O'Hara, Patricia B. Estrogen Receptor Binding

DOI: http://dx.doi.org/10.1016/j.bpj.2011.11.434

Estrogen receptors (ERα  and ER β ) are ligand-binding transcription factors activated by the hormone 17-β estradiol. Ligand binding triggers ER dimerization, translocation of the receptor from the cytoplasm into the nucleus and eventually activation of the genes under control of ER. Studies have revealed a role for estrogen receptors in male and female sexual development, reproductive functions, bone metabolism and regulation of neuroendocrine and cardiovascular systems. ER is also known to bind to other non-native ligands known in pharmacology as receptor agonists or antagonists. Agonists provoke a biological response when bound to the receptor; antagonists inhibit a biological response when bound. Our lab is interested in the promiscuous binding of the estrogen receptor and its ability to activate different genes in different tissues. Fluorescence Resonance Energy Transfer (FRET) assays have previously been performed using ER to study ligand binding affinities for the receptor. However, this technique is unable to determine whether these ligands are agonists or antagonists and allow ER dimerization and gene activation. To investigate these phenomena, an activity assay that measures ER controlled gene expression has been developed which provides the opportunity to gain further insight into the functional activity in living systems. Recombinant yeast cells that express ERα use the green fluorescent protein (GFP) reporter to determine whether ER α , in the presence of a particular ligand, has activated gene of expression. We have correlated the binding to agonist and antagonist behavior of several xeno and phyto estrgens.

DOI: http://dx.doi.org/10.1016/j.bpj.2009.12.203

Estrogen’s role in cell growth and proliferation has long been appreciated both in the normal development of secondary sexual characteristics and in diseased states in cancers of the breast, ovaries and uterus. We are beginning to appreciate estrogen’s expanded role in maintaining such diverse functions as the health of the central nervous system, bone density and cardiac health. Estrogen plays out its roles in varied tissues by binding to two major ligand activated nuclear receptors, estrogen receptor α (ER α) and estrogen receptor β (ER β). Though the ligand binding cavities of the two receptors differ by only two amino acids, the overall degree of homology between ER α and ER β is low. The body uses the receptor selectivity to its advantage by dispersing the receptors in varying ratios to different tissues.

Small molecules have been identified which bind to one or the other receptors with differing binding affinities. These selective estrogen receptor modulators (SERMs) hold the potential to be pharmacologically effective in treating diseases specific to one type of ER while not affecting the other. For example, ER α and ER β are both present in breast tissue, and the ratio of ER β to ER α is being examined as one indicator in determining the likelihood of successful treatment of breast cancer by certain drugs. Here we use changes in the fluorescence polarization of a bound fluorescent estrogen homologue to calculate binding affinities for PPT and DPN, two SERMs, to ER α and ER β.  PPT binds selectively to ER α with a K_i of 47.9 ± 1.8 1 nM and DPN binds selectively to ER β with a K_i of 120 ± 2.4 nM.

Various synthetic compounds ubiquitous in the environment have been implicated as xenoestrogens, or chemicals that can bind to the ERs thus mimicking estrogen.  They have been linked to many health problems including various forms of cancer, sexual and developmental defects, reproductive abnormalities, and behavioral irregularities.  Because both ERs have large binding pockets, a diverse range of chemicals can act as xenoestrogens.  Though these chemicals are not as potent in binding as natural estrogen, the total xenoestrogen burden due to their ubiquity is cause for concern.   This study examined the binding of the plasticizer bisphenol A and three insecticides based on natural pyrethrum from plants: permethrin, fenvalerate and deltamethrin, with both ER α and ER β.  Bisphenol A is a chemical found in plastics and epoxy coating on cans, while the pyrethroids are used in agriculture, ranching and in household insecticides.  None of the pyrethroids were found to bind to ER α or ER β, while bisphenol A exhibited binding to ER α with a K_i of 14.1 ± 3.8 µM and to ER β with a K_i of 850 ± 23 nM. These studies have shown that the fluorescence polarization method can be a powerful tool in understanding the molecular nature of endocrine disruption. 

Using fluorescence polarization, the displacement of a fluorescent estrogen receptor ligand by estradiol (E2), the predominant human estrogen, from ER in the presence or absence of coactivator protein can be tracked over a range of polarizations. This is possible in solution without removing or filtering the ligand-receptor-cofactor complex because the fluorescent tracer molecule emits a unique signal depending on its apparent molecular weight – that is, whether or not the tracer is bound to the receptor. Plasmids encoding the nuclear receptor domain (NRD) of coactivators SRC-1 and SRC-3 were obtained and their encoded proteins were expressed and purified.

No effect of the addition of SRC-1 to the competitive binding assay with full-length ER-α was found, and the results from the same assay with full-length ER-β were inconclusive. Due to resource limitations, new fluorescent tracer molecules and plasmids encoding the ER ligand binding domains (LBDs) were obtained. Expression and purification yields of ER-LBDs were lower than those of SRC-NRDs and the new fluorophore and receptor preparations required reoptimization of the assay for the new reagents. Reoptimization was unsuccessful. Considerable work remains before this assay can reliably be used to investigate the interactions between estrogenic compounds, the estrogen receptors and their cofactors.

DOI: 10.1016/j.bpj.2008.12.2282

The ability of some manmade chemicals to interfere with the endocrine system has recently been the topic of much research.  Many of the chemicals under study have been applied to the environment as agricultural pesticides.  This study looks at the interactions between the estrogen receptor-β, which is a nuclear signaling protein, and four pyrethroid pesticides with the purpose of observing whether or not these chemicals can bind to the receptor.  The structures of the four pyrethroids, permethrin, bifenthrin, deltamethrin and fenvalerate, are shown below.  A fluorescence polarization competitive binding assay was used to observe the effects of the pyrethroids on a complex of the estrogen receptor and a fluorescent estradiol-like ligand called fluormone.  Because this complex is large, it rotates slowly in solution and has a high polarization.  If a pyrethroid binds to the receptor, fluormone is displaced, and its fast rotation free in solution leads to a low polarization.  In this way, the ability of these four pyrethroids to bind the estrogen receptor-β was assessed.

DOI: http://dx.doi.org/10.1016/j.bpj.2008.12.2282

The insecticide dichloro-diphenyl-trichloroethane (DDT) was used heavily in the United States until its ban in 1972.  DDT, an organochloride compound, tends to accumulate in living systems and was found in the lipid tissue of over 99% of women even thirty years after its ban.  Methoxychlor, also an organochloride insecticide, largely replaced DDT and continues to be used to this day.   

Given the present exposure to the two insecticides, the estrogenicity of DDT, methoxychlor, and the insecticides’ metabolites should be investigated.  Studies show that the insecticides as well as their chlorinated metabolites are minimally estrogenic.  However, there is a dearth of research investigating the estrogenicity of the hydroxylated metabolites.  Several hydroxylated metabolites of DDT were isolated by Vernon Feil of North Dakota State University and generously shared for use in the present research.

The work presented here investigates the estrogenicity of DDT, methoxychlor, and their chlorinated and hydroxylated metabolites.  First, a competitive binding assay with a fluorescent estrogen analogue and the estrogen receptor tested the estrogenicity of the compounds in vitro.  The compounds were then administered to ovariectomized female rats to determine their ability to mimic estrogen in vivo by initiating estrus behavior.The findings suggest that methoxychlor and several of the metabolites, both hydroxylated and chlorinated, bind the estrogen receptor in vitro.  In the in vivo test, the same compounds only minimally mimicked estrogen indicating that they may be weakly estrogenic, partial agonists of the estrogen receptor, or perhaps antiestrogens.  The results presented here warrant further investigation if the estrogenicity of insecticides and their metabolites is to be fully understood.

cited in http://www.t3db.ca/toxins/T3D0050, http://www.t3db.ca/toxins/T3D0012, http://www.t3db.ca/toxins/T3D0151