Calf Uterine Estrogen Receptor Research Articles

Comment on “Specific binding of hydroxylated polychlorinated biphenyl metabolites and other substances to bovine calf uterine estrogen receptor: Structure-binding relationships [Kramer and Giesy, Sci Total Environ 1999;233:141–61

We thank Dr. Rayne for the comments on our publication. It should be stated at the outset that none of the comments Dr. Rayne makes challenges the validity of the experimental data indicating that 25 of the 28hydroxylated PCB's tested bind to the calf uterine estrogen receptor withmeasurable affinity. The objectives as set out in our publication were achieved. Regarding the applicability of modeling the protonated form, the literature tells us that this is the form of 17β-estradiol that specifically binds to the estrogen receptor with the phenolic proton forming a key hydrogen bond (Tannenbaum et al., 1998). Thus, themolecular properties of the phenolic forms of the hydroxylated PCB's were modeled and examined for relationships with binding affinity. It is true that the anionic form may dominate in bulk solution for a subset of these molecules. However, receptor binding occurs between one receptor and one ligand and that reaction does not occur in bulk solution (it is a desolvating process). The binding pocket of the receptor is hydrophobic. So, bulk properties thatmay dominate in solution do not necessarily dominate at the level of the individual receptor and ligand binding reactions. Thus, it is appropriate to model the properties of the protonated form for purposes of examining relationships of molecular properties with receptor binding. The molecular modeling of the phenolic structure reflected the ionizability of the oxygen atom as measured by the OCHARGE parameter. So, that parameter does contain information on the potential ionization state of the oxygen atom. The binding data also incorporates the ionization state that was actually achieved in the buffer system used. The ionization potential, which is related to pKa, was incorporated in the QSBR and was discussed in the publication. The bulk solution equilibrium influences the frequency of encounters between protonated ligand and receptor. However, the affinity with which the protonated ligand binds to the receptor is not affected by the bulk solution equilibrium since this is an entirely separate chemical interaction (Eq. (1)).

Specific binding of hydroxylated polychlorinated biphenyl metabolites and other substances to bovine calf uterine estrogen receptor: structure-binding relationships

The objectives of this research were: (1) to survey a wide variety of structurally diverse (and mostly chlorinated) aromatic chemicals for specific binding to the calf uterine estrogen receptor; (2) to develop a quantitative structure-binding relationship (QSBR) for hydroxylated polychlorinated biphenyls (OH-PCBs). This report specifically includes data on substances that did not exhibit specific binding to ER thereby exploring the structural requirements for specific binding to the estrogen receptor. Although several other QSBRs for OH-PCBs have been reported, this study presents data on a larger, environmentally relevant set of OH-PCBs than previously reported. Fifty three chemicals were tested for the ability to bind specifically to calf uterine estrogen receptor. All but three OH-PCBs bound specifically to calf uterine ER. For DDT compounds, receptor binding affinity followed the pattern: o,p'-DDT> o,p'-DDE> o,p'-DDD (Not active). Also exhibiting measurable affinity were 17ß-estradiol (a positive control and the native ligand of the estrogen receptor), 2,4,6-trichlorobiphenyl and 4-chloro-2-isopropyl-5-methyl-phenol. Substances that did not bind to calf uterine estrogen receptor comprised several individual PCB congeners, chlorinated naphthalenes and naphthalenols, chlorinated bibenzyls, chlorinated phenols, and 9-chloro-retene. For 25 hydroxylated PCBs, a five parameter QSBR was developed using multiple linear regression and selection of the most parsimonius model from a total of seven molecular modeling parameters examined. The QSBR model predicted the ER binding log (IC50) to within one log unit.

Hsp70 is not required for high affinity binding of purified calf uterine estrogen receptor to estrogen response element DNA in Vitro

Bovine estrogen receptor (ER) was purified to near homogeneity by estrogen response element (ERE) affinity chromatography, and its ERE binding ability was measured in vitro. Highly purified ER bound EREs with reduced affinity compared to partially purified ER. Partially purified ER contained hsp70, but highly purified ER did not. We examined whether addition of purified recombinant human hsp70 or purified bovine hsp70 would restore the higher ERE binding affinity, stoichiometry, and ligand retention detected with partially purified receptor and how hsp70 affected the rate of ER-ERE association and dissociation. ER-ERE binding was not affected by antibodies to either constitutive or induced forms of hsp70, regardless of ER purity. Addition of purified hsp70, with or without ATP and Mg 2+, did not affect the association or dissociation rates of highly purified liganded ER binding to ERE. hsp70 Did not alter the total amount of ER-ERE complex formed. Similarly, hsp70 did not affect the rate of [ 3H]estradiol (E 2) or [ 3H]4-hydroxytamoxifen (4-OHT) ligand dissociation from ER in the presence or absence of EREs. These data contrast with a report showing that maximal ERE binding by highly purified recombinant human ER required hsp70. We conclude that ER, purified from a physiological source, i.e., calf uterus, does not require hsp70 for maximal ER-ERE binding in vitro. Additionally, once ER is activated and bound by ligand, the receptor assumes its proper tertiary structure, and hsp70 does not impact ER ligand binding domain conformation.

2-Phenylindoles with sulfur containing side chains. Estrogen receptor affinity, antiestrogenic potency, and antitumor activity.

The 2-phenylindole system has been identified as a suitable structure for the design of non-steroidal pure estrogen antagonists [E. von Angerer et al., J. Steroid Biochem. Molec. Biol. 49 (1994) 51-62]. Derivatives with an amide function in the side chain antagonized the stimulatory effect of estrogens both in vitro and in vivo, and showed no agonistic activity when given alone. The findings of other groups who studied steroidal antiestrogens prompted us to replace the amide function by sulfide, sulfoxide, sulfone, sulfonamide and related groups. The compounds with polar sulfur functions retained the high binding affinity for the calf uterine estrogen receptor (RBA: 1-5% of estradiol; ICI 182,780; 6.2%). The estrogenic effect was quantified in a transcription assay using HeLa cells cotransfected with the expression vector HEG0 for the human estrogen receptor and a reporter plasmid that harbored a Vit. A2 ERE and the luciferase gene driven by a thymidine kinase promotor. Pentylsulfide, -sulfinyl, and -sulfonyl groups, linked to the indole nitrogen by a decamethylene spacer, were devoid of any transcriptional activity. These results were confirmed in the mouse uterine weight test. The sulfone (ZK 164,015) completely abolished the effect of a standard dose of estrone at a daily dose of 7 mg/kg. This compound strongly inhibited the growth of hormone-sensitive human MCF-7 breast cancer cells with an IC50-value close to 1 nM. Similar activity was found for the steroidal sulfoxide ICI 182,780. We were also able to demonstrate significant antineoplastic activity in vivo for some of these new 2-phenylindole derivatives.

A-Ring Nitro- and Amino-Substituted Estradiol Analogs Produce a Negative Cooperative or Noncooperative [3H]Estradiol-Estrogen Receptor Binding Mechanism

We have investigated the relation between ligand structure and binding mechanism between the calf uterine estrogen receptor. A series of structurally altered estradiol analogs was used in which either an amino- or a nitro group had been added to the 2 or 4 position on the phenolic A-ring. The binding affinity of both amino analogs and the 4-nitro analog for the estrogen receptor was reduced relative to that of estradiol, as measured by competitive binding assay; the values were between 0.008% and 8% of estradiol's affinity. The slope of the displacement curve for the 4-nitro analog was also significantly different from that of estradiol (p < 0.05), indicating that the binding mechanism of these two ligands was different. The affinity of the 2-nitroestradiol ligand for the receptor was too low to be measured. The binding mechanism was then further investigated by measuring the Hill coefficient of [3H]estradiol binding in the presence of the analog. The presence of a nitro group on C4 eliminated the positive cooperativity of the [3H]estradiol-estrogen receptor interaction; the Hill coefficient of [3H]estradiol binding in the presence of the analog was 0.99 compared with 1.7 for [3H]estradiol alone. Most interestingly, the presence of an amino group on either C2 or C4 brought about a switch from a positive to a negative cooperative binding interaction; the Hill coefficients of [3H]estradiol binding in the presence of the analogs were between 0.6 and 0.7. These results provide additional support for an induced-fit mechanism of ligand-estrogen receptor interactions.

1-carbamoylalkyl-2-phenylindoles: Relationship between side chain structure and estrrogen antagonism

The 2-phenylindole system has proved to be a versatile structure for the design of potent antiestrogens, especially when functional groups have been introduced into the alkyl side chain in position 1. In analogy to steroidal carbamoylalkyl and aminoalkyl side chains were synthetized. They bind to the calf uterine estrogen receptor with relative binding affinities between 2.1 and 21 (estradiol = 100). The antiestrogenic effect of these compounds was demonstrated by the inhibition of transcriptional activity which was measured in a new luciferase assay with the EREwtc luc as reporter plasmid. The derivative with a methyl- n-propyldodecanamide side chain (4h) antagonized the effect of estradiol (10 −9M) completely at concentrations of 10 −7 M and higher. As a sensitive modef for quantification of estrogenic and antiestrogenic effects in vitro we used HeLa-cells cotransfected both with the reporter plasmid and estrogen receptor expression vectors HEG0 and HE0. In cells transfected with these vectors transcriptional activity was strongly dependent on side chain structure. With mutated receptors we were able to show that this activity was mainly due to TAF-1 whereas TAF-2 remained silent. When we studied the effect of some of the new compounds in vivo using the mouse uterine weight assay, we observed a correlation between transciptional activity in transfected HeLA cells and estrogenic effects in mice. Two of the 1-carbamoylalkyl-2-phenylindoles (4f,4h) proved to be “pure” antiestrogens both in vitro and in vivo. In estrogen-sensitive MCF-7 breast cancer cells, they strongly inhibit cellular growth. Some of the IC 50-values were close to 10 −8M.

Antiestrogen-liganded estrogen receptor interaction with estrogen responsive element DNA in vitro

The mechanism whereby antiestrogens alter the ability of the estrogen receptor (ER) to enhance transcription of estrogen-regulated genes is largely unknown. The effect that selected estrogenic and antiestrogenic ligands have on binding of ER to specific DNA sequences, estrogen responsive elements (EREs) has been quantitated. No differences in purification properties of calf uterine ER liganded with 4-hydroxytamoxifen (4-OHT-ER), ICI 164,384 (ICI 164,384-ER) or estradiol (E 2-ER) were detected. A microtiter well plate assay was employed in which liganded ER bound to plasmid DNA is preferentially retained compared to free liganded ER. Binding of E 2-ER, 4-OHT-ER, or ICI 164,384-ER was measured to plasmids containing or lacking a 38bp consensus ERE in vitro. The EREs tested contain an inverted repeat (5′- CAGGTCA GAG TGACCTG -3′). Both E 2-ER and 4-OHT-ER showed similar high affinity specific binding ( K d = 0.24 and 0.16 nM, respectively) to one copy of the ERE. ICI 164,384-ER did not bind to plasmids containing one ERE. At saturation, however, 4-OHT-ER binding was about 50% of that observed for E 2-ER. When the plasmid contained 3 or 4 tandem copies of the ERE, binding E 2-ER, 4-OHT-ER, and ICI 164,384-ER binding was measurable. E 2-ER bound in a cooperative manner as suggested by convex Scatchard plots and Hill coefficients > 1.5. In contrast, 4-OHT-ER binding displayed much reduced cooperativity, and ICI 164,384-ER did not display cooperative binding. From these results, we propose that the conformation of ER induced by 4-OHT reduces its binding capacity to this consensus ERE without altering its affinity of binding. Furthermore, higher order protein-protein interactions between antiestrogen-liganded ER bound to DNA differ from those of E 2-ER bound to ERE.

3-Alkyl-2-phenylbenzo[ b]thiophenes: Nonsteroidal estrogen antagonists with mammary tumor inhibiting activity

A number of 2-(4-hydroxyphenyl)benzo[ b]thiophenes with a hydroxy group in position 5 or 6 and a short alkyl group at C-3 were synthesized and studied for their estrogen receptor affinities. Relative binding affinities (RBA) for the calf uterine estrogen receptor ranged from 3 to 60 (17β-estradiol = 100). The highest RBA values were found with ethyl derivatives [ 3 (5- OH): 60; 7 (6- OH): 28 ]. In accord with their receptor affinity, all benzothiophenes exhibited endocrine activity in the immature mouse uterine weight test. At doses of 0.25–7.0 mg/kg body weight, they showed partial estrogen antagonism and usually weak estrogenic effects. All compounds entered tests with hormone-sensitive human MCF-7 breast cancer cells. At concentrations of 1μM and higher, most of the derivatives displayed significant inhibition of cell growth. These results prompted us to test them in vivo for cytostatic activity using hormone-dependent MXT mouse mammary tumors. The 5-hydroxy derivatives 3 and 4 strongly inhibited the growth of these tumors. After 4 weeks of treatment with 3 × 4.2 mg/kg of compound 3, the average tumor weight was reduced by 83% vs control (tamoxifen at equimolar dose: 74%). The 6-hydroxy derivative 7 required higher doses (25 mg/kg) to give rise to the same effect. At the end of therapy, no increase of uterine weight due to an estrogenic effect was observed. We assume therefore that the antineoplastic activity of these compounds in this tumor model is due mainly to their estrogen antagonism.

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. A stimulatory estrogen response element (ERE) was localized to a 32-bp region between -547 and -516 bp. To determine whether estrogen receptor bound to this region of the gene, we performed DNase I footprinting using purified calf uterine estrogen receptor. DNase I footprinting demonstrates a strong footprint between -567 and -514 bp of the human GnRH gene. In addition, an avidin-biotin complex DNA-binding assay demonstrated that a biotinylated DNA fragment containing -541 to -517 bp of the human GnRH gene bound 35S-labeled estrogen receptor as well as a biotinylated DNA fragment containing the xenopus vitellogenin ERE. On the other hand, the negative control biotinylated DNA fragment derived from adenovirus 5 bound insignificant amounts of 35S-labeled estrogen receptor. Both the GnRH ERE and vitellogenin ERE bound 35S-labeled estrogen receptor with high affinity (approximately 1 nM). These data indicate that the human GnRH gene contains an ERE sufficient to mediate a stimulatory response to estrogen in heterologous cells. Based upon these data we hypothesize that the human GnRH gene might also be directly regulated by estrogen in the hypothalamus, and that this regulation may explain the GnRH hypersecretion observed at the time of the preovulatory luteinizing hormone (LH) surge.

2-Phenylindole-linked [2-(aminoalkyl)pyridine]dichloroplatinum(II): complexes with a selective action on estrogen receptor positive mammary tumors

A number of [2-(aminomethyl)pyridine]dichloroplatinum(II) complexes, linked to 5-hydroxy-2-(4-hydroxyphenyl)indoles by alkyl spacer groups of varying lengths, were synthesized and studied for their binding affinities for the calf uterine estrogen receptor. Their relative binding affinity (RBA) values ranged from 1.0 to 5.2% (estradiol, RBA = 100%). Highest affinities were found with complexes possessing a (CH2)5- or (CH2)6-bridge between the pyridine aminomethyl group and the indole nitrogen. Endocrine activities of the complexes and their ligands, determined in the mouse uterine weight test, were low. All compounds entered comparative tests using estrogen receptor positive and negative mammary tumor models. In cell culture, a growth inhibiting effect was only observed in hormone-sensitive MCF-7 cells, but not in hormone-independent MDA-MB 231 cells. In this assay, there was no significant difference between complexes and their ligands. In vivo, the growth of estrogen receptor positive MXT mouse mammary tumors was strongly inhibited by the complexes whereas the hormone-independent MXT mammary tumors showed only a minor response. At a dose of 3 X 20 mg/kg per week, complexes 10d-g reduced the tumor weight by ca. 80% after 6 weeks of treatment. This effect was generally stronger than that exerted by the ligands. The doses applied were well tolerated. Since the complexes described in this paper require the estrogen receptor for their action, a mechanism similar to that of antiestrogens is assumed.

Estrogen receptor alters the topology of plasmid DNA containing estrogen responsive elements

We have recently used DNA containing estrogen responsive element (ERE) sequences for affinity purification to prepare calf uterine estrogen receptor (ER) at near homogeneity. The capacity of this purified ER to alter DNA topology upon binding was examined. Although the ER is not a topoisomerase, the presence of ER changes the distribution of topoisomers generated by incubation of plasmid DNA with excess wheat germ topoisomerase I. This effect is larger in plasmids containing a consensus ERE sequence. Two dimensional gel electrophoretic analysis suggested that interaction of ER and ERE causes negative supercoiling in regions of the plasmid accessible to topoisomerase I, resulting from overwinding of DNA contacting the ER. The extent of topological alteration was dependent on ER concentration. We suggest that the observed conformational changes in the DNA could have a role in regulation of transcription.

1-(Aminoalkyl)-2-phenylindoles as novel pure estrogen antagonists

A number of 1-(omega-aminoalkyl)-5-hydroxy-2-(4-hydroxyphenyl)indoles were synthesized and studied for their binding affinities for the calf uterine estrogen receptor and estrogen antagonistic activities. Highest binding affinities were found with derivatives bearing a methyl group in position 3 and a hexamethylene chain between the indole and amino nitrogen atoms. Values for relative binding affinity (RBA) are between 20 and 30 for derivatives 5b, 5c, 5f, and 5h (17 beta-estradiol = 100). In the mouse uterine weight test, no significant agonistic (estrogenic) activity was observed up to a daily dose of 125 micrograms/animal, except for derivatives 5c, 5j, and 5l. 2-Phenylindoles with amino (5b), pyrrolidino (5f), piperidino (5h), and morpholino (5k) groups as the amino function completely suppressed estrone-stimulated uterine growth as a dose of 125 micrograms/animal (100% antagonism). Therefore, these derivatives can be considered as first examples of nonsteroidal pure antiestrogens.

Unique Molecular Properties of a Urea- and Salt-Stable DNA-Binding Estrogen Receptor Dimer Covalently Labeled with the Antiestrogen [3H] Desmethylnafoxidine Aziridine. A Comparison with the Estrogen- Receptor Complex.

A new antiestrogen affinity ligand for the covalent labeling of estrogen receptors, [3H]desmethylnafoxidine aziridine, has been used to investigate the salt- and temperature-independent formation of DNA-binding estrogen receptor forms from untransformed (300 kilodaltons) receptor. Calf uterine estrogen receptor proteins labeled with [3H]estradiol or [3H]desmethylnafoxidine aziridine were quantitatively transformed (greater than 90%) to their DNA-binding configuration in low ionic strength buffers by brief exposure to 3 M urea at 0 C. The urea effect was hormone-dependent and partially reversible. The transformed receptors were purified (ca 250-fold) by affinity chromatography on single-stranded DNA-agarose in the continued presence of 3 M urea to prevent transformation reversal. Scatchard analyses revealed a single class of high affinity radioligand binding sites (Kd = 0.34 nM) unchanged by urea-induced transformation and purification. The DNA-binding receptor form labeled with [3H]desmethylnafoxidine aziridine was stable as a probable dimer in 3 M urea with 0.4 M KCl and displayed no evidence of size (Stokes radius 7.3 to 7.5 nm; 4.2 to 4.3 S; Mr = 136,800) heterogeneity. Sodium dodecyl sulfate-polyacrylamide gradient gel electrophoresis indicated the presence of an intact 67 kDa steroid-binding receptor subunit. Reverse-phase chromatography of the covalently labeled receptor on C4 and phenyl stationary phases revealed no evidence of structural heterogeneity. The surface charge of the estrogen- and antiestrogen-receptor complexes, however, was distinctly different in both the presence and absence of 3 M urea. Thus, exposure to urea was an effective salt- and temperature-independent means for achieving the complete transformation of receptor to its stable DNA-binding dimer configuration. The ligand-induced differences in receptor surface charge and the urea effects on DNA-binding (but not hormone-binding) suggest that both electrostatic and hydrophobic or hydrogen bonding receptor domains are influenced by ligand binding.

Reversible, positive cooperative interaction of 11β-chloromethyl-[ 3H]estradiol-17β with the calf uterine estrogen receptor

The binding of 11β-chloromethyl-[ 3H]estradiol-17β [ 3H]CME 2) with the calf uterine estrogen receptor was investigated. The equilibrium binding analysis indicated a positive cooperative interaction yielding curvilinear Scatchard plots and Hill coefficients of 1.4–1.5. This positive cooperative interaction of [ 3H]CME 2 was indistinguishable from the typical cooperative interaction of [ 3H]estradiol with the receptor. The apparent relative association constant and the relative binding affinity of CME 2 for the estrogen receptor measured by competitive binding assay were 146 and 184%, respectively. The dissociation kinetics [ 3H]CME 2 from the receptor was biphasic, composed of a fast dissociating component (15%, t 1 2 = 4 min at 0°C; 9%, t 1 2 = 4 at 28°C) and a slow dissociating component (85%, t 1 2 > 50 h at 0°C; 91 %, t 1 2 > 50 h at 28°C). The dissociation kinetics of [ 3H]estradiol was also biphasic: the t 1 2 of the fast dissociating component was 4 min at 0 and 28°C and ∼ 200 min for the slow dissociating component at both temperatures. The fraction of the slow [ 3H]estradiol dissociating component increased from 56 to 92% upon warming. Ethanol extraction and trichloroacetic acid treatment proved that the binding of [ 3H]CME 2 is fully reversible. The unusual dissociation kinetics and the binding mechanism of CME; are discussed.

Rapid purification of the estrogen receptor by sequence-specific DNA affinity chromatography.

Rapid purification of calf uterine estrogen receptor (ER) to near homogeneity has been accomplished by use of sequence-specific DNA affinity resin. Very high selectivity for the estrogen receptor is achieved through the use of DNA-Sepharose containing eight tandem copies of a consensus estrogen response element (ERE) DNA sequence. The highly purified ER prepared by this new scheme may be labeled economically with ligands of high specific activity. This purification scheme selects for intact receptors retaining function in both estrogen-binding and DNA-binding domains. Purified receptor has an electrophoretic mobility consistent with a molecular weight of 68,000, sediments as a 5S species on sucrose gradients, and reacts with antibody specific to the human estrogen receptor.

The Interaction Site for Tamoxifen Aziridine with the Bovine Estrogen Receptor

Calf uterine estrogen receptor was covalently labeled with [3H]tamoxifen aziridine during affinity chromatography purification. After carboxymethylation, affinity labeled receptor was digested with trypsin under limit conditions and the labeled peptides were fractionated by reversed-phase high performance liquid chromatography into one major and two minor components. Sequence analysis of the dominant labeled fragment indicated the facile cleavage of label during Edman degradation but identified two peptides, both derived from the extreme carboxyl terminus of the steroid-binding domain. The 17 residues of one peptide were fully conserved in all estrogen receptors. This fragment contained five nucleophilic amino acids and was considered as the more favored interaction site for tamoxifen aziridine. A corresponding region of the glucocorticoid receptor has recently been identified as one of three major contact sites for glucocorticoids (Carlstedt-Duke, J., Strömstedt, P.-E., Persson, B., Cederlund, E., Gustafsson, J.-A., and Jörnvall, H. (1988) J. Biol. Chem. 263, 6842-6846). A comparison of amino acid physical characteristics in the hormone-binding domains of human estrogen and glucocorticoid receptors demonstrated an excellent structural correlation between the two regions and delineated elements in the estrogen receptor which may be directly involved in estradiol binding.

Purification of heat shock protein 90 from calf uterus and rat liver and characterization of the highly hydrophobic region

Heat shock protein 90 was purified from calf uterus and rat liver. Both heat shock protein 90s had similar molecular weights, as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, of M r 87 000 and 88 000, isoelectric points of 5.2, and Stokes radii of 6.7 and 6.5 nm, respectively. Heat shock protein 90 bound to phenyl-Sepharose CL-4B even at low ionic strength, and also bound to butyl-Toyopearl at high ionic strength. Heat shock protein 90 bound to phenyl-Sepharose could be eluted with a buffer containing organic solvents or detergents such as 2-propanol, dioxane, dimethylformamide, methyl cellosolve, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate or Triton X-100, but not with ionic salts such as 1 M KCl. These results suggest that heat shock protein 90 possesses a significant hydrophobic region on the surface of the molecule. Hydrophobicities of heat shock protein 90 and 4S calf uterine estrogen receptor were both decreased by formation of a 8S estrogen receptor complex. The role of the hydrophobic region of heat shock protein 90 in the interaction with estrogen receptor and other proteins is discussed.

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.

Synthesis and estrogen receptor affinity of 2,3-diarylindoles.

Abstract2‐Phenylindoles with aromatic substituents at C‐3 and hydroxy functions at the aromatic rings were synthesized and tested for their binding affinity for the calf uterine estrogen receptor. Most of these indoles bind to the estrogen receptor. The highest binding affinity (1,25 % of estradiol) was found with 3‐(4‐hydroxyphenyl)‐2‐phenylindole (3b). The acetate of 3b was studied in vivo. It was devoid of estrogenic or antiestrogenic activity in the mouse and inhibited only weakly the growth of hormone‐dependent DMBA‐induced rat mammary tumors.

Interaction of the calf uterine estrogen receptor with acceptor sites in heterologous chicken target cell nuclei

Estrogen receptor (ER) from chicken liver and calf uterus were used to study the capacity and the characteristics of the receptor binding sites (acceptor sites) in chicken target cell nuclei. Binding studies were performed at a physiological salt concentration of 0.15 M KCl. Binding of liver ER to liver nuclei was temperature-dependent, showing a 9-fold increase between 0 and 28°C. The maximal number of acceptor sites measured in this cell-free system (280 sites/nucleus) was considerably lower than measured in nuclei after in vivo administration of estrogen (820 sites/nucleus). Moreover incubation of nuclei with the liver ER preparation resulted in a substantial breakdown of nuclear DNA, making this ER less suitable for DNA binding studies. The temperature-activated calf uterine receptor bound to liver nuclei at 0°C, at which temperature no DNA degradation was measured. To all chicken cell nuclei tested, the receptor bound with a high affinity ( K d 0.4–1.0 nM). Nuclear binding displayed tissue specificity: oviduct > heart, liver > spleen > erythrocytes and was salt dependent. Calf uterine ER binding in liver nuclei ranged from 3000–6000 acceptor sites per nucleus when assayed under conditions of a constant protein or a constant DNA concentration. Nuclei isolated from estrogen-treated cockerels bound a 2-fold lower number of calf uterine ER complexes when compared to control nuclei. Incubation of nuclei with a fixed concentration of [ 3H]ER from liver and increasing concentrations of uterine non-radioactive-ER also resulted in a reduced binding of the liver receptor. Both types of experiments suggest that liver and uterine ER compete for a common nuclear acceptor site. Our data demonstrate that the ER from calf uterus is very useful as a probe to examine the nature of the acceptor sites in heterologous chicken target cell nuclei. The assay system functions at 0°C, a temperature at which no DNA degradation occurs.