Biochemical and Biophysical Research Communications
Evidence for an association of a ribonucleic acid with the purified, unactivated glucocorticoid receptor
Abstract
The unactivated glucocorticoid-receptor complex (GRC) was purified from rat hepatic cytosol (approximately 4000-fold by specific activity) by a procedure developed in our laboratory. Following elution of unactivated GRC from DEAE-cellulose with a 0.05–0.5 M potassium phosphate gradient, a second gradient of 0.5–1.0 M potassium chloride was started. This gradient eluted material at 0.6 M potassium chloride that incorporated and stained with ethidium bromide. A predominant ethidium bromide stained band of 100–110 nucleotides was observed. The presence of this material was dependent on the presence of highly purified GRC since presaturation of cytosol with 50 μM unlabeled triamcinolone acetonide (TA) precluded the appearance of this material. Experiments with partially purified GRC from CEM-C7 cells incubated with [14C]uridine indicated that the material eluted at 0.6 M potassium chloride incorporated [14C]-uridine. Collectively, these data suggest that a RNA is associated with the purified, unactivated form of the GRC.
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Isolation of untransformed bovine estrogen receptor without molybdate stabilization
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Majority of RNA which co-purifies with unactivated rat hepatic glucocorticoid-receptor complexes is nonspecific and not receptor-associated
1990, Journal of Steroid BiochemistryMolybdate-stabilized, unactivated rat hepatic glucocorticoid-receptor complexes were purified by a three-step procedure which includes affinity chromatography, gel filtration and anion exchange chromatography. Following elution of unactivated steroid-receptor complexes from the final DEAE-cellulose column, RNA which remained bound to the anion exchange resin was eluted with l M KCl. This RNA was small and heterogeneous in size. Equivalent amounts of RNA were detected after a mock purification which was devoid of receptors, suggesting that the presence of this RNA is not dependent on that of receptors. Both a [32P]DNA complementary to the RNA eluted from DEAE-cellulose and a [32P]DNA probe synthesized from total rat liver RNA gave similar results when hybridized to total rat liver RNA. These data indicated that the RNA which co-purified with unactivated receptors through the first two steps was very similar to total RNA in overall composition. Virtually identical hybridization patterns were also detected when end-labeled probes generated from the DEAE-cellulose eluted RNA or total liver RNA were hybridized to total genomic rat DNA, suggesting that the RNA eluted from the anion exchange resin is not specific or unique.
Although these results do not exclude the possibility that there could be specific RNA species associated with the unactivated glucocorticoid receptor, they do indicate that the majority of the RNA eluted from DEAE-cellulose following elution of receptor complexes appears indistinguishable from total rat liver RNA and can be detected in parallel mock purifications.
Evidence that 5 S intermediate state in glucocorticoid receptor transformation contains hsp90 in addition to the steroid-binding protein
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Molybdate-stabilized 5 S GR was apparently re-assembled into the oligomeric nontransfonned state when the salt concentration was reduced. This phenomenon was evident under the low-salt conditions of electrofocusing, by the shift in pI' value of GR from 5.8 to 5.0; and in glycerol density gradients containing 0.15 M KCl, by the shift in the sedimentation of the GR complex from 5 S to 9.5 S.
Glucocorticoid-receptor complexes are associated with small RNA in vitro
1989, Journal of Steroid BiochemistryIdentification of RNA associated with soluble glucocorticoid-receptor complexes of HeLa cells was performed by immunoprecipitation of receptor complexes with a monoclonal antibody raised against rat liver glucocorticoid receptor. Polyacrylamide gel electrophoresis of RNA extracted from immunoprecipitates of cytosolic complexes revealed the presence of eight RNA bands, consisting of 28S, 18S, and small RNAs, including 5.8S, 5S and tRNA. A comparison of RNA species immunoprecipitated by monoclonal anti-glucorcorticoid receptor antibody and IgG purified from normal mouse serum showed that four small RNAs were preferentially recovered after immunoprecipitation with anti-glucocorticoid receptor antibody. When these species were analyzed on sequencing gels, their nucleotide lengths coincided with those of 7-3, 7S, U2, and U1 RNA. Immunoprecipitation of nuclear extracts containing glucocorticoid-receptor-RNA complexes showed that the same set of small RNAs was preferentially immunoprecipitated by anti-glucocorticoid receptor antibody. The four small RNAs we detected represented minor species in whole extracts, and their preferential immunoprecipitation by anti-glucorcorticoid receptor antibody was prevented by removal of glucocorticoid-receptor complexes from HeLa cell extracts. We conclude that 7-3, 7S, U2, and U1 RNA are associated with glucocorticoid-receptor complexes in vitro, and hypothesize that post-transcriptional effects of glucocorticoids may in part be mediated through interaction of receptor complexes with these small RNAs.
Heterogeneity and properties of transformation of corticosteroid receptors in spinal cord and hippocampus
1989, Brain ResearchThe central nervous system contains two classes of corticoid receptors, named types I and II following terminology accepted for the kidney. Phenotypically, type I sites are differentiated into a corticosterone (CORT)-preferring species (Ia) and a mineralocorticoid receptor (Ib). These populations were tentatively compared in the spinal cord and hippocampus. Using [3H]dexamethasone (DEX) and selective blockage of sites, we have observed that type II receptors were comparable in both tissues, while Ia was almost exclusive of the hippocampus. Saturation analysis using [3H]DEX demonstrated that type Ia was a low affinity receptor (Kd ≈ 2–5 nM while type II was a higher affinity site (KdII <KdI). Using [3H]CORT, or [3H]aldosterone (ALDO), as ligand, preferential labeling of type I sites was achieved, always showing higher concentrations in the hippocampus. Therefore, [3H]DEX seems a ligand of choice to visualize types Ia and II receptors. Another difference noted between the spinal cord and hippocampus, pertained to the sensitivity towards the enzyme RNAse A, which increases heat-induced transformation of the bound receptor, according to the results of DNA cellulose affinity chromatography. In these experiments, type I sites of both spinal cord and hippocampus, plus type II of hippocampus, showed sensitivity towards the enzyme, whereas type II of the spinal cord was refractory to RNAse A enhancement of transformation. These results indicate that the dynamics of transformation is different among receptors showing similar affinity and competition, suggesting further heterogeneity due to receptors themselves, or to tissue factors regulating their biochemical properties.
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Present address: Department of Pharmacology, Hershey Medical Center, The Pennsylvania State University, Hershey, PA, 17033
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Present address: The Department of Physiology and Biophysics, University of Iowa, Iowa City, Iowa 52242
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