We have cloned a novel member of the nuclear receptor superfamily. The cDNA of clone 29 was isolated from a rat prostate cDNA library and it encodes a protein of 485 amino acid residues with a calculated molecular weight of 54.2 kDa. Clone 29 protein is unique in that it is highly homologous to the rat estrogen receptor (ER) protein, particularly in the DNA-binding domain (95%) and in the C-terminal ligand-binding domain (55%). Expression of clone 29 in rat tissues was investigated by in situ hybridization and prominent expression was found in prostate and ovary. In the prostate clone 29 is expressed in the epithelial cells of the secretory alveoli, whereas in the ovary the granuloma cells in primary, secondary, and mature follicles showed expression of clone 29. Saturation ligand-binding analysis of in vitro synthesized clone 29 protein revealed a single binding component for 17beta-estradiol (E2) with high affinity (Kd= 0.6 nM). In ligand-competition experiments the binding affinity decreased in the order E2 > diethylstilbestrol > estriol > estrone > 5alpha-androstane-3beta,17beta-diol >> testosterone = progesterone = corticosterone = 5alpha-androstane-3alpha,17beta-diol. In cotransfection experiments of Chinese hamster ovary cells with a clone 29 expression vector and an estrogen-regulated reporter gene, maximal stimulation (about 3-fold) of reporter gene activity was found during incubation with 10 nM of E2. Neither progesterone, testosterone, dexamethasone, thyroid hormone, all-trans-retinoic acid, nor 5alpha-androstane-3alpha,I7beta-diol could stimulate reporter gene activity, whereas estrone and 5alpha-androstane-3beta,17beta-diol did. We conclude that clone 29 cDNA encodes a novel rat ER, which we suggest be named rat ERbeta to distinguish it from the previously cloned ER (ERalpha) from rat uterus.

https://www.pnas.org/content/pnas/93/12/5925.full.pdf

Cloning of a novel receptor expressed in rat prostate and ovary.

The rat estrogen receptor (ER) exists as two subtypes, ER alpha and ER beta, which differ in the C-terminal ligand binding domain and in the N-terminal transactivation domain. In this study we investigated the messenger RNA expression of both ER subtypes in rat tissues by RT-PCR and compared the ligand binding specificity of the ER subtypes. Saturation ligand binding analysis of in vitro synthesized human ER alpha and rat ER beta protein revealed a single binding component for 16 alpha-iodo-17 beta-estradiol with high affinity [dissociation constant (Kd) = 0.1 nM for ER alpha protein and 0.4 nM for ER beta protein]. Most estrogenic substances or estrogenic antagonists compete with 16 alpha-[125I]iodo-17 beta-estradiol for binding to both ER subtypes in a very similar preference and degree; that is, diethylstilbestrol > hexestrol > dienestrol > 4-OH-tamoxifen > 17 beta-estradiol > coumestrol, ICI-164384 > estrone, 17 alpha-estradiol > nafoxidine, moxestrol > clomifene > estriol, 4-OH-estradiol > tamoxifen, 2-OH-estradiol, 5-androstene-3 beta, 17 beta-diol, genistein for the ER alpha protein and dienestrol > 4-OH-tamoxifen > diethylstilbestrol > hexestrol > coumestrol, ICI-164384 > 17 beta-estradiol > estrone, genistein > estriol > nafoxidine, 5-androstene-3 beta, 17 beta-diol > 17 alpha-estradiol, clomifene, 2-OH-estradiol > 4-OH-estradiol, tamoxifen, moxestrol for the ER beta protein. The rat tissue distribution and/or the relative level of ER alpha and ER beta expression seems to be quite different, i.e. moderate to high expression in uterus, testis, pituitary, ovary, kidney, epididymis, and adrenal for ER alpha and prostate, ovary, lung, bladder, brain, uterus, and testis for ER beta. The described differences between the ER subtypes in relative ligand binding affinity and tissue distribution could contribute to the selective action of ER agonists and antagonists in different tissues.

Comparison of the ligand binding specificity and transcript tissue distribution of estrogen receptors alpha and beta

Background and Methods Mutations in the estrogen-receptor gene have been thought to be lethal. A 28-year-old man whose estrogen resistance was caused by a disruptive mutation in the estrogen-receptor gene underwent studies of pituitary-gonadal function and bone density and received transdermal estrogen for six months. Estrogen-receptor DNA, extracted from lymphocytes, was evaluated by analysis of single-strand-conformation polymorphisms and by direct sequencing. Results The patient was tall (204 cm [80.3 in.]) and had incomplete epiphyseal closure, with a history of continued linear growth into adulthood despite otherwise normal pubertal development. He was normally masculinized and had bilateral axillary acanthosis nigricans. Serum estradiol and estrone concentrations were elevated, and serum testosterone concentrations were normal. Serum follicle-stimulating hormone and luteinizing hormone concentrations were increased. Glucose tolerance was impaired, and hyperinsulinemia was present. The bone mineral d...

https://www.nejm.org/doi/pdf/10.1056/NEJM199410203311604

Estrogen Resistance Caused by a Mutation in the Estrogen-Receptor Gene in a Man

All scientific investigations begin with distinct objectives: first is the hypothesis upon which studies are undertaken to disprove, and second is the overall aim of obtaining further information, from which future and more precise hypotheses may be drawn Studies focusing on the generation and use of gene-targeted animal models also apply these goals and may be loosely categorized into sequential phases that become apparent as the use of the model progresses Initial studies of knockout models often focus on the plausibility of the model based on prior knowledge and whether the generation of an animal lacking the particular gene will prove lethal or not Upon the successful generation of a knockout, confirmatory studies are undertaken to corroborate previously established hypotheses of the function of the disrupted gene product As these studies continue, observations of unpredicted phenotypes or, more likely, the lack of a phenotype that was expected based on models put forth from past investigations are noted Often the surprising phenotype is due to the loss of a gene product that is downstream from the functions of the disrupted gene, whereas the lack of an expected phenotype may be due to compensatory roles filled by alternate mechanisms As the descriptive studies of the knockout continue, use of the model is often shifted to the role as a unique research reagent, to be used in studies that 1) were not previously possible in a wild-type model; 2) aimed at finding related proteins or pathways whose existence or functions were previously masked; or 3) the subsequent effects of the gene disruption on related physiological and biochemical systems The alpha ERKO mice continue to satisfy the confirmatory role of a knockout quite well As summarized in Table 4, the phenotypes observed in the alpha ERKO due to estrogen insensitivity have definitively illustrated several roles that were previously believed to be dependent on functional ER alpha, including 1) the proliferative and differentiative actions critical to the function of the adult female reproductive tract and mammary gland; 2) as an obligatory component in growth factor signaling in the uterus and mammary gland; 3) as the principal steroid involved in negative regulation of gonadotropin gene transcription and LH levels in the hypothalamic-pituitary axis; 4) as a positive regulator of PR expression in several tissues; 5) in the positive regulation of PRL synthesis and secretion from the pituitary; 6) as a promotional factor in oncogene-induced mammary neoplasia; and 7) as a crucial component in the differentiation and activation of several behaviors in both the female and male The list of unpredictable phenotypes in the alpha ERKO must begin with the observation that generation of an animal lacking a functional ER alpha gene was successful and produced animals of both sexes that exhibit a life span comparable to wild-type The successful generation of beta ERKO mice suggests that this receptor is also not essential to survival and was most likely not a compensatory factor in the survival of the alpha ERKO In support of this is our recent successful generation of double knockout, or alpha beta ERKO mice of both sexes The precise defects in certain components of male reproduction, including the production of abnormal sperm and the loss of intromission and ejaculatory responses that were observed in the alpha ERKO, were quite surprising In turn, certain estrogen pathways in the alpha ERKO female appear intact or unaffected, such as the ability of the uterus to successfully exhibit a progesterone-induced decidualization response, and the possible maintenance of an LH surge system in the hypothalamus [ABSTRACT TRUNCATED]

Estrogen receptor null mice: what have we learned and where will they lead us?

Our appreciation of the physiological functions of estrogens and the mechanisms through which estrogens bring about these functions has changed during the past decade. Just as transgenic mice were produced in which estrogen receptors had been inactivated and we thought that we were about to understand the role of estrogen receptors in physiology and pathology, it was found that there was not one but two distinct and functional estrogen receptors, now called ERα and ERβ. Transgenic mice in which each of the receptors or both the receptors are inactive have revealed a much broader role for estrogens in the body than was previously thought. This decade also saw the description of a male patient who had no functional ERα and whose continued bone growth clearly revealed an important function of estrogen in men. The importance of estrogen in both males and females was also demonstrated in the laboratory in transgenic mice in which the aromatase gene was inactivated. Finally, crystal structures of the estrogen r...

Mechanisms of Estrogen Action

Estrogen receptor and its ligand, estradiol, have long been thought to be essential for survival, fertility, and female sexual differentiation and development. Consistent with this proposed crucial role, no human estrogen receptor gene mutations are known, unlike the androgen receptor, where many loss of function mutations have been found. We have generated mutant mice lacking responsiveness to estradiol by disrupting the estrogen receptor gene by gene targeting. Both male and female animals survive to adulthood with normal gross external phenotypes. Females are infertile; males have a decreased fertility. Females have hypoplastic uteri and hyperemic ovaries with no detectable corpora lutea. In adult wild-type and heterozygous females, 3-day estradiol treatment at 40 micrograms/kg stimulates a 3- to 4-fold increase in uterine wet weight and alters vaginal cornification, but the uteri and vagina do not respond in the animals with the estrogen receptor gene disruption. Prenatal male and female reproductive tract development can therefore occur in the absence of estradiol receptor-mediated responsiveness.

https://www.pnas.org/content/pnas/90/23/11162.full.pdf

Alteration of reproductive function but not prenatal sexual development after insertional disruption of the mouse estrogen receptor gene

We employed homologous recombination in mouse embryonic stem cells to disrupt the estrogen receptor (ER) gene. Subsequently generated mice that are homozygous for the gene disruption, termed ERKO, possess no demonstrable wild-type ER by Western blot analysis. However, the presence of residual high affinity binding, as detected by [3H]estradiol binding assays and sucrose gradients in uterine extracts from ERKO females prompted further investigation of transcription and translation products from the disrupted ER gene. Analysis of ERKO uterine messenger RNA (mRNA) by reverse transcriptase-polymerase chain reaction demonstrated that although no full-length wild-type ER mRNA was present, two smaller transcripts, labeled E1 and E2, were identified and partially sequenced. Both ERKO transcripts are splicing variants that result in the disrupting NEO sequence being partially or completely removed from the mRNA. In the ERKO-E2 variant, this results in a frame shift and the creation of at least two stop codons downstream. In the ERKO-E1 variant, the ER reading frame is preserved and encodes for a smaller mutant ER that could be the source of the residual estradiol binding. When this mutant form is overexpressed and characterized in vitro, it results in a smaller protein of the predicted size that possesses significantly reduced estrogen-dependent transcriptional activity compared with that of the wild-type ER. Despite residual amounts of an impaired ER variant, estrogen insensitivity in the female ERKOs was confirmed by the failure of estrogen treatment to induce known uterine markers of estrogen action, such as increased DNA synthesis, and transcription of the progesterone receptor, lactoferrin, and glucose-6-phosphate dehydrogenase genes. Furthermore, serum levels of estradiol in the ERKO female are more than 10-fold higher than those in the wild type, consistent with a syndrome of hormone insensitivity.

Analysis of transcription and estrogen insensitivity in the female mouse after targeted disruption of the estrogen receptor gene.

Increased proteolytic degradation of the estrogen receptor (ER) was detected in uterine cytosol of estradioltreated ovariectomized mice compared to saline controls. Estradiol had no direct effect on the proteinase activity or susceptibility of the ER to the enzyme. The proteolytic activity gradually increased after a single injection of estradiol with early increases at 2 and 8 h followed by a progressive increase which reached a maximum at 36 h. The proteinase(s) activity resulted in cleavage of the native ER form of 65,000 (65 K ER) to a product of limited proteolysis having an apparent molecular weight of 54,000 (54 K ER). The pH optimum for this proteinase activity was 6.0. The proteinase was inhibited by 2.5 mM p-chloromercuribenzoic acid and 2.5 mM p-chloromercuriphenylsulfonate and partially inhibited by 2.5 mM iodoacetamide but not by 1 mg/ml leupeptin, 0.1 mg/ml antipain, 0.1 mg/ml chymostatin, 0.1 mg/ml pepstatin, 0.1 mg/ml E-64, 2.5 mg/ml soybean trypsin inhibitor, 2.5 mM phenylmethylsulfonylfl...

Estradiol-stimulated proteolytic cleavage of the estrogen receptor in mouse uterus.

Mouse uterine estrogen receptor (ER) was purified about 11,000-fold from normal mouse uteri by affinity chromatography. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the purified ER demonstrated a major component of 65,000 mol wt with minor fragments of the 54,000- and 37,000-dalton species, as judged by affinity labeling with [3H]tamoxifen aziridine and immunodetection with an ER monoclonal antibody (H-222). The minor fragments were not detected with additional monoclonals (H-226 or D-547), which recognize different domains on the ER molecule. Two-dimensional gel electrophoresis revealed that the major component with a mol wt of 65,000 had a pI of about 6.5. The 54,000- and 37,000-dalton components had similar pI values. Saturation binding and Scatchard plot analysis of purified ER yielded one class of binding sites with an apparent dissociation constant of about 1.4 nM. Changes in the hormonal status resulted in changes in the size of the ER even in the presence of molybdate and leupeptin.

Purification and Characterization of Mouse Uterine Estrogen Receptor under Conditions of Varying Hormonal Status

Holomeric estrogen receptor (ER) prepared from ovariectomized mouse uteri displays heterogeneous electrophoretic mobility when analyzed by NaDodSO4/PAGE. ER derived from nuclei (ERn) appears as a closely spaced doublet having apparent molecular masses of 66.4 and 65 kDa, while ER from the cytosolic compartment (ERc) has a single band of 65 kDa. Both partially purified ERc and the 8S form of unactivated ERc show only the 65-kDa band. The appearance of the ERn doublet is hormonally inducible, and the relative proportions of the two doublet bands are influenced by the type of hormone treatment, with weakly estrogenic compounds yielding the lower band as predominant while potent estrogens increase the proportion of the upper band. Steroid binding of the ERn doublet was determined by [3H]tamoxifen aziridine affinity labeling of both the 66.4- and the 65-kDa peptides; binding to the 65-kDa peptide was predominant. The ERn doublet displays a time dependency after estrogen administration with maximal amounts occurring in a bimodal fashion at 1 and 8 hr.

Thomas S. Golding

Papers

Alteration of reproductive function but not prenatal sexual development after insertional disruption of the mouse estrogen receptor gene

Analysis of transcription and estrogen insensitivity in the female mouse after targeted disruption of the estrogen receptor gene.

Estradiol-stimulated proteolytic cleavage of the estrogen receptor in mouse uterus.

Purification and Characterization of Mouse Uterine Estrogen Receptor under Conditions of Varying Hormonal Status

Nuclear estrogen receptor molecular heterogeneity in the mouse uterus.