Control of estrogen receptor ligand binding by Hsp90

The molecular chaperone Hsp90 interacts with unliganded steroid hormone receptors and regulates their activity. We have analyzed the function of yeast and mammalian Hsp90 in regulating the ability of the human estrogen receptor (ER) to bind ligands in vivo and in vitro. Using the yeast system, we show that the ER expressed in several different hsp82 mutant strains binds reduced amounts of the synthetic estrogen diethylstilbestrol compared to the wild type. This defect in hormone binding occurs without any significant change in the steady state levels of ER protein. To analyze the role of mammalian Hsp90, we synthesized the human ER in rabbit reticulocyte lysates containing geldanamycin, an Hsp90 inhibitor. At low concentrations of geldanamycin we observed reduced levels of hormone binding by the ER. At higher concentrations, we found reduced synthesis of the receptor. These data indicate that Hsp90 functions to maintain the ER in a high affinity hormone-binding conformation.     

The DNA binding domain of estrogen receptor alpha is required for high-affinity nuclear interaction induced by estradiol

Estrogen receptor alpha (ER) is a member of the nuclear hormone receptor family, which upon binding estrogen shows increased apparent affinity for nuclear components (tight nuclear binding). The nuclear components that mediate this tight nuclear binding have been proposed to include both ER-DNA interactions and ER-protein interactions. In this paper, we demonstrate that tight nuclear binding of ER upon estrogen occupation requires ER-DNA interactions. Hormone-bound ER can be extracted from the nucleus in low-salt buffer using various polyanions, which mimic the phosphate backbone of DNA. The importance of specific ER-DNA interactions in mediating tight nuclear binding is also supported by the 380-fold lower concentration of the ERE oligonucleotide necessary to extract estrogen-occupied ER from the nucleus compared to the polyanions. We also demonstrate that estrogen-induced tight nuclear binding requires both the nuclear localization domain and the DNA binding domain of ER. Finally, enzymatic degradation of nuclear DNA allows us to recover 45% of tight nuclear-bound ER. We further demonstrate that ER-AIB1 interaction is not required for estrogen-induced tight nuclear binding. Taken together, we propose a model in which tight nuclear binding of the estrogen-occupied ER is predominantly mediated by ER-DNA interactions. The effects of estrogen binding on altering DNA binding in whole cells are proposed to occur through estrogen-induced changes in ER-chaperone protein interactions, which alter the DNA accessibility of ER but do not directly change the affinity of the ER for DNA, which is similar for both unoccupied and occupied ER.     

Differential DNA binding by calf uterine estrogen and progesterone receptors results from differences in oligomeric states

The studies presented here provided evidence that the calf uterine estrogen and progesterone receptors exhibit different DNA-binding properties in vitro as a result of having different dimerization constants. The affinity of the estrogen and progesterone receptors for DNA was measured by using isocratic elution from DNA-Sepharose. The hormone-free estrogen receptor had a 10-fold higher affinity for DNA than did the hormone-free progesterone receptor when measured at receptor concentrations of 6-12 nM and 180 mM KCl. No effect on DNA binding by binding progesterone to its receptor was detected. This contrasts with the increased affinity for DNA and increased number of ions released upon DNA binding exhibited by the hormone-bound estrogen receptor. Between 2 and 3 ions were released when the progesterone receptor and the diluted estrogen receptor bound DNA. These observations suggested the progesterone receptor was in the monomeric state, whereas the estrogen receptor was in the dimeric state at receptor concentrations of 6-12 nM. When the dimerization constant of the progesterone receptor was measured, the value of approximately 7 nM obtained was 20-fold higher than the value of 0.3 nM reported for the estrogen receptor. This makes it likely the two receptors exist in different forms at the same concentration in vitro. It is also suggested the predominant form of the estrogen and progesterone receptors in vivo could differ.     

The human estrogen receptor hormone binding domain dimerizes independently of ligand activation

High level expression of biochemically active human estrogen receptor hormone binding domain (hER-HBD) was achieved using a Saccharomyces cerevisae expression system. Using dissociation kinetic analysis, density gradient centrifugation and cross-linking studies, a spontaneous dimerization activity of hER-HBD independent of the presence of the DNA binding domain, ligand, and of elevated temperature is demonstrated.     

Two forms of the GABAA receptor distinguished by anion-exchange chromatography

The GABAA receptor complex was solubilized from rat brain membranes in Triton X-100, enriched by 1012-S affinity chromatography, and subjected to DEAE anion-exchange chromatography. Two forms were distinguished by their differential elution during this HPLC with a KCl gradient. They displayed similar [3H]muscimol- and [3H]flunitrazepam-binding characteristics, as well as [3H]flunitrazepam-binding inhibition by CL 218872. Rechromatography of these distinct ionic forms indicated that they were not in dynamic equilibrium during chromatography. Resolution of these two pharmacologically similar populations of GABAA receptor by anion-exchange HPLC suggests that they differ in charge densities, a condition which may reflect differing glycosylation or phosphorylation states of the complex.     

Sequence analysis of the nonsteroid binding component of the calf uterine estrogen receptor

Microbore reversed-phase high performance liquid chromatography has been utilized to fractionate and purify a number of tryptic peptides generated from the 90K nonsteroid binding component of the calf uterine estrogen receptor. Sequence analysis was performed on six peptides yielding 78 unique amino acid assignments, this corresponds to approximately 10% of the molecule. These peptides share sequence similarities with three heat shock proteins, Drosophila hsp 83 (83% homologous), yeast hsp 90 (55%) and chicken hsp 108 (32%). The amino acid composition of the protein indicates a prevalence of charged amino acid residues.     

Identification of the hydrophobic ligand binding pocket of the S1P1 receptor

Sphingosine 1-phosphate (S1P), a naturally occurring sphingolipid mediator and also a second messenger with growth factor-like actions in almost every cell type, is an endogenous ligand of five G protein-coupled receptors (GPCRs) in the endothelial differentiation gene family. The lack of GPCR crystal structures sets serious limitations to rational drug design and in silico searches for subtype-selective ligands. Here we report on the experimental validation of a computational model of the ligand binding pocket of the S1P1 GPCR surrounding the aliphatic portion of S1P. The extensive mutagenesis-based validation confirmed 18 residues lining the hydrophobic ligand binding pocket, which, combined with the previously validated three head group-interacting residues, now complete the mapping of the S1P ligand recognition site. We identified six mutants (L3.43G/L3.44G, L3.43E/L3.44E, L5.52A, F5.48G, V6.40L, and F6.44G) that maintained wild type [32P]S1P binding with abolished ligand-dependent activation by S1P. These data suggest a role for these amino acids in the conformational transition of S1P1 to its activated state. Three aromatic mutations (F5.48Y, F6.44G, and W6.48A) result in differential activation, by S1P or SEW2871, indicating that structural differences between the two agonists can partially compensate for differences in the amino acid side chain. The now validated ligand binding pocket provided us with a pharmacophore model, which was used for in silico screening of the NCI, National Institutes of Health, Developmental Therapeutics chemical library, leading to the identification of two novel nonlipid agonists of S1P1.     

Role of insulin receptor dimerization domains in ligand binding, cooperativity, and modulation by anti-receptor antibodies

To define the structures within the insulin receptor (IR) that are required for high affinity ligand binding, we have used IR fragments consisting of four amino-terminal domains (L1, cysteine-rich, L2, first fibronectin type III domain) fused to sequences encoded by exon 10 (including the carboxyl terminus of the alpha-subunit). The fragments contained one or both cysteine residues (amino acids 524 and 682) that form disulfides between alpha-subunits in native IR. A dimeric fragment designated IR593.CT (amino acids 1-593 and 704-719) bound (125)I-insulin with high affinity comparable to detergent-solubilized wild type IR and mIR.Fn0/Ex10 (amino acids 1-601 and 650-719) and greater than that of dimeric mIR.Fn0 (amino acids 1-601 and 704-719) and monomeric IR473.CT (amino acids 1-473 and 704-719). However, neither IR593.CT nor mIR.Fn0 exhibited negative cooperativity (a feature characteristic of the native insulin receptor and mIR.Fn0/Ex10), as shown by failure of unlabeled insulin to accelerate dissociation of bound (125)I-insulin. Anti-receptor monoclonal antibodies that recognize epitopes in the first fibronectin type III domain (amino acids 471-593) and inhibit insulin binding to wild type IR inhibited insulin binding to mIR.Fn0/Ex10 but not IR593.CT or mIR.Fn0. We conclude the following: 1) precise positioning of the carboxyl-terminal sequence can be a critical determinant of binding affinity; 2) dimerization via the first fibronectin domain alone can contribute to high affinity ligand binding; and 3) the second dimerization domain encoded by exon 10 is required for ligand cooperativity and modulation by antibodies.     

Oncornaviral protein modulation in mouse uterine tissue by estrogen (38467).

Treatment of ovariectomized NIH Swiss mice with estrogens elevated the level of the murine leukemia virus group specific protein and the activity of an RNA-directed DNA polymerase in the uterus. The extent that these markers were raised was dependent on the relative biological potency of the estrogen and on the time interval following treatment. Increases in the levels of both viral marker proteins were evident within 24 hr of treatment and were highest at 48 hr. Subsequently, viral protein levels declined to pretreatment levels.     

Isoelectric heterogeneity of human uterine estrogen binding proteins

Upon Isoelectric Focusing (IEF) of premenopausal uterine myometrial cytosol, specific binding of estradiol (E2) can be shown at elution pH's (EpH) of 4.0-4.4, 5.0-5.2, 5.8-6.2 and 7.5-8.0. Pre-adsorption of premenopausal uterine cytosol by Concanavalin A Sepharose (Con-A) or precipitation with 30% ammonium sulfate results in loss of estradiol binding at EpH's 4.4 and 5.0. The estradiol binding sites that bind to Con-A are present in plasma and have been shown to be Sex Hormone Binding Globulin (EpH = 5.0) and Estrogen Binding Protein (EpH = 4.4). After Con-A adsorption premenopausal cytosol preincubated with 2 nM 3HE2 reveals a single peak on IEF at EpH's congruent to 6.0, while preincubation with 40 nM 3HE2 reveals specific binding peaks at EpH's of congruent to 6.0 and 7.5-8.0. Postmenopausal uterine cytosol preincubated with either 2 or 40 nM 3H-E2 on IEF reveals EpH = 5.8-6.0 binding only. Post-labeling of IEF fractions with 20 nM 3HE2 demonstrates one peak at EpH 5.8-6.0 in postmenopausal tissue and two peaks (5.8-6.2 and 7.5-8.0) in premenopausal tissue. Scatchard analysis of postmenopausal cytosol demonstrates a single population of binding sites with a dissociation constant (Kd) of 10(-10) M. Premenopausal cytosol on Scatchard analysis contains two estradiol binding populations with Kd's of 10(-10) and 10(-9) M. The data suggest that the 10(-10) M E2 binding population has a EpH of 5.8-6.2, while the 10(-9) M component has an EpH of 7.5-8.0.     

Sensitive detection of estradiol based on ligand binding domain of estrogen receptor and gold nanoparticles

With increasing concerns of estrogenic effects of endocrine disrupting compounds, the development of simple detection assay for these compounds is an ongoing need. Herein, a simple, rapid, and highly sensitive assay for estradiol (E2) detection was developed using the ligand binding domain of estrogen receptor α (LBD-ERα), the receptor interacting domain of steroid receptor co-activator 1 (RID-SRC1), and gold nanoparticles (AuNPs). The colloidal AuNPs could be stabilized against a salt-induced aggregation by adding LBD-ERα protein. However, with the presence of E2, the specific binding of LBD-ERα protein and E2 led to a salt-induced aggregation of AuNPs as seeing from a color change from red to blue. This developed assay exhibited a high sensitivity for E2 detection with the limit of detection (LOD) of 2.62 × 10⁻¹⁴ M. When the RID-SRC1 protein was included, the detection sensitivity was increased, which the LOD for E2 was at 1.20 × 10⁻¹⁵ M. This assay was specific for a detection of E2 but not progesterone, the negative control ligand. Results of this work clearly showed the efficiency of developed assay for E2 detection, which possibly further developed for an onsite monitoring of E2.     

Differential modulation of gonadotropin secretion by selective estrogen receptor 1 and estrogen receptor 2 agonists in ovariectomized ewes

The objectives of this study were to determine whether activation of estrogen receptor 1 (ESR1; also known as ERalpha), or estrogen receptor 2 (ESR2; also known as ERbeta), or both are required to: 1) acutely inhibit secretion of LH, 2) induce the preovulatory-like surge of LH, and 3) inhibit secretion of FSH in ovariectomized (OVX) ewes. OVX ewes (n = 6) were administered intramuscularly 25 micrograms estradiol (E2), 12 mg propylpyrazoletriol (PPT; a subtype-selective ESR1 agonist), 21 mg diaprylpropionitrile (DPN; a subtype-selective ESR2 agonist), or PPT + DPN. Like E2, administration of PPT, DPN, or combination of the two rapidly decreased (P < 0.05) secretion of LH. Each agonist induced a gradual, prolonged rise in secretion of LH after the initial inhibition, but neither agonist alone nor the combined agonists was able to induce a "normal" preovulatory-like surge of LH similar to that induced by E2. Compared with E2-treated ewes, the beginning of the increase in secretion of LH occurred earlier (P < 0.01) in DPN-treated ewes, later (P < 0.05) in PPT-treated ewes, and at a similar interval in ewes receiving the combined agonist treatment. Like E2, PPT decreased (P < 0.05) secretion of FSH, but the duration of suppression was much longer in PPT-treated ewes. DPN did not alter secretion of FSH in this study. Modulation of the number of GnRH receptors by PPT and DPN was examined in primary cultures of ovine pituitary cells. In our hands, both PPT and DPN increased the number of GnRH receptors, but the dose of DPN required to stimulate synthesis of GnRH receptors was 10 times higher than that of PPT. We conclude that in OVX ewes: 1) ESR1 and ESR2 mediate the negative feedback of E2 on secretion of LH at the level of the pituitary gland, 2) ESR1 and ESR2 do not synergize or antagonize the effects of each other; however, they do interact to synchronize the beginning of the stimulatory effect of E2 on secretion of LH, 3) ESR1 and ESR2 may mediate at least partially the positive feedback of E2 on LH secretion by increasing the number of GnRH receptors, and 4) only ESR1 appears to be involved in the negative feedback of E2 on secretion of FSH.