT [1,1,1-trichloro-2(p-chlorophenyl)-2-(o-chlorophenyl)ethane], and their metabolites p,p'-DDD [1,1-dichloro-2,2-bis(p-chlorophenyl)ethane], o,p'-DDD [1,1-dichloro-2-(p-chlorophenyl)-2-(o-chlorophenyl)ethane], p,p'-DDE [1,1,-dichloro-2,2-bis(p-chlorophenyl)ethylene], o,p'-DDE [1,1-dichloro-2-(p-chlorophenyl)-2-(o-chlorophenyl)ethylene], and p,p'-DDA [2,2-bis(p-chlorophenyl)acetic acid], could bind to and transcriptionally activate the human estrogen receptor (hER). Novel results from competitive binding assays showed that o,p'-DDD, o,p'-DDE, and p,p'-DDT, as well as the established environmental estrogen o,p'-DDT, were able to bind specifically to the hER with approximately 1000-fold weaker affinities for the hER than that of estradiol. In contrast, only o,p'-DDT, but not p,p'-DDT, bound to the rat estrogen receptor.

Estradiol-binding mechanism and binding capacity of the human estrogen receptor is regulated by tyrosine phosphorylation.

We have investigated the effects of tyrosine phosphorylation on the estradiol-binding mechanism and binding capacity of the human estrogen receptor (hER). The wild type hER and a point mutant form of the hER, in which tyrosine 537 was mutated to phenylalanine (Y537F hER), were expressed in Sf9 insect cells. The wild type hER, but not the Y537F hER, reacted with a anti-phosphotyrosine monoclonal antibody, indicating that tyrosine 537 was the only tyrosine phosphorylated on the hER.

A yeast estrogen screen for examining the relative exposure of cells to natural and xenoestrogens.

Xenoestrogens, such as o,p'-DDT and octyl phenol (OP), have been associated with reproductive abnormalities in various wildlife species. Xenoestrogens mimic the natural estrogen 17 beta-estradiol and compete for binding to the estrogen receptor. Even though the affinity of o,p'-DDT and OP for the estrogen receptor is approximately 1000-fold lower than 17 beta-estradiol, the actions of xenoestrogens could be enhanced if their bioavailability in serum were greater than 17 beta-estradiol.

Kinetic analysis of the interaction of human estrogen receptor with an estrogen response element.

The kinetics of the interaction between recombinant human estrogen receptor and chicken vitellogenin gene II estrogen response element (ERE) were determined by ERE-Sepharose chromatography. The association constant of the interaction between the ERE and the human estrogen receptor was dependent on receptor concentration, estradiol binding and temperature. The highest association constant (80-100 x 10(6)M-1) was measured for the estradiol-bound receptor prepared at 25 degrees C and at concentrations higher than 7 nM.

Phosphorylation of tyrosine 537 on the human estrogen receptor is required for binding to an estrogen response element.

We report here that the phosphorylation of tyrosine 537 on the human estrogen receptor (hER) controls the receptor's dimerization and DNA binding ability. The DNA-binding form of both the hER from human MCF-7 mammary carcinoma cells and the hER overexpressed in Sf9 insect cells was isolated using estrogen response element (ERE) affinity chromatography. Western blot analyses demonstrated that the DNA-binding form of the hER from MCF-7 or Sf9 cells was (i) phosphorylated at tyrosine 537, (ii) localized in the nucleus of estradiol-treated MCF-7 cells with an apparent molecular mass of 67 kDa, and (iii) hyperphosphorylated at serine residue(s).

Phosphorylation of the human estrogen receptor by mitogen-activated protein kinase and casein kinase II: consequence on DNA binding.

We determined the amino acid and radiolabel sequences of tryptic [32P]phosphopeptides of the purified human estrogen receptor (hER) from MCF-7 cells and Sf9 cells. Serine 118 was identified as a site that was phosphorylated independently of estradiol-binding in MCF-7 cells. Proline is on the carboxy terminus of serine 118, which suggests that the serine-proline may be a consensus phosphorylation site motif for either the mitogen-activated protein (MAP) kinase or p34cdc2 kinase.

An antiestrogen: a phosphotyrosyl peptide that blocks dimerization of the human estrogen receptor.

We have previously identified tyrosine-537 as a constitutively phosphorylated site on the human estrogen receptor (hER). A 12-amino acid phosphotyrosyl peptide containing a selected sequence surrounding tyrosine-537 was used to investigate the function of phosphotyrosine-537. The phosphotyrosyl peptide completely blocked the binding of the hER to an estrogen response element (ERE) in a gel mobility shift assay.

In vivo and in vitro phosphorylation of the human estrogen receptor.

We report here that the human estrogen receptor (hER) overexpressed in Sf9 insect cells is phosphorylated similarly to hER from the human MCF-7 mammary carcinoma cell line. The recombinant and native hER labeled to steady-state with [32P]phosphate were purified to homogeneity using specific DNA-affinity chromatography followed by SDS-gel electrophoresis. Resolution of the hER tryptic digests by reverse phase-high performance liquid chromatography revealed that five [32P]phosphopeptides from the hER expressed in the Sf9 cells had retention times identical to five of the seven [32P]phosphopeptides from the hER in MCF-7 cells.

Phosphorylation of the human estrogen receptor on tyrosine 537 in vivo and by src family tyrosine kinases in vitro.

Its reactivity to the antiphosphotyrosine 4G10 monoclonal antibody by Western blot analysis demonstrated that the human estrogen receptor (hER) from human MCF-7 cells and the recombinant hER expressed in Sf9 insect cells were phosphorylated on tyrosine(s). Reverse phase-HPLC separation of a tryptic digest of the 32P-labeled purified hER from Sf9 and MCF-7 cells followed by amino acid and radiolabel sequencing revealed that tyrosine-537 was phosphorylated. The phosphorylation on tyrosine-537 was independent of estradiol treatment of MCF-7 cells, indicating that tyrosine-537 is a basal phosphorylation site.

Serine 167 is the major estradiol-induced phosphorylation site on the human estrogen receptor.

Serine 167 has been identified by radiolabel and amino acid sequencing as the major estrogen-induced phosphorylation site on the human estrogen receptor (hER) from human MCF-7 mammary carcinoma cells. The phosphorylation of the hER on serine 167 was estrogen-dependent, increasing 4-fold upon estradiol treatment of MCF-7 cells and accounted for almost half of the total [32P]phosphate incorporated into the recombinant hER from Sf9 insect cells and the native hER from MCF-7 cells. Casein kinase II was found to phosphorylate the purified recombinant hER on serine 167 in vitro.

Demonstration of estrogen and vitamin D receptors in bone marrow-derived stromal cells: up-regulation of the estrogen receptor by 1,25-dihydroxyvitamin-D3.

We have shown earlier that 17 beta-estradiol inhibits cytokine-induced interleukin-6 (IL-6) production by bone marrow-derived stromal cells as well as osteoblasts, two types of cells with a critical influence on osteoclast development, and that ovariectomy causes an IL-6-mediated up-regulation of osteoclastogenesis in mice. Prompted by this, we have searched here for the presence of estrogen receptors (ERs) in two murine bone marrow-derived stromal cell lines, +/+ LDA11 and MBA 13.2, and the osteoblast-like cell line MC3T3-E1.

Estrogen-responsive elements contain non-B DNA.

The estrogen receptor, a hormone-regulated transcription factor, regulates gene expression by interacting with a specific nucleotide sequence called the estrogen-responsive element (ERE). In this report we demonstrate by potassium permanganate, osmium tetroxide and diethylpyrocarbonate reactivity and S1 nuclease sensitivity that the nucleotides either within or in the immediate region of imperfect and perfect EREs are in a non-B DNA conformation. The presence of nucleotides in a non-B DNA conformation in the ERE is an intrinsic property of the DNA and is independent of whether the ERE is in linear or supercoiled DNA.

Hormone- and DNA-binding mechanisms of the recombinant human estrogen receptor.

We have investigated the hormone- and DNA-binding mechanisms of the wild-type human estrogen receptor (hER) overproduced in insect cells using a baculovirus expression system. The recombinant hER was indistinguishable in size (67 kDa) and immunogenically from the native human estrogen receptor in MCF-7 breast carcinoma cells. The recombinant hER was purified to 70-80% homogeneity with a two-step procedure that included ammonium sulfate precipitation and oligonucleotide affinity chromatography using a unique Teflon affinity matrix.

Analysis of estrogen receptor interaction with tertiary-structured estrogen responsive elements.

An initial crucial step in estrogen activation of gene expression is the interaction of the estrogen receptor with a specific nucleotide sequence [estrogen responsive element (ERE)]. Previously, we found that the estrogen receptor binds preferentially and with high affinity to the lower strand of the rat prolactin imperfect ERE which contains tertiary structure (Lannigan DA and Notides AC, Proc Natl Acad Sci USA 86: 863-867, 1989). Using perfect and imperfect EREs from the upstream region of the chicken vitellogenin II gene, we have now extended our findings and have determined that the estrogen receptor preferentially interacts with either perfect or imperfect EREs which contain tertiary structure.

The binding of estrogen and estrogen antagonists to the estrogen receptor.

The model of the estrogen receptor as a dimer of identical, interacting subunits and data obtained by Sasson and Notides (1988, Mol. Endocrinol. 2, 307-312) were used to find the standard free energy changes that describe the binding of estradiol and 4-hydroxytamoxifen to the estrogen receptor.

Estrogen receptor phosphorylation. Hormonal dependence and consequence on specific DNA binding.

We have shown that the 32P-phosphorylation of the nuclear estrogen receptor from human MCF-7 cells or the calf uterus is estrogen-dependent. Within 2 min of estradiol treatment the phosphorylation of the estrogen receptor from MCF-7 cells doubled, and increased 4-fold within 20-40 min of estradiol treatment. Progesterone was ineffective in stimulating the phosphorylation of the estrogen receptor.

Evidence for direct estrogen regulation of the human gonadotropin-releasing hormone gene.

This study is an attempt to determine whether estrogen could directly regulate human gonadotropin-releasing hormone (GnRH) gene expression. Human GnRH expression vectors were constructed by fusing various 5' flanking regions of the human GnRH gene upstream of the luciferase reporter gene (LUC) or the thymidine kinase promoter linked to the chloramphenicol acetyltransferase reporter gene (CAT). These constructs were transiently transfected into a human choriocarcinoma cell line (JEG-3) and LUC or CAT activity was measured after either no treatment or treatment with various concentrations of estradiol.

Phosphate-sensitive binding of the estrogen receptor to its response elements.

Although the nucleotide bases that constitute the consensus DNA sequence of the estrogen response element (ERE) have been identified, the involvement of electrostatic contacts between the sugar-phosphate backbone of the ERE and the estrogen receptor (ER) is not known. Moreover, the contribution of these contacts to sequence-specific DNA binding has not been determined. Therefore, the interactions of highly purified ER with the phosphate residues of the ERE derived from the chick vitellogenin (cVit)-II gene were examined by phosphate ethylation interference.

Estradiol inhibits transcription of the human glycoprotein hormone alpha-subunit gene despite the absence of a high affinity binding site for estrogen receptor.

Chronic administration of estradiol inhibits transcription of the gene encoding the alpha-subunit of pituitary glycoprotein hormones. Here, we show, using transfection analyses and a filter binding assay, that 1500 basepairs of proximal 5' flanking sequence of the human alpha-subunit gene lack a functional estrogen response element when transfected into heterologous cell lines, and fail to bind estrogen receptor purified from calf uterus. Yet, this same region of the alpha-subunit gene confers estradiol responsiveness (transcriptional suppression) to the bacterial chloramphenicol acetyltransferase gene in transgenic mice.