Donald E. Frail

Expression of Estrogen Receptor-β Protein in Rodent Ovary

Estrogen is an essential hormone for the LH surge and ovulation. The primary source of estrogen is from ovarian granulosa cells and in rats, estrogen, in turn, increases granulosa cell number and enhances FSH-stimulated gene expression in these cells. Thus, rat granulosa cells both respond to and synthesize estrogen. To further elucidate the mechanisms mediating the actions of estrogen in granulosa cells, we have identified and characterized the estrogen receptor-β (ER-β) subtype in rodent granulosa cells. ER-β protein was localized to the nuclei of rat granulosa cells in preantral and antral follicles by immunocytochemistry, coincident with the location of ER-β messenger RNA (mRNA). Immunoprecipitation and Western blot analysis using ER-β specific antisera demonstrated a protein of approximately 60 kDa in granulosa cells prepared from PMSG-primed immature mice and estrogen-treated immature rats. Extracts from granulosa cells specifically bound an estrogen response element and the complex was recognized b...

The ligand binding profiles of estrogen receptors α and β are species dependent

Abstract Estrogens and selective estrogen receptor modulators are used for the treatment and prevention of conditions resulting from menopause. Since estrogens exert their activity by binding to nuclear receptors, there is intense interest in developing new ligands for the two known estrogen receptor subtypes, ER-α and ER-β. Characterization assays used to profile new estrogen receptor ligands often utilize receptors from different species, with the assumption that they behave identically. To test this belief, we have profiled a number of estrogens, other steroids, phytoestrogens and selective estrogen receptor modulators in a solid phase radioligand binding assay using recombinant protein for human, rat, and mouse ER-α and ER-β. Certain compounds show species dependent binding preferences for ER-α or ER-β, leading to differences in receptor subtype selectivity. The amino acids identified by crystallography as lining the ligand binding cavity are the same among the three species, suggesting that as yet unidentified amino acids contribute to the structure of the binding site. We conclude from this analysis that the ability of a compound to selectively bind to a particular ER subtype can be species dependent.

Allosteric Activation of the Follicle-stimulating Hormone (FSH) Receptor by Selective, Nonpeptide Agonists

The pituitary glycoprotein hormones, luteinizing hormone and follicle-stimulating hormone (FSH), act through their cognate receptors to initiate a series of coordinated physiological events that results in germ cell maturation. Given the importance of FSH in regulating folliculogenesis and fertility, the development of FSH mimetics has been sought to treat infertility. Currently, purified and recombinant human FSH are the only FSH receptor (FSH-R) agonists available for infertility treatment. By screening unbiased combinatorial chemistry libraries, using a cAMP-responsive luciferase reporter assay, we discovered thiazolidinone agonists (EC50s = 20 μm) of the human FSH-R. Subsequent analog library screening and parallel synthesis optimization resulted in the identification of a potent agonist (EC50 = 2 nm) with full efficacy compared with FSH that was FSH-R-selective and -dependent. The compound mediated progesterone production in Y1 cells transfected with the human FSH-R (EC50 = 980 nm) and estradiol production from primary rat ovarian granulosa cells (EC50 = 10.5 nm). This and related compounds did not compete with FSH for binding to the FSH-R. Use of human FSH/thyroid-stimulating hormone (TSH) receptor chimeras suggested a novel mechanism for receptor activation through a binding site independent of the natural hormone binding site. This study is the first report of a high affinity small molecule agonist that activates a glycoprotein hormone receptor through an allosteric mechanism. The small molecule FSH receptor agonists described here could lead to an oral alternative to the current parenteral FSH treatments used clinically to induce ovarian stimulation for both in vivo and in vitro fertilization therapy.

Identification of novel estrogen receptor α antagonists

Abstract We have identified novel estrogen receptor alpha (ERα) antagonists using both cell-based and computer-based virtual screening strategies. A mammalian two-hybrid screen was used to select compounds that disrupt the interaction between the ERα ligand binding domain (LBD) and the coactivator SRC-3. A virtual screen was designed to select compounds that fit onto the LxxLL peptide-binding surface of the receptor, based on the X-ray crystal structure of the ERα LBD complexed with a LxxLL peptide. All selected compounds effectively inhibited 17-β-estradiol induced coactivator recruitment with potency ranging from nano-molar to micromolar. However, in contrast to classical ER antagonists, these novel inhibitors poorly displace estradiol in the ER-ligand competition assay. Nuclear magnetic resonance (NMR) suggested direct binding of these compounds to the receptors pre-complexed with estradiol and further demonstrated that no estradiol displacement occurred. Partial proteolytic enzyme digestion revealed that, when compared with 17-β-estradiol- and 4 hydroxy-tamoxifen (4-OHT) bound receptors, at least one of these compounds might induce a unique receptor conformation. These small molecules may represent new classes of ER antagonists, and may have the potential to provide an alternative for the current anti-estrogen therapy.

Characterization of a membrane-associated estrogen receptor in a rat hypothalamic cell line (D12).

The ability of estrogens to produce rapid changes in cellular function has been firmly established. The question remains whether these changes are mediated by a modified form of the nuclear estrogen receptor (ER) that is associated with the plasma membrane (mER) or by a completely novel membrane receptor. Therefore, we characterized the biochemical properties of the nuclear and membrane-associated ERs expressed endogenously in a rat hypothalamic endothelial cell line (D12). Radioligand binding experiments using D12 membrane fractions showed that these cells exhibit properties consistent with a binding site specific for estrogens (mER). Equilibrium binding assays using [125I]16-α-iodo-3,17-β-estradiol revealed saturable binding to mER, an affinity value similar to nuclear ER, with differing receptor expression levels. Competition assays revealed that 9 of 12 ER ligands tested had comparable affinities for mER and ER. For example, 17-α-estradiol and estrone had similar binding characteristics for both receptors while differences were noted for raloxifene, 17β-estradiol (E2), and genistein. Western blot and immunocytochemical analyses using antibodies specific for ERα confirmed that D12 cells expressed a membrane-associated protein with a molecular mass (67 kDa) similar to that of ERα that colocalized with caveolae-enriched membranes. A rapid increase in intracellar Ca2+ levels in the presence of E2 suggests that mER can mediate physiologic changes through calcium mobilization. These data support the expression of mER in these brain-derived endothelial cells that is similar to, but biochemically distinguishable from, nuclear ERα.

Pharmacological characterization of soluble human FSH receptor extracellular domain: facilitated secretion by coexpression with FSH.

Follicle-stimulating hormone (FSH) is a member of the glycoprotein hormone family that regulates gametogenesis and steroidogenesis. Glycoprotein hormones signal through a unique class of G-protein-coupled receptors (GPCRs) that have a long extracellular domain (ECD), which is the primary site for hormone binding. The hFSHR-ECD was expressed in insect cells as a C-terminal, epitope-tagged protein resulting in production of soluble active receptor in the intracellular compartment and in the secreted culture medium. Coexpression of hFSHR-ECD with FSHβ or FSHα/β increased the secretion of the truncated receptor. Pharmacological studies to assess ligand-receptor interactions without the transmembrane domains showed higher affinity values (KDs) for [125l]hFSH using mammalian-expressed full-length receptor, secreted hFSHR-ECD, or secreted hFSHR-ECD coexpressed with FSHβ, whereas the KD value for hFSHR-ECD coexpressed with FSHα/β subunits showed lower affinity. Competition of other glycoprotein hormones for secreted hFSHR-ECD coexpressed with FSHβ or mammalian full-length hFSHR resulted in similar binding profiles, indicating analogous pharmacology. Finally, we have demonstrated that a small molecule, suramin, which has been reported to interact with the mammalian full-length FSHR, competes for the binding of [1251]hFSH by interacting directly at the hFSHR-ECD.