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.     

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.     

Recognition of two forms of the estrogen receptor in the guinea-pig uterus at different stages of development by a monoclonal antibody to the human estrogen receptor. Dynamics of the translocation of these two forms to the nucleus

Two forms of the estrogen receptor were recognised in the cytosol fraction of fetal uterus of guinea pig by a monoclonal antibody (D547Spγ) to the human estrogen receptor. It was observed that 60–65% of the total cytosol estrogen receptor (the α form) was bound to the antibody, increasing its sedimentation coefficient in a high ionic strength sucrose gradient (10–30% w/v sucrose, 0.4 M KCl) from 4.5 S to 7.4 S. The remaining fraction (the β form) has the classical sedimentation coefficient of 4.5 S. Dynamic studies of the translocation in vitro of the cytosol receptor to the nucleus as a function of time have shown that the a form decreases sharply while the β form is slightly affected when the cytosol was incubated with the nuclei. In contrast only one form, which is bound totally to the antibody, was found in the nuclear fraction. In addition, the presence of these two forms of the cytosol estrogen receptor was also demonstrated in newborn and immature animals.     

Oligomeric structures of cytosoluble estrogen-receptor complexes as studied by anti-estrogen receptor antibodies and chemical crosslinking of intact cells

The structure of estrogen-receptor complexes recovered in cytosolic extracts of MCF-7 cells treated with hormone at 2°C was probed by chemical crosslinking of intact cells and sample analysis with four monoclonal anti-estrogen receptor antibodies. When MCF-7 cells were treated with either glutaraldehyde or dithiobis(succinimidyl propionate), cytosoluble estrogen-receptor complexes consisted of two major forms sedimenting as 4 S monomers and 8–9 S salt-resistant oligomers. By high salt sucrose density gradient centrifugation, we could observe that the four monoclonal anti-estrogen receptor antibodies bound different forms of receptor complexes from crosslinked cells. While H222 and H226 antibodies could interact with any form we detected, the D75 and D547 monoclonals could only recognize those showing sedimentation coefficients lower than 7 S. When cytosolic extracts from [³⁵S]-methionine-labeled cells were subjected to immunoprecipitation with H222 and D75 anti-estrogen receptor antibodies, electrophoretic analysis of material extracted from immunoprecipitates revealed the presence of 65 kDa estrogen receptors. If extracts were prepared from crosslinked cells, instead, two more components with estimated molecular masses of 220 and 100 kDa were specifically immunoprecipitated by the H222 antibody, whereas only the 100 kDa component and the estrogen receptor were found in immunoprecipitates obtained with the D75 monoclonal. When estrogen-receptor complexes were immunopurified from extracts prepared after cells had been crosslinked with dithiobis(succinimidyl propionate), and the oligomers were dissociated by treatment with β-mercaptoethanol, electrophoretic analysis of our samples showed that only the 65 kDa estrogen receptor and a 50 kDa protein were selectively immunoprecipitated by anti-estrogen receptor antibodies. We concluded that the structures of cytosoluble estrogen-receptor complexes in MCF-7 cells treated with hormone at 2°C, include oligomeric forms which contain a 50 kDa non-steroid binding protein.     

Studies on the non-activated and activated forms of the estrogen receptor.

The activation of the steroid receptor is a necessary process for the biological role of the receptor. Many factors are involved in this mechanism; in addition to time, temperature, and salt concentration, RNA and RNAase can also affect the transformation of the non-activated to the activated form of the receptor. Using as a model the estrogen receptor of fetal uterus of guinea-pig, the studies of the interaction with three different monoclonal antibodies (D547, H222 and H226) reveal structural transformation during the process of the receptor activation. These conformational transformations suggest that a change in the exposure of the functional domains of the estrogen receptor occurs during activation.     

Immunorecognition of the active form of the oestrogen receptor by using a monoclonal antibody.

In previous studies, two forms (alpha and beta) of the oestrogen receptor, with different immunological characteristics, were observed in the cytosol fraction of fetal guinea-pig uterus, by using a monoclonal antibody to the human oestrogen receptor (D547Sp gamma). Only the alpha form was recognized by the antibody, shifting its sedimentation coefficient in high-salt sucrose gradients. The present work investigated the effect of several factors (time, temperature, high salt concentrations and Na2MoO4) on the interconversion of these two forms. Only the beta form was observed when cytosol was incubated with oestradiol for only 2-3 h, but 20 h later, 40-60% of the total oestradiol-receptor complexes were found as the alpha form. The transformation from the beta to the alpha form was accelerated by temperature (25 degrees C, 15 min) and exposure to high salt concentrations (0.4 M-KCl). On the other hand, Na2MoO4 completely blocked the transformation induced by time and temperature, but had little effect on that induced by KCl. The appearance of the alpha form always correlated with an increase in receptor binding to nuclei and DNA-cellulose. Finally, it was found that the isolated beta form, recovered from the gradient, was transformed into the alpha form after overnight dialysis under reduced pressure. The present data suggest that the alpha form, which is recognized by the monoclonal antibody, is the activated form of the oestrogen receptor.     

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.     

Antibodies to steroid receptor deoxyribonucleic acid binding domains and their reactivity with the human glucocorticoid receptor

Functional properties of the DNA-binding domain of the human glucocorticoid receptor were investigated using high titer polyclonal antibodies produced against single synthetic peptides or a mixture of peptides whose sequences were derived from the DNA-binding domain of steroid receptor proteins. Three of seven antisera recognized both native and denatured forms of the glucocorticoid receptor, although considerably lower antisera dilutions were required for antibody binding to native receptor. Activation of the glucocorticoid receptor to its DNA-binding form was required for antibody recognition of the native receptor. Antisera to the second finger region of the DNA-binding domain caused a portion of the activated 4S glucocorticoid receptor to sediment as 7 or 9S in sucrose gradients containing 0.4 M KCl, but did not alter the sedimentation of the nontransformed 8S receptor. Specificity of the glucocorticoid receptor-antibody interaction was demonstrated by loss of reactivity after preabsorption with peptide antigens. Antisera that interacted specifically with the glucocorticoid receptor inhibited DNA binding of the activated receptor by as much as 80%. Thus, antibody probes directed against DNA-binding domain sequences provide immunological evidence that glucocorticoid receptor activation exposes the DNA-binding region of the receptor.     

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.     

Tyrosine phosphorylation of the receptor for insulin-like growth factor II is inhibited in plasma membranes from insulin-treated rat adipocytes.

We have identified a factor from rat liver cytosol that enhances the DNA-cellulose-binding ability of the glucocorticoid receptor and lowers the sedimentation value from 9-10 S to 4-5 S. Cytosol is prepared in the presence of molybdate, and unactivated receptor is isolated by chromatography on DEAE-cellulose in the presence of molybdate. This receptor sediments at 9-10 S and has little affinity for DNA. If the molybdate is removed and the receptor is incubated at 25 degrees C with the low-salt wash of the DEAE-cellulose column, DNA binding is enhanced by 50-600% relative to controls incubated with buffer only. In addition, the factor present in the low-salt wash converts the 9-10 S receptor into a mixture of 5 S and 4 S forms. The factor must be present during the incubation in order to exert its maximal effect. Factor added after the incubation has only marginal effects on the DNA-binding ability of the receptor, indicating that the factor does not increase the DNA-binding ability of activated receptor. Moreover, the factor is significantly less effective on receptor that has been activated before incubation with the factor. These results suggest that the factor acts as an activation enhancer. Preliminary characterization indicates that the activation enhancer is a trypsin-sensitive protein of approx. 70,000 Da, whose activation-enhancing properties are inhibited by ATP. RNAase A, which has effects similar to those described above on the 7-8 S receptor, does not mimic the effects of the activation enhancer on the 9-10 S receptor.     

Characteristics of different cytoplasmic and nuclear estrogen receptors appearing with continuous hormonal exposure

Biochemical properties of cytosol estrogen receptor (ERC) and nuclear estrogen receptor (ERN) from rat uteri continuously exposed in vivo to 17 beta-[2,4,6,7-3H] estradiol ( [3H]E2) for 6 h have been studied on the basis of immunological recognition and chromatographic elution patterns. Overall concentrations of ERC and ERN did not change during this time period when receptor-saturating concentrations of [3H]E2 were maintained (Jakesz, R., Kasid, A., and Lippman, M. E. (1983) J. Biol. Chem. 258, 11798-11806); however, biochemical characteristics were different in ERC and ERN after short or long term hormonal exposure. When ERC from rats treated with estradiol for 30 min was applied to HAP or DEAE columns, two different ER binding components were seen. DNA binding in a cell-free system revealed that these binding components represented an activated and a nonactivated ERC population. After long term hormonal exposure (6 h), only one component of ERC with low DNA binding could be shown despite the preservation of an equivalent quantity of cytoplasmic binding activity. This binder does not react with a monoclonal antibody directed against extranuclear estrogen receptor species. These data suggest disappearance of the activated ERC population, with appearance of a new, immunologically nonrecognizable ERC species with 6 h of continuous hormonal exposure. Elution profiles of ERN on HAP chromatography reveal 2 different binding components at 30 min and at 6 h of continuous [3H]E2 exposure. There is an increase of the population eluted at higher molarity after 6 h of in vivo treatment. This later eluting binding component is the major DNA binder in vitro. ERN from both time points are recognized immunologically by monoclonal antibody. After reaction with the antibody, the sedimentation coefficient shifted to 8-9 S on sucrose gradients, but the previously described faster sedimentation of ERN extracted 6 h after injection persisted. We conclude that ER in both cellular compartments undergoes time-dependent alterations, which may be involved in the initiation of hormone action.     

The inhibition of prolactin synthesis in GH3 rat pituitary tumor cells by monohydroxytamoxifen is associated with changes in the properties of the estrogen receptor

E2 (1 nM) stimulated the synthesis of PRL in GH3 cells. OH TAM (100 nM) did not affect basal PRL synthesis, but completely inhibited the increase produced by 1 nM E2. [3H]E2 and [3H]OH TAM both bound to the cytosolic 8S ER and these were split into 4S subunits on sucrose gradients containing 0.4 M KCl. By comparison, ER complexes extracted from nuclei of GH3 cells cultured in media containing [3H]E2 or [3H]OH TAM both sedimented at 5S on sucrose gradients containing 0.4 M KCl. Both 4S and 5S ER complexes were recognized by the monoclonal antibody D547 which increased their sedimentation coefficients to 8-9S. In contrast, a polyclonal antibody raised to calf uterine ER in the goat, interacted with the cytosolic ER so that the binding of [3H]E2 was inhibited but the binding of [3]OH TAM was only slightly reduced. A molecular model is proposed to describe the binding of E2 and OH TAM to the ER that might contribute to an understanding of estrogen and antiestrogen action.     

Characterization of estrogen-binding sites associated with the endoplasmic reticulum of rat uterus

Microsomes prepared from rat uterine homogenates harbor high-affinity (Ka = 10(10) M-1), low-capacity binding sites for estrogens. Previous work from our laboratory has demonstrated that these estrophiles are located on endoplasmic reticulum and are not cytosolic contaminants of the membrane preparation. Subfractionation of microsomes into granular and agranular membranes and polysomes revealed approximately equal distribution of estrogen-binding activity among each of these constituents. These binding sites were fully extractable with 0.6 M KCl. Microsomal estrophiles solubilized under conditions of low ionic strength and complexed with estradiol migrated as 8S forms on continuous sucrose gradients. In the presence of 0.4 M KCl, the solubilized binding sites exhibit a sedimentation coefficient of 4S. Extracted binding sites do not undergo heat-induced transformation from a 4S to 5S species. The monoclonal antibody JS34/32 interacted with the endoplasmic reticulum-associated estrogen-binding sites when present in 50-fold molar excess, but not at lower antibody to binding site ratios. In comparison, the rat uterine cytosolic estrogen receptor formed complexes with JS34/32 at antibody to receptor ratios as low as 2:1. These results suggest that the endoplasmic reticulum possesses estrogen-binding sites with biochemical properties that differ from those of the classically described cytosolic (loosely associated nuclear) estrogen receptor.     

Polyanionic-binding properties of the receptor for 2,3,7,8-tetrachlorodibenzo-p-dioxin. A comparison with the glucocorticoid receptor

The interaction of the rat hepatic receptor for 2,3,7,8-tetrachlorodibenzo-p-dioxin (dioxin) with immobilized heparin (heparin-Sepharose) or DNA (DNA-cellulose) has been compared to the polyanionic-binding properties of the rat hepatic glucocorticoid receptor. Both the nonoccupied and in vitro occupied forms of the receptors interacted with heparin-Sepharose but with varying strength, as determined by ligand binding assays or an enzyme-linked immunosorbent assay based on a monoclonal antibody against the steroid- and DNA-binding Mr approximately 94,000 glucocorticoid receptor protein. In the absence of ligand, both the dioxin and glucocorticoid receptors eluted from heparin-Sepharose at 0.1-0.2 M KCl, in contrast to the in vitro occupied receptor forms which eluted at 0.3-0.4 M KCl. Following elution of the in vitro occupied dioxin receptor from heparin-Sepharose, it was efficiently retained on DNA-cellulose and eluted at an ionic strength of approximately 0.2 M KCl. In the presence of 20 mM sodium molybdate which is known to inhibit the activation of steroid hormone receptors to a DNA-binding form, both the dioxin and glucocorticoid receptors eluted at 0.1-0.2 M KCl from heparin-Sepharose. In analogy to what has previously been shown for the glucocorticoid receptor, sodium molybdate stabilized a large dioxin-receptor complex with a sedimentation coefficient, S20,w, of 9-10 S, a Stokes radius of approximately 7.5 nm, and a calculated Mr of 290,000-310,000. Limited proteolysis of both the dioxin and glucocorticoid receptors with trypsin which is known to eliminate the DNA-binding property of both receptor forms also resulted in a decreased strength in the interaction of both in vitro occupied receptors with heparin-Sepharose (elution at 0.1-0.2 M KCl). In line with these data, calf thymus DNA in solution competed for receptor binding to heparin-Sepharose. In conclusion, the chromatographic properties of the dioxin receptor on heparin-Sepharose are indistinguishable from those of the glucocorticoid receptor, and both receptors appear to be structurally and functionally closely related proteins.     

Monoclonal antibodies to chick intestinal receptors for 1,25-dihydroxyvitamin D3. Interaction and effects of binding on receptor function

Monoclonal antibodies, developed against the chick intestinal receptor for 1,25-dihydroxyvitamin D3 (1,25(OH)2D3), were characterized with respect to their interaction with this protein and for their effects on the polypeptide's hormone-binding and nuclear-binding functions. Antibodies, internally labeled with [35S]methionine, react directly with hormone-labeled receptor, as identified by comigration of both isotopes during sedimentation on hypertonic 10-30% sucrose gradients. Antibodies bound both the unoccupied and occupied forms of the receptor, the latter with equilibrium dissociation constants of 10(-10)-10(-11) M at 4 degrees C. Excess antibody, added to unoccupied receptors prior to incubation with 1,25(OH)2D3, did not affect the receptor's apparent affinity for the hormone (Kd approximately equal to 6 X 10(-11) M). In contrast, all three antibodies, complexed with occupied receptors, significantly reduced the extent of the receptor's association with isolated nuclei (48-64% inhibition). This inhibition most likely represents a general reduction in the affinity of the protein for nuclei under the conditions tested, since the affinity of the occupied 1,25(OH)2D3 receptor for DNA, as well as the ionic strength necessary to elute receptor from both cation and anion exchange resins was significantly reduced by prior incubation with excess antibody. These findings suggest that the epitopes for each of the three monoclonal antibodies may be located in or near the DNA or nuclear binding domain of the 1,25(OH)2D3 receptor. Taken cumulatively, these results indicate that the monoclonal immunoreagents utilized here should prove useful in delineating important biochemical features of this unique sterol hormone receptor.     

A monoclonal antibody to the estrogen receptor inhibits in vitro criteria of receptor activation by an estrogen and an anti-estrogen

The effect of an IgM class monoclonal antibody (B36) (Greene, G. L., Fitch, F. W., and Jensen, E. V. (1980) Proc. Natl. Acad. Sci. U. S. A. 77, 157-161) raised against the calf uterine estrogen receptor was tested in vitro on certain parameters of estrogen receptor activation by estradiol or 4-hydroxytamoxifen, a potent anti-estrogen. The following results were obtained. The antibody prevented the decrease in the dissociation rate of the receptor-estradiol complex which results from activation of the complex, whereas it did not affect the dissociation rate of the receptor-4-hydroxytamoxifen complex, which remains unchanged upon activation. The antibody also increased the dissociation rate of the preactivated receptor-estradiol complex. The antibody protected the naked estrogen receptor against heat-inactivation. B36 partially inhibited the binding of the estradiol- and 4-hydroxy-tamoxifen-receptor complexes to DNA adsorbed onto cellulose, but did not reverse the receptor-DNA binding. This inhibition was not overcome by higher DNA concentrations and was more pronounced for the receptor interacting with estrogen than with anti-estrogen. All these effects were specific since they were related to antibody/antigen recognition and were dose-dependent. These results indicate that the binding of the antibody to the estrogen-activated receptor induces a conformational change in the receptor and that the antibody can prevent and overcome the effect of activation whatever its mechanism. They also confirm that the conformations of the estrogen receptor differ when bound to estradiol or to 4-hydroxytamoxifen.     

Regulation of nuclear binding of the avian oviduct progesterone receptor. Changes during estrogen-induced oviduct development, withdrawal, and secondary stimulation

The progesterone receptors from various stages of estrogen induced oviduct development, estrogen withdrawal, and secondary stimulation with estrogen were examined. The progesterone receptors were characterized for their biological function (i.e. capacity for nuclear translocation, nuclear binding, and effects on RNA polymerase II activity) as well as certain physical properties. The progesterone receptors from the undeveloped or partially developed oviducts (0 to 8 days of estrogen treatment) displayed little or no nuclear translocation and binding in vivo or in vitro. Similarly, progesterone showed little or no effect in vivo on RNA polymerase II activity at the early stages of development. As development progressed from 8 to 12 days of estrogen treatment, the above parameters rapidly increased to maximal levels and plateaued through day 23 of estrogen treatment. A marked decrease in these parameters occurred within 1 day of estrogen withdrawal. The reverse series of events occurred during secondary estrogen stimulation of 10-day-old withdrawn chicks. While the receptor concentrations increased rapidly to maximum values by 2 days of restimulation, receptor function did not return until day 4. Similarly, the effects of progesterone on RNA polymerase II activity reached maximal values by day 4. The progesterone receptor isolated from oviducts during development, estrogen withdrawal, and restimulation, displayed similar patterns of cell-free binding to chromatin and nucleoacidic protein as that observed in vivo supporting the nativeness of the in vitro binding assay. In contrast, the cell-free binding of these same progesterone receptor to pure DNA were not similar to the in vivo binding, i.e. no patterns (differences) in progesterone receptor binding were observed. These data support that protein DNA complexes and not pure DNA represent the native acceptor sites for oviduct progesterone receptor. Comparison of the progesterone receptor between the functional and nonfunctional states revealed no differences in the steroid affinity for the receptor, in the apparent pI of the species, or in the sedimentation of the receptor under high salt conditions. However, the nonfunctional receptors consistently displayed a deficiency in one of the two monomer molecular species (the B species) as determined by isoelectric focusing. These results suggest that both monomer species of progesterone receptor are required for biological activity. Interestingly, the 7S "aggregate" species of the progesterone receptor was constantly detected even when only one of the monomer species was present.(ABSTRACT TRUNCATED AT 400 WORDS)     

Interactions of nuclear estrogen receptor with DNA and RNA

The interaction of the nuclear estrogen receptor from hen oviduct with nucleic acids were studied by competition assay using DNa-cellulose centrifugation. We demonstrated that the estradiol-receptor complex binds similarly well to poly(A) RNA and denatured DNA. The estrogen receptor was found to interact more strongly with poly(G), poly(U) than with poly(A), poly(C). The receptor complex binds similarly to poly(A) and poly(dA), and to poly(U) and poly(dU). However, the receptor complex shows stronger binding to poly(G) than to poly(dG) and to poly(C) than to poly(dC). Studies with heteropolyribonucleotides indicated that poly(U1G1) is more effective in competing for the estrogen receptor, and poly(AC) and poly(AUG) are moderately effective, whereas poly(ACU) is least effective. GMP and dGMP showed some competition for the nuclear receptor at 300-fold higher nucleotide concentrations than that of the synthetic poly(G). Observations that the nuclear estrogen receptor binds to poly(A) RNA and interacts selectively with polyribonucleotides suggest that the estrogen receptor-RNA interaction may play a role for the function of estrogens in gene regulation.     

Characterization of a monoclonal antibody that probes the functional domains of the glucocorticoid receptor.

Monoclonal antibodies to the rat hepatic glucocorticoid receptor (GR) were produced by using 4000-fold-purified unactivated rat hepatic GR as the immunogen in an immunization in vitro. Hybridomas were screened for anti-GR antibody production by using an enzyme-linked immunosorbent assay. The antibody, 3A6, described here, is an IgM (lambda). The interaction of 3A6 with the purified GR was explored by sedimentation analysis, where a shift of the 9 S GR to a form with a higher s20,w value was demonstrated. Binding specificity and sensitivity were demonstrated by protein immunoblotting. 3A6 cross-reacted with all rat tissue glucocorticoid receptors (GRs) examined, except those of the brain. Species cross-reactivity was observed with other mammalian GRs (from human CEM-C7 cells and from pig and mouse liver). Immunocytochemical localization of the GR was assessed by indirect immunofluorescence in intact fixed cells, which demonstrated intense cytoplasmic staining in the absence of pretreatment with glucocorticoids and nuclear localization when cells were pretreated with glucocorticoids. This monoclonal antibody significantly inhibited steroid binding to unoccupied receptor and DNA binding of activated steroid-receptor complexes. Furthermore, preincubation of the purified activated GR complex with 3A6 prevented phosphorylation of the GR in vitro. Thus 3A6 differs from previous monoclonal antibodies to the GR in its capacity to cross-react with the human GR and by its specificity for an epitope on or near a functional domain of the GR.     

Characterization of the Ah receptor for 2,3,7,8-tetrachlorodibenzo-p-dioxin: use of chemical crosslinking and a monoclonal antibody directed against a 59-kDa protein associated with steroid receptors

The Ah receptor regulates induction of cytochrome P450IA1 (aryl hydrocarbon hydroxylase) by "3-methylcholanthrene-type" compounds and mediates the toxic effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin and related halogenated aromatic hydrocarbons. Hepatic Ah receptor from untreated rodents is localized in the cytosol and has an apparent molecular mass of 250 to 300 kDa. This large form can be dissociated into a smaller ligand-binding subunit upon exposure to high ionic strength. The Ah receptor displays many structural similarities to the receptors for steroid hormones. Two non-ligand-binding proteins have been identified to be associated with the cytosolic forms of the steroid hormone receptors. The first is a 90-kDa heat shock protein (hsp 90); the second is a 59-kDa protein (p59) of unknown function. The cytosolic Ah receptor ligand-binding subunit previously has been shown to be associated with hsp 90. In the present study, we used a monoclonal antibody, KN 382/EC1, generated against the 59-kDa protein which is associated with rabbit steroid receptors to determine if p59 also is a component of the large cytosolic Ah receptor complex. Cytosolic forms of rabbit progesterone receptor, glucocorticoid receptor, and Ah receptor were analyzed by velocity sedimentation on sucrose gradients under low-ionic-strength conditions and in the presence of molybdate. Progesterone receptor from rabbit uterine cytosol and glucocorticoid receptor from rabbit liver each had a sedimentation coefficient of approximately 9 S. In the presence of KN 382/EC1 antibody the progesterone receptor and the glucocorticoid receptor both underwent a shift in sedimentation to a value of approximately 11 S. The increase in sedimentation velocity is an indication that the receptor-protein complexes are interacting with the antibody. Under low-ionic-strength conditions the Ah receptors from rabbit uterine cytosol and liver cytosol had a sedimentation coefficient of approximately 9 S. However, in contrast to the steroid receptors, the Ah receptor showed no change in its sedimentation properties in either tissue in the presence of KN 382/EC1, indicating that the antibody is not interacting with the Ah receptor. Multimeric Ah receptor complexes that were chemically crosslinked still did not show any interaction with KN 382/EC1. These data indicate that the 59-kDa protein either is not associated with the Ah receptor or is present in an altered form which the antibody cannot recognize.