Properties of the estrogen receptors contained in the MtTF4 tumor whose growth is inhibited by estradiol: 17 beta-estradiol, 17 alpha-estradiol and DNA bindings

The steroid and the DNA bindings of the estrogen receptor of the MtTF4 tumor whose growth is inhibited by estradiol where characterized and compared to those of uterine estrogen receptors. In the tumor cytosol: E protects its binding sites against thermal denaturation, depending on the effects of sodium molybdate upon the dissociation rate of [3H]E at 20 degrees C and the ability of receptor to bind to DNA, the activation (or transformation) process, supposed to be necessary for the full action of estrogen ligand, occurs on estrogen receptor complexes and the calf thymus DNA interacts with estrogen receptor with an affinity similar to that of uterine estrogen receptor. Kinetic and equilibrium studies with 17 alpha-[3H]E both in uterus and tumor indicate that this ligand is fast-associating, fast-dissociating and that its affinity for ER is 2- to 4-fold lower than that of 17 beta-[3H]estradiol one. Competition experiments between 17 beta-[3H]estradiol and the unlabelled 17 alpha epimer reveal, in both uterus and tumor, a time-dependent decrease of the apparent potency of 17 alpha-E to inhibit the binding of [3H]E. It is concluded that the estrogen receptors are very similar in MtTF4 tumor and uterus and the diversity of the response of cell growth to E is due rather to differences at the post-receptor level.     

Transformation (4S to 5S) of the nuclear estrogen receptor is reversible but not accompanied by a change in the affinity for DNA

The nuclear estrogen receptor from calf uterus was used to investigate the possible relationship between receptor transformation (4S to 5S) and receptor activation (DNA binding). Receptors extracted from nuclei after exposure of uterine tissue tc [3H]estradiol sedimented at 5.2S, the characteristic value of the transformed receptor. After storage at -20 degrees C the receptor sedimented at 4.0S, indicating conversion of the 5S form into the non-transformed 4S form. Upon reincubation at 28 degrees C the 4S form transformed into the 5S form following second-order kinetics. The rate constant obtained was 4.3 x 10(7) M-1 min-1, a value identical to that reported for the cytosol receptor. These data show that receptor transformation is reversible. Molybdate (10-50 mM) was not able to prevent receptor transformation in the nuclear extract, but was inhibitory in cytosol. This suggests that molybdate does not prevent receptor transformation, but rather inhibits disaggregation of the 8S oligomer into the 4S monomer. In DNA-binding assays (DNA-cellulose or nuclei) the non-transformed (4S) and transformed (5S) states of the nuclear estrogen receptors displayed identical affinities for DNA. The present data show that 4S to 5S transformation of nuclear receptors follows a readily reversible process, but this process is not an essential step for the exposure of the receptors' DNA-binding site. Although the physiological function of the 5S form remains unclear it may be important for the recognition of specific gene regulatory sites.     

A nuclear transforming factor that converts the goat uterine nonactivated estrogen receptor to nuclear estrogen receptor II

A 62-kDa nuclear protein that transforms the goat uterine nonactivated estrogen receptor (naER) to nuclear estrogen receptor II (nER II) has been isolated and purified. This is being identified as the naER-transforming factor (naER-TF). The transformation is achieved through deglycosylation of the naER. It is observed that the naER-TF action on the naER introduces significant changes in the structural and functional features of the naER. The capacity of the naER to bind estradiol increases 8- to 10-fold, while its hormone binding affinity reduces to a considerable extent following its exposure to naER TF. There is a critical ratio in the concentration of the two proteins, the TF and the naER, that would ensure an optimum transformation process. The transformed naER is incapable of dimerization with the estrogen receptor activation factor (E-RAF).     

A kinetic analysis of the estrogen receptor transformation.

The rate of the 4 to 5 S estrogen-binding protein (EBP) in vitro transformation was measured by sucrose gradient centrifugation analysis. The temperature-activated 4 to 5 S EBP transformation is found to be highly reproducible without loss of [3H]estradiol-binding activity in a buffer containing an excess of [3H]estradiol, 40 mM Tris, 1 mM dithiothreitol, and 1 M urea at pH 7.4. The presence of [3H]estradiol is necessary for the 4 to 5 EBP transformation. A kinetic analysis of the 4 to 5 EBP transformation shows that it is a bimolecular reaction, the dimerization of the 4 S EBP with a second (similar or dissimilar) monomer or subunit. In buffers containing 0.4 M KCl the apparent second order rate constant is 2.3 plus or minus 0-2 times 10-7 M minus 1 min minus 1 at 28 degrees. The reaction is independent of the initial receptor concentration, suggesting that the 4 S EBP is dissociated into monomeric units in buffers of high ionic strength. In buffers without KCl or with 0.1 M KCl the apparent second order rate constant of receptor transformation increases with decreasing receptor concentration. This suggests that the 4 S EBP is associated weakly with another macromolecule (or macromolecules) in buffers of low ionic strength. The rate of 4 to 5 S EBP transformation shows a 200-fold increase between 0 and 35 degrees. The Arrhenius energy of activation is 21.3 kcal mol minus 1 in buffer without KCl and 19.1 kcal mol minus 1 in buffer with 0.4 M KCl. Following the temperature-activated dimerization, the avidity of binding between the 4 S EBP and its complementary subunit is increased, 0.4 M KCl can no longer cause dissociation, and the 5 S EBP dimer appears. This kinetic analysis indicates that the avidity of binding between the subunits of the estrogen receptor is modulated by estradiol, temperature, and ionic strength. We propose that these interactions of the estrogen receptor's subunits reflect conformational changes involved in receptor activation.     

Analysis of monomeric-dimeric states of the estrogen receptor with monoclonal antiestrophilins

We studied the antibody-combining properties of 3 forms of the estrogen receptor found in buffers of high ionic strength. Shifts to a faster sedimenting peak on sucrose gradients or a faster eluting peak on a gel filtration column with antibody addition allowed us to determine whether a given form contained one, two or more antibody-binding sites. The monomeric cytosolic estrogen receptor, ERC, contained one antibody binding site for each of 2 monoclonal antiestrophilins (H222 and H165, provided by Abbott Laboratories). Both the heat-transformed cytosolic estrogen receptor, ERC*, and a major fraction of the estrogen receptor extracted from nuclei, ERN, contained two sites for H165, but only one for H222. A minor fraction of ERN had only one site for each antibody. The kinetics of transformation of ERC to a species with two H165 binding sites were appropriate to a dimerization of ERC*. Addition of H222, but not H165, before the onset of the heat-induced transformation blocked the formation of ERC to ERC. These data suggest that ERC* and a major form of ERN are comprised of two immunologically similar subunits identical to ERC. Also, the antigenic determinant for H222, but not H165, appears to be located close to the dimerization domain. The minor form of ERN appears to contain an altered or dissimilar subunit.     

Transformation of the estrogen receptor detected by two monoclonal antibodies

The estrogen receptor from fetal guinea-pig uterus is recognised by two monoclonal antibodies (H222 and H226) developed against the human estrogen receptor but it interacts differently with each of them. The H222 antibody, whose epitope is located in the hormone-binding domain of the receptor, shifts the sedimentation coefficient of the nonactivated oligomeric receptor in low salt sucrose gradients from 9S to 11S. When this oligomeric receptor-H222 complex is centrifuged in high salt gradients, it dissociates to an 8S monomer-H222 complex, indicating that all the estradiol-binding units present in the nonactivated receptor can bind the H222 antibody. In contrast, the H226 antibody, whose epitope is located close to the DNA-binding domain, shifts the sedimentation coefficient of the nonactivated receptor only to 9.4S and when this complex sediments in high salt gradients, it dissociates to a 7S monomer-H226 complex plus a 4.5S monomeric receptor not bound to the antibody. This observation suggests that not all the H226 epitopes are accessible in the nonactivated receptor. On the other hand, the temperature-activated receptor reacts with the H226 antibody to form two complexes sedimenting at 7S and 9S in high salt gradients. This 9S complex indicates the formation of a homodimer that binds two molecules of the H226 antibody. However, only one H222 epitope seems to be accessible in this dimeric form of the receptor, since only one 8S complex is observed when the activated receptor reacts with the H222 antibody. In addition, binding to the H222 antibody before activation prevents the dimerisation. This suggests that the H222 epitope is near or directly involved in the dimerisation domain. Interaction of the H222 and H226 antibodies with the estrogen receptor reveals modifications of its structure during activation, and consequently of the exposure of its functional domains.     

Activation and immunorecognition by a monoclonal antibody of the tamoxifen-estrogen receptor complex

The interaction of tamoxifen with the estrogen receptor of fetal guinea pig uterus, the activation of the tamoxife-estrogen receptor complex and its immunorecognition by a monoclonal antibody raised against the human estrogen receptor is described in the present paper. The results show that: (1) the tamoxifen-receptor complex sediments at 8 S in low-salt and at 4.5 S in high-salt sucrose gradients, (2) this complex is partially recognized by the monoclonal antibody allowing the differentiation of two forms: the α form, which binds to the monoclonal antibody, and the β form, which does not react with it; (3) several factors such as time, temperature and high salt concentrations were capable of activating the tamoxifen-receptor complex, as determined by the increase of its binding to DNA-cellulose; (4) these factors also induced a partial transformation of the β form to the α form; (5) sodium molybdate inhibited both activation and transformation of the β into the α form. The correlation between activation and induction of the α form suggests that the monoclonal antibody recognizes selectively the activated form of the tamoxifen-receptor complex. These results indicate similar properties of the estrogen receptor when bound to either tamoxifen or estradiol; however, the differences observed in the behavior of the tamoxifen-receptor complex as compared with the estradiol-receptor complex. though quantitative rather than qualitative, suggest that the estrogen receptor is affected differently by tamoxifen and estradiol.     

Immunological difference between ribonuclease and temperature, time and salt-induced forms of the estrogen receptor detected by a monoclonal antibody

The effect of RNAase A on the activation of the estrogen receptor from fetal guinea pig uterus was studied by DNA-cellulose binding assay and immunorecognition of the estradiol-receptor complex by the monoclonal antibody D547 raised against the human estrogen receptor. After RNAase treatment at 4 degrees C or 25 degrees C the binding of the receptor to DNA-cellulose doubled. This stimulation was partially prevented by sodium molybdate. RNAase treatment did not modify the interaction of the receptor with the monoclonal antibody D547; this antibody, as was demonstrated previously, selectively recognizes the activated form of the receptor when activation has been induced by temperature, time or high salt concentrations. In addition, RNAase had little or no effect on the transformation of the 8-9 S receptor to more slowly sedimenting forms under low salt concentrations. These observations suggest that even if RNAase induces receptor activation, which can be inferred from the increase in its binding to DNA-cellulose, the conformational modifications of the receptor molecule involved in this process are apparently different from those induced by factors such as temperature, time or high-salt concentrations.     

Dopaminergic activation of estrogen receptors induces fos expression within restricted regions of the neonatal female rat brain

Steroid receptor activation in the developing brain influences a variety of cellular processes that endure into adulthood, altering both behavior and physiology. Recent data suggests that dopamine can regulate expression of progestin receptors within restricted regions of the developing rat brain by activating estrogen receptors in a ligand-independent manner. It is unclear whether changes in neuronal activity induced by dopaminergic activation of estrogen receptors are also region specific. To investigate this question, we examined where the dopamine D1-like receptor agonist, SKF 38393, altered Fos expression via estrogen receptor activation. We report that dopamine D1-like receptor agonist treatment increased Fos protein expression within many regions of the developing female rat brain. More importantly, prior treatment with an estrogen receptor antagonist partially reduced D1-like receptor agonist-induced Fos expression only within the bed nucleus of the stria terminalis and the central amygdala. These data suggest that dopaminergic activation of estrogen receptors alters neuronal activity within restricted regions of the developing rat brain. This implies that ligand-independent activation of estrogen receptors by dopamine might organize a unique set of behaviors during brain development in contrast to the more wide spread ligand activation of estrogen receptors by estrogen.     

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.     

Follicle-stimulating hormone promotes the transformation of cholesterol to estrogen in mouse adipose tissue

Follicle-stimulating hormone (FSH) levels increase estrogen biosynthesis in obese menopausal women. Ovariectomized mice and 3T3-L1 cells were used to explore estrogen biosynthesis in the decline of ovarian function. After ovariectomy, lipid deposition, and FSH and estrogen levels changed, and feed intake increased significantly. In mouse adipose tissue, FSH was found to have a role in accelerating lipid deposition via the peroxisome proliferators-activated receptor pathway, and in inducing estrogen biosynthesis via the steroid hormone metabolism pathway. Furthermore, FSH bound to the FSH receptor promoted CREB phosphorylation, which was activated by cAMP-PKA. Moreover, pCREB could up-regulate PPARγ and SREBP2 mRNA levels, resulting in an increased transformation of cholesterol to estrogen. Overall, this study shows that FSH induces fat deposition and promotes the transformation of cholesterol to estrogen through CREB activation by cAMP-PKA in mouse adipose tissue. Our findings provide a new understanding of menopause treatment.     

Gene expression analysis reveals a signature of estrogen receptor activation upon loss of Pten in a mouse model of endometrial cancer

Loss of PTEN is the earliest detectable genetic lesion in the endometrioid subtype of endometrial cancer (EEC), a tumor thought to be associated with an increase in unopposed estrogen activity. Pten(+/-) mice develop endometrial neoplastic lesions with full penetrance, despite having normal estrogen levels. We have utilized oligonucleotide arrays to identify the alterations in gene expression patterns associated with loss of Pten and consequent neoplastic transformation of the endometrium. We show that 487 and 330 genes are substantially up- and downregulated, respectively, in Pten(+/-) mice. Several genes whose expression levels are impacted by loss of Pten are associated with pathways and functions that are relevant to the transformation and progression processes. Strikingly, we found that the expression levels of over 100 genes known to be regulated by estrogen receptor alpha (ERalpha) are also altered in the neoplastic uterus from Pten(+/-) mice, thus mimicking a hyperestrogenic environment. These results provide in vivo evidence supporting the hypothesis that loss of Pten and subsequent Akt activation result in the activation of several ERalpha-dependent pathways that, mimicking increased estrogen signaling, may play a pivotal role in the neoplastic process.     

Estrogenic Regulation of Histamine Receptor Subtype H1 Expression in the Ventromedial Nucleus of the Hypothalamus in Female Rats

Female sexual behavior is controlled by central estrogenic action in the ventromedial nucleus of the hypothalamus (VMN). This region plays a pivotal role in facilitating sex-related behavior in response to estrogen stimulation via neural activation by several neurotransmitters, including histamine, which participates in this mechanism through its strong neural potentiating action. However, the mechanism through which estrogen signaling is linked to the histamine system in the VMN is unclear. This study was undertaken to investigate the relationship between estrogen and histamine receptor subtype H1 (H1R), which is a potent subtype among histamine receptors in the brain. We show localization of H1R exclusively in the ventrolateral subregion of the female VMN (vl VMN), and not in the dorsomedial subregion. In the vl VMN, abundantly expressed H1R were mostly colocalized with estrogen receptor α. Intriguingly, H1R mRNA levels in the vl VMN were significantly elevated in ovariectomized female rats treated with estrogen benzoate. These data suggest that estrogen can amplify histamine signaling by enhancing H1R expression in the vl VMN. This enhancement of histamine signaling might be functionally important for allowing neural excitation in response to estrogen stimulation of the neural circuit and may serve as an accelerator of female sexual arousal.     

Cytosol and nuclear estrogen receptor binding in the preoptic area and hypothalamus of female rats during pregnancy and ovariectomized, nulliparous rats after steroid priming: correlation with maternal behavior

In a previous study, high nuclear estrogen receptor concentrations in the preoptic area (POA) were found on Day 16 of pregnancy to prime females to respond to a subsequent low dose of estradiol benzoate (EB) after hysterectomy-ovariectomy by exhibiting maternal behavior in 48 hr. Receptor concentrations in the POA were found to be higher than those in the hypothalamus (HYP). The present study investigated when nuclear estrogen receptors increase during pregnancy in POA and when the difference in receptor concentrations between POA and HYP occurs. An attempt was made to reproduce these pregnancy changes with a 16-day treatment of estrogen and progesterone in ovariectomized (OVX), nulliparous rats. In Experiment 1, we measured cytosol and nuclear estrogen receptor concentrations in the POA and HYP of female rats during pregnancy. Nuclear receptor concentrations in the POA increased beginning on Day 10, increased again on Day 16, and continued at this high level for the remainder of pregnancy. Nuclear estrogen receptor concentrations in the HYP remained at a lower level throughout most of pregnancy until Day 22 when they increased significantly. In Experiment 2, we tested the maternal behavior and measured estrogen receptor concentrations in OVX, steroid-primed, nulliparous rats after hysterectomy (H) and EB treatment. While 90% of estradiol (E) + progesterone (P)-primed females displayed short-latency maternal behavior 48 hr after H and EB treatment, 46% of E + vehicle (V)-treated controls were maternal. At 0 hr (prior to H and EB treatment), there was a significantly larger nuclear receptor accumulation in the POA but significantly attenuated receptor binding in the HYP. P treatment significantly affected cytosol and nuclear estrogen receptor dynamics. Differences in nuclear estrogen receptor concentrations were shown to be based on the number of available binding sites and not to changes in receptor affinity for estradiol.     

Immunological differences between the estradiol-, tamoxifen- and 4-hydroxy-tamoxifen-estrogen receptor complexes detected by two monoclonal antibodies

Two monoclonal antibodies (D547 and H222), obtained against the estrogen receptor from MCF-7 breast cancer cells, were used to study the estrogen receptor from fetal guinea-pig uterus bound to estradiol or to the antiestrogens tamoxifen and 4-hydroxytamoxifen. The estradiol-receptor complex binds partially to the monoclonal antibody D547, shifting its sedimentation coefficient in high salt sucrose density gradients from 4.5S to 7.5S. Recently, we demonstrated that the form selectively recognized by this monoclonal antibody is the activated form of the receptor. The estrogen receptor complexed with tamoxifen or 4-hydroxytamoxifen is also partially recognized by this monoclonal antibody but the fraction of total receptor bound to the antibody is significantly less than for the receptor complexed with estradiol. Another series of experiments showed that the monoclonal antibody H222, which recognizes a different antigenic site on the receptor molecule, binds all the estradiol-receptor complex (independently of the degree of activation), shifting its sedimentation coefficient to 7.5S. However, even if all the 4-hydroxytamoxifen-receptor complex is bound by this antibody, only a fraction of the receptor is recognized when it is complexed with tamoxifen. These data show different interactions between the estradiol-, tamoxifen- and 4-hydroxytamoxifen-receptor complexes and the two monoclonal antibodies tested and suggest that these compounds induce different conformational modifications of the estrogen receptor molecule.     

Hormone independent activation of rat uterine estrogen receptor by exposure of isolated uteri to anaerobic conditions

Incubation of isolated rat uteri under anaerobic conditions, which consisted of either an atmosphere of carbon monoxide or nitrogen, caused an increase in nuclear estrogen binding which was not dependent on added estrogen. The incubation of uteri in the absence of added estrogen under aerobic conditions (atmosphere of oxygen or oxygen-carbon dioxide [95-5%]) did not increase uterine nuclear estrogen binding levels. High salt (0.5-M KCl) extracts of the nuclear estrogen binding moiety induced by anaerobiosis were shown to possess a sedimentation coefficient on sucrose-glycerol gradients of 4.8S, a binding specificity restricted to estrogens and an apparent affinity constant of 1.35 nM. These data confirm that the nuclear binding moiety induced by anaerobiosis possesses the characteristics of an estrogen receptor. The enhanced nuclear estrogen receptor retention induced under anaerobic conditions could be accounted for by a significant increase in nuclear receptor extracted by high salt (0.5 M KCl) and by ethanol (salt resistant fraction). Furthermore, sequential extraction of nuclear estrogen receptor from uteri exposed to aerobic conditions in the presence of added estradiol paralleled the results obtained with anaerobiosis. Total receptor retained under anaerobiosis represented 25% of that observed under aerobic conditions in the presence of estrogen. These results indicate that anaerobic conditions can cause an activation of uterine estrogen receptor. This activation process represents a pathway for receptor activation which does not require steroid.     

Raloxifene and desmethylarzoxifene block estrogen-induced malignant transformation of human breast epithelial cells

There is association between exposure to estrogens and the development and progression of hormone-dependent gynecological cancers. Chemical carcinogenesis by catechol estrogens derived from oxidative metabolism is thought to contribute to breast cancer, yet exact mechanisms remain elusive. Malignant transformation was studied in MCF-10A human mammary epithelial cells, since estrogens are not proliferative in this cell line. The human and equine estrogen components of estrogen replacement therapy (ERT) and their catechol metabolites were studied, along with the influence of co-administration of selective estrogen receptor modulators (SERMs), raloxifene and desmethyl-arzoxifene (DMA), and histone deacetylase inhibitors. Transformation was induced by human estrogens, and selectively by the 4-OH catechol metabolite, and to a lesser extent by an equine estrogen metabolite. The observed estrogen-induced upregulation of CYP450 1B1 in estrogen receptor negative MCF-10A cells, was compatible with a causal role for 4-OH catechol estrogens, as was attenuated transformation by CYP450 inhibitors. Estrogen-induced malignant transformation was blocked by SERMs correlating with a reduction in formation of nucleobase catechol estrogen (NCE) adducts and formation of 8-oxo-dG. NCE adducts can be formed consequent to DNA abasic site formation, but NCE adducts were also observed on incubation of estrogen quinones with free nucleotides. These results suggest that NCE adducts may be a biomarker for cellular electrophilic stress, which together with 8-oxo-dG as a biomarker of oxidative stress correlate with malignant transformation induced by estrogen oxidative metabolites. The observed attenuation of transformation by SERMs correlated with these biomarkers and may also be of clinical significance in breast cancer chemoprevention.     

Conformational and electrostatic properties of unoccupied and liganded estrogen receptors determined by aqueous two-phase partitioning

The technique of aqueous two-phase partitioning (ATPP) has been used to characterize conformational and electrostatic properties of unoccupied and liganded rat uterine estrogen receptors. The adaptation of the hydroxylapatite receptor assay with ATPP systems has permitted estrogen receptor (ER) partition coefficients to be accurately determined, even when the partitioning process results in significant loss of ER binding capacity. The pH and salt dependences of estrogen receptor partition coefficients indicate that the theory governing partitioning behavior can be accurately applied to partitioning data obtained with crude cytosols. This technique has revealed a ligand-induced change in the properties of the unoccupied receptor that precedes the process of heat-induced transformation in vitro. The difference in partitioning behavior between unoccupied and nontransformed estrogen receptor is observed in all combinations of buffers and salts tested and is of equal magnitude as the difference between partition coefficients of nontransformed and transformed ER. The partition coefficients of both unoccupied and nontransformed ER are constant over the ER concentration range in which binding cooperativity has been previously demonstrated. The combined effects of salt and pH on ER partition coefficients indicate a pI of approximately 5.5 for both unoccupied and nontransformed estrogen receptors. However, the partition coefficients at the pI differ. It is concluded that estradiol binding to its unoccupied receptor results in a change in surface properties of the ER monomer that is independent of receptor transformation and makes the receptor less hydrophobic.