Biomedical subjects

B S Katzenellenbogen

Publications and source records attributed to B S Katzenellenbogen.

At least 91 records · Page 5Linked to original sources

PubMed1994

Phosphorylation of the human estrogen receptor. Identification of hormone-regulated sites and examination of their influence on transcriptional activity.

We have used a transient transfection system with a cytomegalovirus-based vector expressing high levels of biologically active human estrogen receptor (ER) in COS-1 cells to study the phosphorylation of human ER and to identify major hormone-regulated phosphorylation sites. The features of phosphorylation of the wild-type ER were very similar to those previously observed for the endogenous ER in uterine cells: The ER exhibited a basal level of phosphorylation which was increased approximately 3-4-fold by estrogen (estradiol) and by antiestrogens (hydroxytamoxifen and ICI164,384), and phosphorylation was increased to an almost similar extent by activation of either protein kinase A or C signal transduction pathways with cholera toxin plus isobutyl methylxanthine (CT+IBMX) or phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA), respectively. Phosphoamino acid analysis revealed that the phosphorylation occurred exclusively on serine residues in all cases. Tryptic phosphopeptide analysis of ER, using a two-dimensional peptide mapping procedure, revealed similar patterns for ER in cells treated with estradiol, antiestrogens or TPA; with CT+IBMX treatment, the same phosphopeptides were seen, but the relative phosphorylation of the different ER phosphotryptic peptides differed. In ER deleted of the NH2-terminal A and B (A/B) domains, estrogen and antiestrogen-stimulated phosphorylations were abolished, while the phosphorylation induced by CT+IBMX was maintained. This suggests that sites of phosphorylation enhanced by estradiol and antiestrogen, but not those induced by CT+IBMX, are located in the A/B domain. These results were further confirmed by comparing the tryptic phosphopeptide patterns of wild-type and A/B-deleted receptor upon estradiol and CT+IBMX treatments, and then by site-directed mutagenesis, by substituting alanines for the serine residues in the A/B domain (Ser104, Ser106, Ser118, Ser154, and Ser167) involved in known protein kinase consensus sequences. Comparison of the tryptic phosphopeptide patterns of wild-type ER and these mutant ERs allowed us to identify serine 104 and/or serine 106 and serine 118, all three being part of a serine-proline motif, the preferred substrate of proline-directed protein kinase, as major ER phosphorylation sites. When tested with two estrogen-responsive reporter gene constructs in several cell types, the mutant S104A, S106A, S118A showed a approximately 40% reduction in transactivation activity in response to E2, while the mutants S118A and S104A, S106A alone showed a approximately 15% decrease in transactivation. Our studies identify several serines in the NH2-terminal portion of the human ER as being major hormone-regulated phosphorylation sites.(ABSTRACT TRUNCATED AT 250 WORDS)

Consensus Sequence↗

PubMed1994

Bivalent ligands as probes of estrogen receptor action.

The estrogen receptor (ER) is a hormone-regulated transcription factor which is thought to bind to specific DNA sequences as a homodimer. In order to better understand structural requirements for dimerization and its functional role in ER action, we synthesized a series of bivalent ligands based on the non-steroidal estrogen hexestrol. These molecular probes join two hexestrol molecules of the erythro (E, active) configuration with either 4 or 8 carbon linkers (designated E-4-E and E-8-E series, respectively), or with longer linkers comprised of ethylene glycol units (E-eg-E series). Several other bi- and monovalent control compounds were prepared. The bivalent ligands bind to ER with a relative affinity 1-7% that of estradiol. While most of the ligands demonstrated normal monophasic displacement curves in competitive binding assays with [3H]estradiol, uncharacteristic biphasic competitive binding curves were seen for some of the ligands, indicating possible structure-specific, negative site-site interaction. In ER-deficient Chinese hamster ovary (CHO) cells transfected with an expression vector encoding ER, one series of bivalent ligands (E-4-E) had little stimulatory activity and inhibited transcription stimulated by hexestrol, as determined by a transient transfection assay using an estrogen-responsive reporter gene construct [(ERE)2-TATA-CAT, containing two estrogen response elements linked to a TATA promoter and the chloramphenicol acetyl transferase reporter gene]. Monovalent or control bivalent ligands failed to antagonize hexestrol-stimulated activity and were as fully active as hexestrol itself. Studies performed in MCF-7 human breast cancer cells, which contain endogenous ER, yielded similar bioactivity profiles for the E-4-E bivalent inhibitory ligands, showing them to be effective estrogen antagonists, when using either induction of progesterone receptor or (ERE)2-TATA-CAT transcriptional activation as the endpoint. The E-8-E ligand, however, acted as a partial agonist/antagonist of ERE-reporter gene transactivation and a full agonist of progesterone receptor induction in MCF-7 cells, thus showing cell- and response-specific differences in the effects of this bivalent ligand. These bivalent ligands for ER do not show enhanced potency or receptor binding affinity; however, some of them display binding properties that suggest the possibility of structure-specific negative site-site interaction, and some of them function as quite effective estrogen antagonists.

Animals↗

PubMed1994

Regulation of progesterone receptor gene expression in MCF-7 breast cancer cells: a comparison of the effects of cyclic adenosine 3',5'-monophosphate, estradiol, insulin-like growth factor-I, and serum factors.

We have compared regulation of progesterone receptor (PR) gene expression in MCF-7 human breast cancer cells by cAMP and that by estradiol (E2) and insulin-like growth factor-I (IGF-I). Treatment of cells with 8-bromo-cAMP or agents known to activate protein kinase-A, namely cholera toxin plus 3-isobutyl-1-methylxanthine (CT plus IBMX; which increased intracellular cAMP > 10-fold) evoked an increase in PR protein levels as did treatment with IGF-I or E2. Increases in PR caused by IGF-I were not accompanied by increases in PR mRNA, whereas PR mRNA levels were markedly induced by E2 and CT plus IBMX, showing regulation at different levels by these agents. The increases in PR mRNA evoked by E2 or CT plus IBMX were almost completely abolished by treatment with antiestrogen. Treatment with cycloheximide inhibited CT- plus IBMX-mediated PR mRNA stimulation, but not the induction of E2, indicating that the PR mRNA response to cAMP is not a primary one and probably requires de novo protein synthesis. Distinct effects of serum were observed on the expression of PR in MCF-7 cells. PR mRNA and protein were consistently elevated when cells were cultured under low serum conditions (0-0.5% charcoal dextran-treated calf serum) and were reduced as the serum concentration was increased, suggesting that a serum factor(s) repress constitutive PR levels. Also, the ability of CT plus IBMX to stimulate PR declined markedly for cells grown in medium containing higher (5%) serum levels; by contrast, the ability of E2 to induce PR was increased at the higher serum concentration. Thus, unknown serum factors interfere with the action of cAMP in up-regulating PR, whereas serum factors are important for the effectiveness of E2 in stimulating PR. These observations indicate that regulation of PR occurs at different levels by several factors (cAMP, E2, and IGF-I) and imply that cAMP, serum factors, and growth factors, such as IGF-I, in addition to E2 will be of importance in determining PR levels and, hence, cell sensitivity to progestins.

1-Methyl-3-isobutylxanthine↗

PubMed1994

Activation of transcriptionally inactive human estrogen receptors by cyclic adenosine 3',5'-monophosphate and ligands including antiestrogens.

We show that some transcriptionally inactive human estrogen receptor (ER) mutants can be activated by 17 beta-estradiol (E2), and sometimes by antiestrogens, in the presence of elevated levels of intracellular cAMP. ER-deficient Chinese hamster ovary or 3T3 mouse fibroblast cells were transfected with mutant ERs (the point mutant L540Q, the frameshift mutant S554fs, or the carboxy-terminal truncated receptor ER1-530) and various estrogen response element-containing reporter genes. Individual treatments with E2, the antiestrogens trans-hydroxytamoxifen and ICI 164,384, or with 3-isobutyl-1-methyl-xanthine plus cholera toxin (IBMX plus CT) which raise intracellular cAMP, generally do not activate the mutant receptors. However, cotreatment with IBMX/CT and one of the three ligands (E2, trans-hydroxytamoxifen, or ICI164,384) results in the unexpected recovery of strong activation of the L540Q or S554fs receptors, the magnitude of which is dependent upon promoter- and cell-contexts. Unlike L540Q and S554fs, the transcriptionally inactive ER1-530 is not activated by any combination of ligands and IBMX/CT. These data demonstrate that some ER mutants that form transcriptionally nonproductive ER-E2 complexes can be successfully activated by the combination of an agonist or antagonist ligand and an agent thought to act via phosphorylation pathways. Also highlighted is the promoter- and cell-specific nature of the transcriptional response to different ligand-ER complexes. Lastly, the enhanced transcriptional activity of wild type ER and some ER mutants in the presence of antiestrogens and elevated intracellular cAMP may provide a partial explanation of the ability of some estrogen-dependent human breast tumors to resist antiestrogen therapies currently employed.

1-Methyl-3-isobutylxanthine↗

PubMed1994

Alteration in the agonist/antagonist balance of antiestrogens by activation of protein kinase A signaling pathways in breast cancer cells: antiestrogen selectivity and promoter dependence.

We find that stimulation of the protein kinase A (PKA) signaling pathway in MCF-7 human breast cancer cells changes the agonist/antagonist activity of tamoxifen and related antiestrogens; it activates or enhances their estrogen agonist activity and reduces their ability to antagonize the effects of estradiol (E2). In MCF-7 human breast cancer cells which contain high levels of endogenous estrogen receptor (ER), the antiestrogen trans-hydroxy-tamoxifen (TOT) fails to stimulate transcription of the estrogen-responsive promoter-reporter constructs estrogen response element (ERE)-TATA-chloramphenicol acetyl transferase (CAT), (ERE)2-TATA-CAT, and pS2-CAT. However, when cells are treated with isobutyl methylxanthine plus cholera toxin (which increases intracellular cAMP approximately 10-fold), or with 8-bromo-cAMP, or are transfected with expression vectors for the PKA catalytic subunits, the transcriptional activity of the antiestrogen-ER complex is now increased, to levels 20-75% that of E2, and TOT also becomes much less effective in antagonizing the stimulation of transcription by E2. Although this alteration in the agonist and antagonist activity of TOT is observed with three promoter-reporter constructs, containing a simple TATA promoter or a more complex, pS2 promoter, elevation of cAMP did not enhance the transcription by either TOT or E2 of the reporter plasmid ERE-thymidine kinase-CAT. Thus, this phenomenon is promoter specific. The maximal stimulatory effects of isobutylmethylxanthine plus cholera toxin and PKA catalytic subunits on TOT and E2 transcriptional enhancement were not additive, consistent with the hypothesis that they are both acting via stimulation of the same signal transduction pathway.(ABSTRACT TRUNCATED AT 250 WORDS)

1-Methyl-3-isobutylxanthine↗

PubMed1994

Identification of multiple, widely spaced estrogen-responsive regions in the rat progesterone receptor gene.

Progesterone receptors (PRs) mediate the actions of progestin hormones and play important roles during the reproductive cycle and pregnancy. Since PR expression is known to be regulated by estrogen, we have undertaken studies to examine the mechanisms underlying this regulation. We have identified multiple distinct regions of the rat PR gene, widely spaced and spread throughout the 5'-flanking region, the 5'-untranslated region, and the first exon (between -2264 and +2241), that can form a strong estrogen-responsive enhancer when linked together. Estrogen-responsive activities for two of the regions in isolation (+461/+636 and +2176/+2241) were demonstrated in one or more homologous or heterologous promoter contexts. The contributions of the other regions (-2264/-1970, -1167/-957 and +2088/+2110) to the overall activity of the assembled enhancer were cryptic in that they were only observed in the context of the other PR gene fragments, not in isolation. We identified four weak, but functional, imperfect estrogen response elements (EREs) in these regions of the PR gene, each differing from the consensus by 2 base pairs. In addition, we identified four ERE half-sites in the PR gene, three of which are paired (i.e. < 150 base pairs away) with the EREs in the estrogen-responsive regions. Competitive gel shift assays demonstrated weak, but detectable, binding of estrogen receptor to the EREs. Of note, the estrogen-responsive enhancer assembled from the five regions of the PR gene exhibited promoter specificity; it conferred estrogen responsiveness of the distal PR gene promoter, but it failed to enhance the endogenous estrogen responsiveness of the proximal PR gene promoter. The positioning of response elements in the rat PR gene, which we show to be unique among steroid hormone-regulated genes, may have functional consequences for the regulation of the magnitude and timing of PR gene expression by estrogen.

3T3 Cells↗

PubMed1993

Structure-function analysis of the hormone binding domain of the human estrogen receptor by region-specific mutagenesis and phenotypic screening in yeast.

To investigate the structural requirements for recognition and response to ligands by the human estrogen receptor (hER), a series of point mutations were generated in the hormone binding domain (HBD) of the receptor using a limited formic acid treatment of its cDNA. Receptors having a reduced sensitivity to the estrogen, 17 beta-estradiol (E2), or to the antiestrogen, trans-hydroxytamoxifen, were selected from a library of intact hER cDNAs containing these mutant HBDs by expression and phenotypic screening in yeast (Saccharomyces cerevisiae). Several were sequenced, and the encoded receptors were characterized in both yeast and mammalian (Chinese hamster ovary) cells using hormone-binding and transactivation assays. In general, parallel phenotypes were observed in yeast and in Chinese hamster ovary cells following estrogen exposure. We report on 15 receptors having point mutations located at various positions throughout the HBD. Four categories of mutants were identified: 1) those showing no change from wild type in their response to E2; 2) those showing a greatly reduced transactivation response over the range of ligand concentrations tested; 3) those requiring much higher concentrations of E2 for maximal transactivation, indicating a reduced sensitivity to ligand; and 4) those showing reduced response to E2, but little change in response to trans-hydroxytamoxifen in yeast. Two mutations in the carboxyl terminus of the HBD eliminated hormone-dependent transactivation despite the continued ability to bind E2 with high affinity. Therefore, our results show a separation of the transactivation and hormone-binding functions of the hER, and indicate that the integrity of many regions throughout the large, approximately 250-amino acid HBD is important for these functions. Our studies also demonstrate the advantages of using regional mutagenesis combined with phenotypic screening in yeast to complement site-directed mutagenesis when investigating a large, functionally complex region.

Animals↗

PubMed1993

Powerful dominant negative mutants of the human estrogen receptor.

We have identified and characterized three human estrogen receptor (ER) mutants, which, at low concentrations, are capable of blocking the intracellular activity of wild type ER. The mutants, a truncated ER (ER1-530), a point mutant (L540Q), and a frameshift (S554fs), were generated by random chemical mutagenesis of the ER hormone binding domain and screened first for low activity in a yeast selection system. In transient co-transfection assays using ER-deficient Chinese hamster ovary cells, all three mutants exhibited less than 10% of the transcription activation activity of wild type ER, and when co-expressed with wild type ER, each of the mutants effectively suppressed the ability of wild type ER to activate transcription of an estrogen-regulated reporter plasmid. When equal amounts of plasmid encoding the ER mutants and wild type ER were used, S554fs, ER1-530, and L540Q suppressed the activity of wild type ER by 80, 55, and 75%, respectively. At a ratio of 1 part S554fs to 10 parts wild type ER, transcription was still inhibited by 40%. Western blot analysis showed that all three mutants were expressed at approximately the same level as wild type ER. Suppression of transcription was specific for ER, since the mutants did not inhibit progesterone receptor-mediated transcription. Not all mutations leading to inactive ER confer the dominant negative phenotype, as five ER mutants rendered transcriptionally inactive by point mutations between residues 516 and 524 of the ER hormone binding domain were poor inhibitors of wild type ER activity. Binding studies showed that the L540Q and S554fs dominant negative mutants bound 17 beta-estradiol with wild type affinity (Kd = 0.3-0.5 nM), whereas ER1-530 exhibited a 15-fold reduction in affinity for estradiol. The three dominant negative ERs showed significant ability to interact with the estrogen response element (ERE) in promoter interference assays, but ER1-530 and S554fs displayed little or no binding to the ERE in gel mobility shift assays where higher affinity for the DNA may be required for the receptor-ERE complex to remain associated during the electrophoresis. These data support the idea that, in all three mutants, it is loss of function of the COOH-terminal transactivation domain which leads to the dominant negative phenotype. S554fs, a powerful dominant negative mutant, is a good candidate for further studies aimed at suppressing the estrogen-dependent growth of human breast cancer cells.

Amino Acid Sequence↗

PubMed1993

William L. McGuire Memorial Symposium. Estrogen receptors: ligand discrimination and antiestrogen action.

We have used affinity labeling, site-directed mutagenesis and regional chemical mutagenesis in order to determine regions of the estrogen receptor (ER) important in hormone binding, ligand discrimination between estrogens and antiestrogens, and transcriptional activation. Affinity labelling studies with the antiestrogen, tamoxifen aziridine and the estrogen, ketononestrol aziridine have identified cysteine 530 in the ER hormone binding domain as the primary site of labeling. In the absence of a cysteine at 530 (i.e. Cys530A1a mutant), C381 becomes the site of estrogen-compatible tamoxifen aziridine labeling. Hence these two residues, although far apart in the primary linear sequence of the ER protein, must be close in the three-dimensional structure of the protein, in the ER ligand binding pocket, so that the ligand can reach either site. Site-directed and region-specific chemical mutagenesis have identified a region around C530 important in discrimination between estrogens and antiestrogens, and other mutants have allowed identification of residues important in hormone-dependent transcriptional activation. Some transcriptionally inactive ER mutants also function as potent dominant negative ERs, suppressing the activity of wild-type ERs at low concentrations. These studies are beginning to provide a more detailed picture of the ER hormone binding domain and amino acids important in ligand binding and discrimination between different categories of agonist and antagonist ligands. Such information will be important in the design of maximally effective antiestrogens. In addition, since there is now substantial evidence for a mixture of wild-type and variant ERs in breast cancers, our studies should provide insight about the bioactivities of these variant receptors and their roles in modulating the activity of wild type ER, and should lead to a better understanding of the possible role of variant receptors in altered response or resistance to antiestrogen and endocrine therapy in breast cancer.

Amino Acid Sequence↗

PubMed1993

Hormone binding and transcription activation by estrogen receptors: analyses using mammalian and yeast systems.

We have used affinity labeling, site-directed mutagenesis and regional chemical mutagenesis in order to determine regions of the human estrogen receptor (ER) important in hormone binding, ligand discrimination between estrogens and antiestrogens, and transcriptional activation. Affinity labeling studies with the antiestrogen, tamoxifen aziridine and the estrogen, ketononestrol aziridine have identified cysteine 530 in the ER hormone binding domain as the primary site of labeling. In the absence of a cysteine at 530 (i.e. C530 mutant), C381 becomes the site of estrogen-compatible tamoxifen aziridine labeling. Hence these two residues, although far apart in the primary linear sequence of the ER protein, must be close in the three-dimensional structure of the protein, in the ER ligand binding pocket, so that the ligand can reach either site. Site-directed mutagenesis of selected residues in the ER and region-specific chemical mutagenesis of the ER hormone binding domain with initial phenotypic screening in yeast have enabled the identification of a region near C530 important in discrimination between estrogens and antiestrogens and of other residues important in hormone-dependent transcriptional activation. Some ER mutants with alterations in the carboxy-terminal portion of the hormone binding domain are transcriptionally inactive yet bind hormone and also function as potent dominant negative ERs, suppressing the activity of wild-type ER at low concentrations. These studies reveal a separation of the hormone binding and transcription activation functions of the ER. They are also beginning to provide a more detailed picture of the ER hormone binding domain and amino acids important in ligand binding and discrimination between different categories of agonist and antagonist ligands. Such information will be important in the design of maximally effective antiestrogens. In addition, since there is now substantial evidence for a mixture of wild-type and variant ERs in breast cancers, our studies should provide insight about the bioactivities of these variant receptors and their roles in modulating the activity of wild type ER, and should lead to a better understanding of the possible role of variant receptors in altered response or resistance to antiestrogen and endocrine therapy in breast cancer. In addition, some dominant negative receptors may prove useful in examining ER mechanisms of action and in suppressing the estrogen-dependent growth of breast cancer cells.

Affinity Labels↗

PubMed1993

An assessment of the role of domain F and PEST sequences in estrogen receptor half-life and bioactivity.

The estrogen receptor (ER) is a rapidly turning over protein, with a half-life of ca. 3-4 h in estrogen target cells. Sequence analysis of the human ER reveals a putative PEST sequence, sequences rich in proline (P), glutamic acid (E), serine (S) and threonine (T), in the carboxy-terminal F domain of the protein. Since PEST sequences have been implicated in the rapid turnover of some proteins, we have used site-directed mutagenesis to investigate the role of the F region containing PEST residues in the stability and bioactivity of the receptor. A truncated form of ER lacking the last 41 amino acids of the protein and encompassing the PEST sequences (amino acids 555 to 567) was made by mutagenesis of the ER cDNA. Pulse-chase experiments, involving immunoprecipitation of [35S]methionine/[35S]cysteine labeled receptors or of receptors covalently labeled with tamoxifen aziridine followed by gel electrophoresis, were used to determine the half-life of the wild-type and truncated ERs. These experiments showed that the turnover rate of the receptors expressed in Chinese hamster ovary and monkey kidney (COS-1) cells was 3 to 5 h and that elimination of the PEST residues did not have a significant effect on the degradation rate of the protein. Moreover, deletion of the last 41 amino acids (F domain) of the ER did not affect transactivation ability, ligand binding affinity, or the phosphorylation pattern of the receptor. Therefore, the role of domain F in ER function remains unclear, but it is not a determinant of the relatively rapid rate of ER turnover in cells.

Algorithms↗

PubMed1993

Regulation of progesterone receptor gene expression and growth in the rat uterus: modulation of estrogen actions by progesterone and sex steroid hormone antagonists.

Although the rat uterus has often been used as a model to study estrogen action, relatively little is known of the mechanism(s) by which estrogen regulates uterine progesterone receptor (PR) levels in this species. In the present study, we used immature ovariectomized rats to examine the regulation of PR gene expression and growth in the uterus by estradiol (E2) as well as hormonal modulators of E2 action, namely progesterone (P), the antiestrogen LY117018 (LY), and the antiprogestin RU486. Northern blot analyses revealed eight PR mRNA species ranging in size from 3.3-14 kilobases, with the most abundant being 7.1 kilobases. E2 treatment caused rapid time- and dose-dependent increases in the steady state levels of PR mRNA, which peaked at 24 h (6-fold increase) and declined thereafter. All eight PR mRNA transcripts increased proportionally in response to E2. Immunoblot analyses indicated that these changes were accompanied by increases in PR protein (6-fold increase by 48 h), which continued to accumulate over time, unlike PR mRNA, which decreased despite continued E2 exposure. In contrast to the stimulatory effect of E2 on PR, the levels of immunoreactive estrogen receptor were reduced to about 15% of the control value by E2 within 48 h and remained low throughout the remaining treatment period. Treatment with P blocked the stimulatory effects of E2 on both PR mRNA and protein. These antagonistic actions of P were prevented by simultaneous administration of RU486. LY, which caused a slight (approximately 2.5-fold) increase in PR mRNA when administered alone, was an effective antagonist of E2-stimulated increases in PR mRNA. However, LY was incapable of completely antagonizing E2-stimulated increases in PR protein. The differences between the profiles of the time-dependent increases in PR mRNA and protein in response to E2, as well as the different sensitivities of these two end points to the antagonistic actions of LY, highlight the lack of direct correspondence between these two end points and suggest that E2 may be acting through distinct mechanisms (transcriptional and posttranscriptional, for example) to increase the levels of PR in the rat uterus. Our results indicate that E2 rapidly increases uterine PR expression and growth, and that P as well as sex steroid hormone antagonists are important modulators of these E2 actions in the rat uterus.

Animals↗

PubMed1993

Identification of charged residues in an N-terminal portion of the hormone-binding domain of the human estrogen receptor important in transcriptional activity of the receptor.

We have shown that charged amino acids near C530 of the human estrogen receptor (ER) are involved in receptor discrimination between estrogen and antiestrogen. We now examine the role of charged residues, adjacent to the three other cysteines (381, 417, and 447) in the hormone-binding domain of the human ER, in the hormone-binding, DNA-binding, and transcription activation abilities of the receptor. Mutation of the one charged amino acid nearest to C381 gave a mutant receptor (E380Q) requiring two to three times less estradiol (E2) than wild type (WT) ER to achieve maximal activity and having activity in the absence of added estrogen that was 6-fold higher than that of WT receptor. The enhanced ability of this mutant to bind to estrogen response element DNA in the absence and presence of estrogen may, at least in part, explain its elevated, seemingly constitutive trans-activation activity and its increased sensitivity to estrogen. While more sensitive to E2, this E380Q mutant was less sensitive than WT ER to antiestrogen for suppression of transcriptional activity. Mutation of all three charged residues nearest to C381 (the triple mutant D374N, E380Q, and E385Q) resulted in a greatly reduced potency of the receptor in trans-activation with no change in estrogen-binding affinity. When K449 (near C447), highly conserved among steroid receptors, was mutated to Q, 400-fold more E2 was required for maximal reporter gene trans-activation due to an unstable, temperature-sensitive hormone-receptor complex. In contrast, the mutant K416Q (near C417) was unaltered in E2-binding or receptor transcriptional activity. These studies reveal a region in the N-terminal portion of the hormone-binding domain (ca. amino acids 374-385) where alterations in charged residues result in either increases or decreases in receptor transcriptional activity with no change in receptor affinity for hormone. Our findings suggest that this region may be important in DNA binding and protein-protein interactions that modulate transcriptional activity of the ER. In addition, the region near C447, which is well conserved among steroid receptors, appears to be important in maintaining the receptor in a conformation that is stable at physiological (37 C) temperatures. To our knowledge, this is the first report of an ER (E380Q) with a sensitivity to E2 for trans-activation greater than that of WT receptor and having high trans-activation activity in the absence of added hormone.

Amino Acid Sequence↗

PubMed1993

Cloning of the rat progesterone receptor gene 5'-region and identification of two functionally distinct promoters.

To examine some of the molecular mechanisms controlling transcription of the rat progesterone receptor (PR) gene, we have cloned and sequenced the 5'-region of the gene. Northern blot analyses with a series of probes identified two regions where distinct subsets of the multiple PR gene transcripts initiated, suggesting the presence of two promoters in the gene. Promoter activities for two gene fragments encompassing these regions, -131/+65 (P; distal) and +461/+675 (P'; proximal), were demonstrated in transient transfection experiments using reporter constructs containing the gene fragments linked individually upstream of the chloramphenicol acetyltransferase (CAT) gene. Cotransfection of P-CAT or P'-CAT constructs containing two upstream GAL4 binding sites into primary cultures of rat uterine cells with a vector expressing a GAL4 DNA binding domain-VP16 activating region fusion protein resulted in a 10-fold increase in CAT activity relative to cells transfected with either reporter and a vector expressing only the GAL4 DNA binding domain. The estrogen inducibility of the promoter-CAT constructs was assessed by transfection into MCF-7 breast cancer cells, which contain high levels of estrogen receptor (ER). P'-CAT, but not P-CAT, was induced by estradiol (E2; 8-fold). In primary rat uterine cells, which contain lower levels of ER, P'-CAT required the addition of one upstream consensus estrogen response element (ERE) to be estrogen inducible, whereas P-CAT required the addition of two EREs. Point and deletion mutants of the proximal promoter region in the P'-CAT reporter, screened in MCF-7 cells, were used to identify a 20-base pair fragment (+617/+636) that retained the promoter activity and 50% of the estrogen inducibility of P'. This fragment contained an ERE-like sequence conserved in 8 of 10 positions relative to the consensus ERE. Two copies of this sequence conferred estrogen inducibility (4-fold) when placed upstream of the distal promoter in P-CAT. To examine ER-dependent stimulation of the two PR gene promoters by cAMP, P-CAT and P'-CAT reporter constructs containing two upstream consensus EREs were cotransfected into ER-negative 3T3 cells with an ER expression vector. Induction by E2 was greater than 50-fold for both constructs. Treatment of the cells with agents that increase intracellular cAMP levels, namely cholera toxin plus isobutyl methylxanthine, resulted in CAT activity that was 8% and 51% of the E2-stimulated activity for the P and P' constructs, respectively.

3T3 Cells↗

PubMed1993

Synergistic activation of estrogen receptor-mediated transcription by estradiol and protein kinase activators.

Since we have observed effects of growth factors and cAMP as well as estradiol (E2) on regulation of expression of some genes stimulated by the estrogen receptor (ER), we have undertaken studies to examine directly whether activators of protein kinases can modulate transcriptional activity of the ER. We find that activators of protein kinase-A [cholera toxin plus 3-isobutyl-1-methylxanthine (CT+IBMX)] and protein kinase-C [12-O-tetradecanoylphorbol-13-acetate (TPA)] markedly synergize with E2 in ER-mediated transcriptional activation. When a reporter plasmid [with a minimal promoter containing a TATA region and estrogen-responsive elements (ERE) linked to a chloramphenicol acetyltransferase (CAT) reporter gene] was transfected into MCF-7 human breast cancer cells, which contain endogenous ER, E2 evoked a dose-dependent increase in CAT activity. While treatment with protein kinase-A or -C activator alone evoked only very low CAT activity, the maximal (approximately 25-fold) CAT activity stimulated by E2 alone was increased 2- to 3-fold (to approximately 60 times the control value) upon cotreatment with either of the protein kinase activators. Interestingly, antiestrogen abolished all of the CAT activity induced by E2 and protein kinase activators. Immunoblots showed that TPA reduced ER levels to 30% of control values after 24 h, while CT+IBMX increased levels about 1.5-fold. Scatchard binding analysis revealed no change in the binding affinity of E2 to ER by these agents. Gel mobility shift competition assays with extracts prepared from cells that had been treated with E2 and protein kinase activators did not reveal any quantitative or qualitative changes in the binding of ER to the ERE in vitro. In ER-deficient Chinese hamster ovary (CHO) cells transfected with the reporter gene and varying amounts of an ER expression vector, the level of CAT activity obtained by cotreatment with E2 and CT+IMBX was 3-fold higher than that observed with E2 alone over the range of different ER amounts tested. This ER-mediated synergism was still retained in an amino-terminal A/B-domain-deleted ER mutant lacking the hormone-independent transcriptional activation function (TAF-1), but was greatly reduced in two hormone-binding domain (region E) mutants that exhibit significantly diminished ligand-dependent transcriptional activation. TPA did not show any synergistic activation with E2 in CHO cells, indicating differences in responses between cell types.(ABSTRACT TRUNCATED AT 400 WORDS)

1-Methyl-3-isobutylxanthine↗

PubMed1993

Stimulation of estrogen receptor-mediated transcription and alteration in the phosphorylation state of the rat uterine estrogen receptor by estrogen, cyclic adenosine monophosphate, and insulin-like growth factor-I.

We have shown previously that exposure of rat uterine cells in primary culture to estradiol (E2), insulin-like growth factor-I (IGF-I), or agents which alter intracellular cAMP levels, such as cholera toxin plus isobutylmethylxanthine (CT + IBMX) and 8-Br-cAMP, results in the up-regulation of cellular levels of the progesterone receptor, an effect believed to be mediated through the activation of estrogen receptor (ER) and phosphorylation pathways. We have therefore undertaken studies using transient transfection of these uterine cell cultures with a simple estrogen-responsive reporter gene in order to determine the ability of these agents to stimulate ER-mediated gene transcription directly. We also compared the ability of these same agents to alter the phosphorylation state of the endogenous uterine ER protein. Plasmid DNA containing two tandem estrogen responsive elements and a TATA box linked to the chloramphenicol acetyl transferase (CAT) gene was introduced into immature rat uterine cells grown in primary culture. Treatment of transfected cells with 10(-9) M E2, CT (1 micrograms/ml) + IBMX (10(-4) M), 8-Br-cAMP (10(-4) M), or IGF-I (20 ng/ml) resulted in an 8- to 10-fold induction of CAT activity. CAT activity stimulated by all agents was nearly completely suppressed by coincubation with the antiestrogen ICI 164,384 (ICI) or the protein kinase (PK) inhibitor H8. CAT activity induced by 8-Br-cAMP was more readily suppressed by ICI than that induced by E2, indicating that ER in cells exposed to 8-Br-cAMP is either unoccupied or minimally occupied by ligand. The level of ER phosphorylation in uterine cells was increased 3- to 5-fold upon exposure to E2, CT + IBMX, 8-Br-cAMP, or IGF-I. Of interest, the antiestrogen ICI also elicited a similar increase in overall ER phosphorylation. The PK inhibitors H8 and PKI suppressed the increase in overall ER phosphorylation stimulated by these agents by 50-75%. The results of our study indicate that E2, IGF-I, and agents which raise intracellular cAMP are able to stimulate ER-mediated trans-activation and ER phosphorylation. The fact that antiestrogen (ICI) evokes a similar increase in ER phosphorylation without a similar increase in transcription activation indicates that an increase in overall ER phosphorylation does not necessarily result in increased transcriptional activity.(ABSTRACT TRUNCATED AT 400 WORDS)

1-Methyl-3-isobutylxanthine↗

PubMed1992

Characterization of a temperature-sensitive mutation in the hormone binding domain of the human estrogen receptor. Studies in cell extracts and intact cells and their implications for hormone-dependent transcriptional activation.

A previous report from this laboratory (Reese, J.C., and Katzenellenbogen, B. S. (1991) J. Biol. Chem. 266, 10880-10887) identified an estrogen receptor (ER) mutant which had a similar binding affinity for estradiol as wild-type ER but displayed a dose-response shift for estradiol in transactivation studies. In this study, we have utilized hormone binding, DNA binding, and gene transfer experiments to further characterize this mutant, which contains an alanine substitution for a cysteine at amino acid 447 in the hormone binding domain of the receptor. Hormone binding studies indicate that the C447A receptor is a temperature-sensitive mutant, whose instability is only apparent at elevated temperatures, and that ligand can stabilize the mutant receptor. Western blot analysis reveals that the temperature-sensitive loss of hormone binding is not attributable to a degradation of receptor protein, but rather is an inactivation of the receptor's hormone binding ability. In addition to the loss in the hormone binding capacity of the C447A mutant, this mutant shows a temperature-sensitive loss in the DNA binding ability of the receptor. Transactivation profiles of the mutant and wild-type receptors demonstrate that incubation of transfected cells with increasing concentrations of estradiol at more ambient temperatures shifts the mutant receptor's dose-response curves to the left, converging on the wild-type curve. Hence, these transactivation studies reveal that the dose-response shift observed for this mutant in cells reflects the measured instability of the hormone binding and DNA interaction of the C447A mutant that can be demonstrated in vitro. In addition, this temperature-sensitive ER mutant is of interest in that its DNA binding is now ligand-dependent with the result that transcriptional activation now parallels receptor occupancy by ligand, which is similar to other steroid hormone receptors.

Alanine↗

PubMed1992

Human estrogen receptor mutants with altered estrogen and antiestrogen ligand discrimination.

A structural characteristic of many antiestrogens (AEs) is a bulky side chain with basic or polar functional groups that may interact with charged and polar amino acids near the hormone-binding site in the estrogen receptor. Recently, we have identified Cys530 as the residue of the human estrogen receptor that is the site of covalent labeling by aziridine analogs of estrogens (Es) and AEs (Harlow, K. W., Smith, D. N., Katzenellenbogen, J. A., Greene, G. L., and Katzenellenbogen, B. S. (1989) J. Biol. Chem. 264, 17476-17485). Since the aziridine function is on the bulky side chain of these ligands, Cys530 must be at or near the site of those side chain interactions. To probe these interactions, we have, by site-directed mutagenesis, made mutant human estrogen receptors in which charged and polar amino acids near Cys530 are changed (Glu523 to Gln, Lys529 and Lys531 to Gln, Asn532 to Asp, and Asp538 to Asn) so as to alter charge with minimal steric alteration. These receptors were expressed in mammalian (Chinese hamster ovary or COS-1) cells and assayed for their binding affinity for Es and AEs, their interaction with estrogen-responsive element DNA, and their ability to activate or suppress transcription of estrogen-responsive reporter genes. Two of the estrogen receptor mutants, KKN-QQD (mutation of Lys529, Lys531, and Asn532 to Gln529, Gln531, and Asp532, respectively) and KK-QQ (mutation of Lys529 and Lys531 to Gln529 and Gln531, respectively), in which the local charge is changed from +2 to -1 or 0, respectively, display an affinity for estradiol (E2) 5-10 times lower than that of the wild-type receptor, which is attributable to an enhanced rate of E2 dissociation. Although these mutant receptors have reduced affinity for a variety of Es, they retain unaltered affinity for AE. The profiles of transcriptional activation of reporter genes by various concentrations of E2 show that these two mutants (KKN-QQD and KK-QQ) require 40- and 15-fold higher E2 concentrations, respectively, to achieve half-maximal activity. In contrast, mutants E523Q and D538N, with changes at amino acids further from Cys530, were unaltered in their hormone binding and transactivation activity by E or AE. Interestingly, the AEs 4-hydroxytamoxifen, LY 117018, U 23469M, and ICI 164384 were 15-30-fold more effective in inhibiting E2-stimulated transcription by mutants KKN-QQD and KK-QQ compared to the wild-type receptor.(ABSTRACT TRUNCATED AT 400 WORDS)

Amino Acid Sequence↗