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1.
Biochem J ; 256(1): 47-52, 1988 Nov 15.
Article in English | MEDLINE | ID: mdl-3223910

ABSTRACT

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.


Subject(s)
Liver/metabolism , Proteins/metabolism , Receptors, Glucocorticoid/metabolism , Adenosine Triphosphate/pharmacology , Animals , Centrifugation, Density Gradient , Chromatography, DEAE-Cellulose , Chymotrypsin/pharmacology , Cytosol/metabolism , DNA/metabolism , Rats , Rats, Inbred Strains , Receptors, Glucocorticoid/drug effects , Ribonucleases/pharmacology , Trypsin/pharmacology
2.
Mol Endocrinol ; 2(10): 952-8, 1988 Oct.
Article in English | MEDLINE | ID: mdl-2460750

ABSTRACT

We have reported that the 7-8S form of the rat liver glucocorticoid receptor is associated with RNA. Whether the unactivated 9-10S form of the glucorticoid receptor is also associated with RNA is less clear. Here we provide evidence that the unactivated 9-10S receptor is indeed associated with RNA. Unactivated 9-10S receptor was partially purified by diethylaminoethyl (DEAE)-cellulose chromatography in the presence of molybdate, an activation inhibitor. This preparation was then bound to BuGR-2, a mouse monoclonal antibody of the immunoglobulin G (IgG)-2 class to the rat liver glucocorticoid receptor, or to nonspecific mouse IgG-2. The antibody-antigen complex was then bound to protein A sepharose and washed to remove extraneous RNA. When the receptor was dissociated from the antibody and the RNA extracted and end-labeled, a distinct band of approximately 170 nucleotide (nt) was found that was specific for the BuGR-2 purified receptor. This band could also be found in DEAE-cellulose receptor that had been isolated from sucrose gradients. The DEAE-cellulose receptor was then cross-linked with formaldehyde before mixing with BuGR-2 in order to permit more vigorous washing of the antigen-antibody complex. In addition to the 170 nt RNA band, another distinct band at approximately 400 nt was seen that was specific to the BuGR-2 derived isolate. These results provide evidence that the 9-10S form of the glucocorticoid receptor from rat liver is associated with RNA.


Subject(s)
Liver/analysis , RNA/isolation & purification , Receptors, Glucocorticoid/analysis , Animals , Antibodies, Monoclonal , Centrifugation, Density Gradient , Cross-Linking Reagents , Electrophoresis, Polyacrylamide Gel , RNA/analysis , Rats , Receptors, Glucocorticoid/isolation & purification
3.
Biochim Biophys Acta ; 930(1): 114-21, 1987 Aug 19.
Article in English | MEDLINE | ID: mdl-2441756

ABSTRACT

The DEAE-cellulose-purified 4 S form of the rat liver glucocorticoid receptor can associate with cytosolic factors, as evidenced by an alteration of the sedimentation value of the 7-8 S form. On the basis of sedimentation profile, this form is indistinguishable from the activated, low-salt 7-8 S form isolated from rat liver cytosol. In addition, both the endogenous and reconstituted 7-8 S receptor can bind DNA as the 7-8 S form. In keeping with our reports that the endogenous form of the 7-8 S receptor is sensitive to RNAase digestion, treatment of the cytosol with RNAase prior to mixing with the 4 S receptor prevents the formation of the 7-8 S material. Moreover, warming the cytosol to 50 degrees C prior to mixing with the 4 S receptor also eliminates the ability to form the heavier material. Since RNA is heat-stable, this suggests that other factors may be involved. Treatment of the cytosol with N-ethylmaleimide, a reagent reported to be specific for sulfhydryl groups, also eliminates 7-8 S generating ability. These observations suggest that a protein may be a component of the 7-8 S generating material. This is substantiated by the observation that trypsin or chymotrypsin treatment of the cytosol mitigates the ability of the cytosol to form the 7-8 S material and results in the appearance of a form of the receptor that sediments at approximately 6 S. Protease treatment of partially purified material eliminates the 7-8 S generating activity entirely. We conclude that the 7-8 S form of the receptor can be reconstituted from the 4 S receptor via association with at least two other cytosolic factors, a protein and an RNA.


Subject(s)
Liver/metabolism , Proteins/metabolism , RNA/metabolism , Receptors, Glucocorticoid/metabolism , Animals , Centrifugation, Density Gradient , Chymotrypsin/metabolism , Cytosol/metabolism , Ethylmaleimide/pharmacology , Hot Temperature , Macromolecular Substances , Rats , Rats, Inbred Strains , Receptors, Glucocorticoid/drug effects , Ribonucleases/metabolism , Trypsin/metabolism
4.
Biochim Biophys Acta ; 888(3): 296-305, 1986 Oct 10.
Article in English | MEDLINE | ID: mdl-2428406

ABSTRACT

The glucocorticoid receptor from rat liver displays a differential sensitivity toward digestion by chymotrypsin and RNAase A that is dependent on its activation state. Unactivated (9-10 S) receptor is not digested by these enzymes, while activated 7-8 S receptor is. Chymotrypsin treatment yields an approx. 3 S form, while RNAase treatment yields a 4.9 S form that is distinct from the high-salt 4 S form. To firmly establish that the results are due to specific hydrolytic activities of the particular enzymes, we show that the chymotrypsin effect is inhibited by diisopropylfluorophosphate and not RNAasin, while the reverse is true for RNAase A. We further show that the differential sensitivity toward chymotrypsin is due to the association of a proteinase-resistant, heat-stable low molecular weight factor with the unactivated glucocorticoid receptor. When this factor is removed by warming, dialysis or molecular sieving of the receptor complex, the complex becomes sensitive to chymotrypsin. We also show that moderate chymotrypsin treatment yields a 6-7 S form of the receptor which is composed of, at least, RNA and the 4 S receptor. On the basis of these results, we propose that the 9-10 S receptor is composed of a low molecular weight stabilizing factor whose presence apparently alters the conformation of the complex such that the RNA and the RNA-binding site of the receptor are protected, a chymotrypsin-sensitive factor, RNA and the 4 S receptor itself.


Subject(s)
Chymotrypsin/pharmacology , Liver/metabolism , Receptors, Glucocorticoid/drug effects , Ribonucleases/pharmacology , Animals , Isoflurophate/pharmacology , Macromolecular Substances , Male , RNA/metabolism , Rats , Rats, Inbred Strains , Receptors, Glucocorticoid/metabolism , Trypsin/pharmacology
5.
J Steroid Biochem ; 23(3): 299-306, 1985 Sep.
Article in English | MEDLINE | ID: mdl-2413281

ABSTRACT

A small (Mr less than 500) anionic, heat-stable molecule has been identified in rat liver cytosol which prevents the RNase-induced decrease in the glucocorticoid receptor sedimentation properties which we described previously. This factor, which can be removed by dialysis, molecular exclusion chromatography, or ultrafiltration, functions as a true stabilizer of the RNA-glucocorticoid receptor association, and not as a RNase inhibitor. Preliminary characterization shows that the factor is not a protein, nucleic acid, or nucleotide, is not absorbed by activated dextran-charcoal, and is unaffected by extraction with organic solvents. This factor prevents activation of the glucocorticoid receptor by dilution. The relationship of this stabilization factor to a low molecular weight activation inhibitor described by others is discussed.


Subject(s)
RNA/metabolism , Receptors, Glucocorticoid/metabolism , Animals , Centrifugation, Density Gradient , Chromatography/methods , Cytosol/metabolism , Liver/metabolism , Male , Molecular Weight , Rats , Rats, Inbred Strains , Ribonucleases/antagonists & inhibitors , Solubility
6.
Biochim Biophys Acta ; 846(2): 193-9, 1985 Aug 30.
Article in English | MEDLINE | ID: mdl-2411295

ABSTRACT

Certain types of RNA can prevent the association of the rat liver glucocorticoid receptor with DNA. This inhibition of receptor binding to DNA cannot be mitigated by increasing amounts of DNA, suggesting that the RNA is not merely acting as a competitive inhibitor. Treatment of partially purified receptor with low concentrations of chymotrypsin eliminates the inhibitory effects of some RNAs without negatively affecting the DNA-binding ability of the receptor. Potent inhibitors of the receptor-DNA association, such as poly(G) and poly(X), still inhibit DNA binding of the treated receptor, although to a lesser extent than the untreated controls. However, moderate inhibitors, such as tRNA and poly(U), no longer inhibit the receptor-DNA association at low concentrations. We take these findings to suggest that RNA inhibition of DNA binding is due to the interaction of the RNA at a distinct RNA-binding site. This site may serve as a regulator site for the receptor-DNA association.


Subject(s)
Chymotrypsin/pharmacology , DNA/metabolism , RNA/pharmacology , Receptors, Glucocorticoid/metabolism , Receptors, Steroid/metabolism , Animals , Binding Sites , Centrifugation, Density Gradient , Male , Poly U/pharmacology , RNA, Transfer/pharmacology , Rats , Rats, Inbred Strains , Receptors, Glucocorticoid/drug effects , Ribonucleases/metabolism
7.
Arch Biochem Biophys ; 230(1): 345-54, 1984 Apr.
Article in English | MEDLINE | ID: mdl-6712244

ABSTRACT

The 7-8 S form of the [3H]dexamethasone (9 alpha-fluoro-11 beta,17,21-trihydroxy-16 alpha-methylpregna-1,4-diene-3, 20-dione) receptor from rat liver cytosol can be converted to the 3-4 S form by RNase treatment or high salt, suggesting a salt-sensitive association between the receptor protein and RNA. In DNA-cellulose column assays, the gradient-purified 3-4 S form bound DNA more efficiently than the 7-8 S form, though the 7-8 S form was also capable of binding to DNA-cellulose to a significant extent. Activated 7-8 S dexamethasone receptor could be released from its association with soluble DNA by treatment with DNase I. Sucrose gradient analysis showed that the released receptor sedimented as the 7-8 S form and was sensitive to RNase treatment, which induced a conversion to the 3-4 S form. Activated RNase-generated 3-4 S receptor again displayed a higher degree of binding to soluble DNA and was recovered in the 3-4 S form following DNase extraction. The fact that the 3-4 S form bound immobilized or soluble DNA more efficiently suggests that the associated RNA of the 7-8 S form interferes directly or indirectly with the receptor association with DNA. The observation that the receptor binds to DNA in its 7-8 S form suggests that the receptor complex is capable of binding RNA and DNA concurrently.


Subject(s)
DNA/metabolism , Liver/metabolism , Receptors, Glucocorticoid/metabolism , Receptors, Steroid/metabolism , Animals , Centrifugation, Density Gradient , Cytosol/metabolism , Protein Binding , Rats , Rats, Inbred Strains , Ribonucleases , Solubility
8.
Endocrinology ; 112(1): 142-9, 1983 Jan.
Article in English | MEDLINE | ID: mdl-6847812

ABSTRACT

The ability of the dexamethasone (9 alpha-fluoro-11 beta, 17,21-trihydroxy-16 alpha-methylpregna-1,4-diene-3,20-dione)-receptor complex to bind to DNA-cellulose is stimulated by RNase treatment of the activated receptor. Both RNase A and RNase T1 can induce the stimulation. The enhancement of the DNA binding ability occurs concomitantly with an alteration of the sedimentation profile of the dexamethasone-receptor complex from the 7-8S form to the 3-4S form in low salt sucrose gradients. If RNase treatment occurs in the presence of sodium molybdate, both the increase in DNA binding ability and the alteration in sedimentation profile fail to occur. Treatment of the receptor with high salt suggests that the 3-4S form can reversibly combine with a factor in a salt-sensitive association. These experiments indicate that the 7-8S form of the dexamethasone-receptor complex is associated with a RNA molecule(s) that can be removed by RNase treatment or salt dissociation, and that this RNA inhibits the binding of the receptor to DNA.


Subject(s)
DNA/metabolism , Liver/metabolism , Receptors, Glucocorticoid/metabolism , Receptors, Steroid/metabolism , Ribonucleases/metabolism , Adrenalectomy , Animals , Centrifugation, Density Gradient , Molybdenum/pharmacology , Potassium Chloride/pharmacology , Rats , Rats, Inbred Strains
9.
J Steroid Biochem ; 16(5): 595-8, 1982 May.
Article in English | MEDLINE | ID: mdl-7098474

ABSTRACT

The ability of polyribonucleotides to affect the association of [3H]-Dexamethasone-receptor complex with DNA-cellulose has been investigated. Certain ribopolymers can release the receptor complex DNA. This release is not dependent on the charge of the polymer only, but displays some specificity with regard to the base composition of the ribopolymer. Of the polyribonucleotides examined, polyguanylic acid and polyxanthylic acid were most effective at promoting release of the steroid-receptor from the DNA. Polyadenylic acid and polycytidylic acid had no effect on the association. For a polymer to display activity it must possess a base with an oxygen or sulfur atom at the C-6 position of purines or C-4 position of pyrimidines. Simple nucleotides possessing this structural feature are inactive.


Subject(s)
DNA/metabolism , Polyribonucleotides/pharmacology , Receptors, Glucocorticoid/drug effects , Receptors, Steroid/drug effects , Animals , Dose-Response Relationship, Drug , Hot Temperature , Osmolar Concentration , Rats , Rats, Inbred Strains , Receptors, Glucocorticoid/metabolism
11.
J Reprod Fertil Suppl ; (24 suppl): 147-62, 1976 Sep.
Article in English | MEDLINE | ID: mdl-794465

ABSTRACT

In the rat ventral prostate, various antiandrogens can interfere with the normal formation of an androgen-receptor complex which appears to be required for androgen action. Some of the antagonists may act by inhibiting the enzymatic formation of 5 alpha-dihydrotestosterone or by competing with androgens for the binding sites on the receptors, whereby interacting with and altering the receptor molecules themselves. If the action of an androgen-receptor complex can indeed occur at the stage of gene transcription as well as in certain post-transcriptional processes, the molecular site of antiandrogen action may be in the nuclei and/or cytoplasm of the target cells.


Subject(s)
Androgen Antagonists , Prostate/drug effects , Receptors, Androgen/drug effects , Receptors, Steroid/drug effects , Animals , Carrier Proteins/analysis , Contraceptive Agents, Male/pharmacology , Cyproterone/pharmacology , Cytosol/analysis , Dihydrotestosterone/biosynthesis , Flutamide/pharmacology , Genes , Male , Molecular Conformation , Prostate/physiology , Protein Binding/drug effects , RNA/biosynthesis , Rats , Transcription, Genetic
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