Rat Liver Glucocorticoid Receptor Research Articles

Immunochemical characterization of the rat kidney glucocorticoid receptor. The role of proteolysis in the formation of corticosteroid binder IB.

Glucocorticoid receptors of rat kidney and liver were compared by physicochemical and immunochemical methods to investigate the role of proteolysis in the formation of corticosteroid binder IB. Kidney cytosol prepared in the presence of sodium molybdate contained receptor forms comparable to rat liver glucocorticoid receptor; [3H]triamcinolone acetonide-labeled receptors eluted from Sephacryl S-300 as a multimeric 6.1 nm component in the presence of molybdate and as a monomeric 5.7 nm component in the absence of molybdate. Both forms were recognized by the monoclonal antibody BUGR-1 which was raised against rat liver glucocorticoid receptor. When kidney cytosol was prepared in the absence of molybdate, labeled receptor complexes eluted from Sephacryl S-300 as a 5.8 nm component in the presence of molybdate. However, in the absence of molybdate, the receptor eluted as a smaller 3.4 nm component which was identical with the size of activated kidney glucocorticoid receptor chromatographed in either the presence or absence of molybdate. The 3.4 nm activated kidney glucocorticoid receptor did not bind to DEAE-cellulose under conditions where activated liver receptor was retained. These properties of the activated kidney receptor are characteristic of corticosteroid binder IB. Incubation of the activated kidney receptor complex with BUGR-1 resulted in a shift in apparent Stokes radius from 3.4 nm to 5.4 nm, indicating immunochemical similarity with rat liver receptor. Identification of the immunoreactive receptor subunit by Western blotting demonstrated that kidney cytosol prepared in the presence of molybdate contained a major 94-kDa immunoreactive component which co-migrated with rat liver glucocorticoid receptor, while cytosol prepared in the absence of molybdate contained principally a 44-kDa immunoreactive species. These results suggest that corticosteroid binder IB can be generated by in vitro proteolysis and does not represent a polymorphic form of the glucocorticoid receptor.

Interaction of rat liver glucocorticoid receptor with lectins: Is the glucocorticoid receptor a glycoprotein?

Although glucocorticoid receptors have been extensively studied in a variety of tissues, the precise nature of the receptor protein still remains unknown. To further characterize this protein we assessed the effects of various lectins on [ 3H]dexamethasone binding to prepurified preparations of rat liver glucocorticoid receptor. Among the lectins tested only Ulex europeus and Lens culinaris induced a concentration-related decrease in [ 3H]dexamethasone binding. Following Ulex europeus or Lens culinaris exposure Scatchard analysis showed that these lectins led to a 3-fold reduction in receptor affinity without influencing the concentration of binding sites. These results provide new experimental evidence that rat liver glucocorticoid receptor would possess α- l-fucosyl and α- d-mannopyranoside residues in close proximity to the glucocorticoid receptor binding domain.

Biochemical characterization of the rat liver receptor for 2,3,7,8-TCDD - a comparison to the rat liver glucocorticoid receptor

An assessment of the physicochemical characteristics of the rat hepatic receptors for 2,3,7,8-TCDD and for corticosteroid hormones reveals striking analogies between both receptor systems.

Activation of rat liver glucocorticoid receptor bound to the antiglucocorticoid Ru38486

The kinetics of steroid binding to rat liver glucocorticoid receptor (GR) and receptor denaturation were dependent upon the nature of the molecule occupying GR. Both the agonist [triamcinolone acetonide (TA)] and the antagonist (Ru38486) however competed for the same saturable binding site. Despite opposing physiological action, both steroid analogues permitted receptor activation as evident by binding to DNA-cellulose and 9S to 4S shift on sucrose gradient sedimentation. It therefore seems necessary to reevaluate a current notion that antagonist action of RU38486 in rat liver is a result of impaired receptor activation.

Mapping of glucocorticoid receptor immunoreactive neurons in the rat tel- and diencephalon using a monoclonal antibody against rat liver glucocorticoid receptor.

By means of a monoclonal antibody against the rat liver glucocorticoid receptor (GR) in combination with the indirect immunoperoxidase technique it has been possible to demonstrate GR-immunoreactive nerve and glial cell nuclei all over the tel- and diencephalon of the male rat. Strongly GR-immunoreactive nerve cell nuclei were only present in the parvocellular part of the paraventricular hypothalamic nucleus, in the anterior periventricular hypothalamic nucleus, in the ventral part of the mediobasal hypothalamus, and in the CA1 and CA2 subregion of the hippocampal formation. Within the paraventricular hypothalamic nucleus a substantial overlap exists between the GR-immunoreactive area and the CRF-immunoreactive area. Medium to high densities of moderately GR-immunoreactive nerve cell nuclei were present all over the cortical hemispheres. Medium densities of moderately GR-immunoreactive nerve cells were demonstrated in many thalamic nuclei and in the central amygdaloid nucleus. After adrenalectomy the GR immunoreactivity was predominantly located in the pericaryon. Upon acute corticosterone treatment of adrenalectomized male rats, the GR immunoreactivity was again mainly demonstrated in the nerve cell nuclei indicating that corticosterone can translocate GR from the cytoplasm to the cell nuclei. It is suggested that the hypothalamic GR may be involved in the regulation of especially CRF secretion but also in the secretion of other anterior pituitary hormones such as TRH and somatostatin.

Characterization of the purified molybdate-stabilized glucocorticoid receptor from rat liver. An in vitro transformable complex.

Rat liver glucocorticoid receptor was purified in the presence of molybdate by a three-step procedure comprising protamine sulfate precipitation, affinity chromatography on a dexamethasone matrix and high-performance size-exclusion chromatography (HPSEC) on a TSK G 3000 SW column. The [3H]triamcinolone-acetonide-receptor complex was obtained in 20% yield with an overall 11 800-fold purification. The dissociation rate constant of this complex was 1.6 X 10(-4) min-1. The purified receptor sedimented at 8.3 S in high-salt and 9.4 S in low-salt sucrose gradients containing molybdate. A 7.0-nm Stokes radius was determined by HPSEC on a TSK G 4000 column in high-salt buffer. The calculated Mr was 278000. Dodecyl sulfate/polyacrylamide gel electrophoresis revealed an almost homogeneous 90 000-Mr band. Three minor bands with Mr of 78 000, 72 000 and 48 000 were also inconstantly seen. An apparent pI = 5.1 was observed for the [3H]steroid complex by isoelectric focusing in agarose gel. Furthermore high-performance ion-exchange chromatography of the purified complex on a DEAE 545 LKB column (DEAE HPLC) yielded a sharp peak eluted at a 315 mM potassium ion concentration. This peak was shown to contain almost all the 90 000-Mr protein. Moreover the purified receptor complex appeared to be transformable to a DNA-binding state after molybdate removal followed by warming 30 min at 25 degrees C in presence of 0.2% bovine serum albumin: 50-78% transformation yield could be demonstrated by DNA-cellulose chromatography. Partial transformation could also be obtained at 0 degrees C in the absence of any added protein and was followed by DEAE HPLC. The transformed complex was eluted by 180 mM potassium.

Immunocytochemical studies on the localization of glucocorticoid receptor immunoreactive nerve cells in the lower brain stem and spinal cord of the male rat using a monoclonal antibody against rat liver glucocorticoid receptor

By means of the indirect immunoperoxidase method glucocorticoid receptor (GR) immunoreactive nerve cells of the lower brain stem and the spinal cord have been mapped out in the rat, using a monoclonal antibody against rat liver GR. The GR immunoreactivity was predominantly located within the nuclei of these nerve cell bodies but also in glial cells of the gray and white matters. Strongly GR immunoreactive nerve cells were mainly found in the area of the noradrenaline, adrenaline and 5-hydroxytryptamine (5-HT) cell groups of the lower brain stem, and of the substantia gelatinosa of the nuc. tractus spinalis nervi trigemeni and spinal cord. The results suggest that glucocorticoids control transmitter and metabolic functions in discrete areas of the brain stem and spinal cord.

Monoclonal antibody to the rat glucocorticoid receptor. Relationship between the immunoreactive and DNA-binding domain.

The region of the glucocorticoid receptor that reacted with a monoclonal antibody (BUGR-1) was identified. In order to identify the immunoreactive region, the rat liver glucocorticoid receptor was subjected to limited proteolysis; immunoreactive fragments were identified by Western blotting. The monoclonal antibody reacted with both the undigested Mr approximately 97,000 receptor subunit and a Mr approximately 45,000 fragment containing the steroid-binding and DNA-binding domains. Digestion by trypsin also produced two steroid-binding fragments of Mr approximately 27,000 and 31,000 which did not react with the antibody and an immunoreactive Mr approximately 16,000 fragment. This Mr approximately 16,000 fragment was shown to bind to DNA-cellulose, indicating that it contained a DNA-binding domain of the receptor. The undigested receptor must have steroid associated with it to undergo activation to a DNA-binding form. However, the Mr approximately 16,000 immunoreactive fragment binds to DNA-cellulose even if it is obtained by digestion of the steroid-free holoreceptor which does not itself bind to DNA.

Chymotrypsin treatment of glucocorticoid receptor attenuates RNA-dependent inhibition of DNA binding. Evidence for a distinct RNA-binding site

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.

Characterization of non-liganded glucocorticoid receptor in rat liver cytosol using indirect competitive enzyme-linked immunosorbent assay

We have previously shown that the purified or unfractionated cytosolic, activated glucocorticoid receptor of rat liver consists of a polypeptide with a Stokes radius of ~ 6 nm, a sedimentation coefficient of 4 S and a molecular mass of ~ 90,000 Daltons. We have confirmed previous observations by other authors that if sodium molybdate is introduced into the cytosol preparation buffer the non-activated glucocorticoid receptor appears as an 8 nm, 9 S species with an apparent molecular mass of 330,000 Daltons. In order to study the physicochemical parameters of the glucocorticoid receptor prior to ligand binding, we have used an enzyme-linked immunosorbent assay (ELISA) based on antibodies raised in rabbits against the purified activated glucocorticoid receptor. In isotonic buffer, the non-liganded glucocorticoid receptor was shown to have a Stokes radius of 6 nm in the absence and 8 nm in the presence of molybdate. Furthermore, experimental conditions known to result in activation of the glucocorticoid receptor complex (increased ionic strength, increased temperature) did not lead to activation of the 6 nm non-liganded glucocorticoid receptor as judged from the lack of binding of the treated, non-liganded receptor to DNA-cellulose. The existence of both 6 and 8 nm forms of nonactivated, non-liganded glucocorticoid receptor in vitro suggests that dissociation of an 8 nm form to a 6 nm form, if it occurs in vivo, is probably not the only molecular event constituting the activation of the glucocorticoid receptor.

Effects of D- and L-thyroxine on the glucocorticoid binding capacity of adult rat liver

Administration of either D- or L-thyroxine (T 4) significantly increased the glucocorticoid binding capacity of cytosol of the livers of adrenalectomized adult rats. Administration of up to 0.5 mg/100 g body wt. of L-T 4 was more effective than that of D-T 4, but higher doses (0.8–3 mg/100 g body wt.) of D-T 4 increased the binding capacity markedly to more than that with L-T 4. T 4-administration did not alter the apparent dissociation constant of glucocorticoid binding proteins for glucocorticoid binding, or their behavior on DEAE-cellulose chromatography either before or after thermal activation (23°C for 40 min). Thus the increased binding capacity seemed to be due to increase in the level of glucocorticoid receptor in rat liver.

Characterization of wild type and mutant glucocorticoid receptors from rat hepatoma and mouse lymphoma cells.

Using a combination of immunological blotting techniques and hormone affinity labeling, we have characterized the glucocorticoid receptors present in wild type and mutant rat hepatoma (HTC) and mouse thymoma (S49 and WEHI7) cells. Mutant HTC and WEHI7 cells of the receptorless phenotype, which contain greatly reduced amounts of glucocorticoid hormone binding activity, show parallel decreases in immunoreactive material using a monoclonal antibody raised against the rat liver glucocorticoid receptor. This indicates that these receptorless mutant cells harbor defects in either the production or accumulation of receptor protein. Quantitation of immunoreactivity and hormone binding activity present in wild type and mutant S49 cells indicates that these cells contain significantly more immunoreactive material than hormone binding activity. We conclude that S49 cells produce, in addition to their well characterized wild type or mutant receptors, a mutant receptor from a second allele which is of wild type size, is immunologically reactive, but is unable to bind hormone. The S49 mutant cell line nti (nuclear transfer increase) contains a glucocorticoid receptor which has a molecular weight of 40,000, while the wild type receptor has a molecular weight of 94,000. Affinity labeling of glucocorticoid receptors in nti cells with [3H]dexamethasone mesylate indicates that nti cells do not contain wild type sized precursor molecules which bind hormone, nor do they contain immunoreactive fragments of a molecular mass smaller than 94 kDa. It is proposed that the 40-kDa nti receptor is produced as a truncated protein most likely resulting from a nonsense mutation or from a truncated messenger RNA.

Phosphorylation of rat liver glucocorticoid receptor.

Rat liver glucocorticoid-receptor complex (GRc) was purified 2000-fold by a combination of methods including (NH4)2SO4-fractionation and phosphocellulose and DNA-cellulose chromatography. The purified glucocorticoid receptor preparation contained a major peptide of Mr = 90,000 and the GRc sedimented as 4 S in 5-20% sucrose gradients. An additional peptide of Mr = 45,000 (45K) was also observed. Some preparations yielded only the Mr = 90,000 (90K) peptide suggesting that the 45K peptide may be a proteolyzed portion of the 90K protein. The purified GRc was incubated with [gamma-32P]ATP in the presence of cAMP-dependent kinase. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the above preparation revealed the presence of two 32P-containing bands with apparent Mr = 90,000 and 45,000. The 32P incorporation was dependent on the availability of divalent cation (Mg2+). GRc in cytosol labeled with [3H]dexamethasone mesylate and purified as above co-migrated with 32P-containing bands. GRc was also purified from cytosol obtained from livers of rats injected with [32P]orthophosphate. Both 32P and 3H bands were associated with 90K and 45K peptides. Our results indicate that rat liver glucocorticoid receptor is a phosphoprotein and that both the phosphorylated peptides 90K and 45K also contain the steroid and the DNA binding regions of the glucocorticoid receptor.

Glucocorticoid and progesterone receptors bind to the same sites in two hormonally regulated promoters.

The glucocorticoid receptor of rat liver recognizes nucleotide sequences near the promoter of mouse mammary tumour virus (MMTV) required for hormonal induction in gene transfer experiments. Similar nucleotide sequences have been found in the human metallothionein gene IIA and in the chicken lysozyme gene, the later induced also by oestrogen, progesterone and androgens. In microinjection experiments, deletion of only 44 base pairs (bp) of the lysozyme promoter (from -208 to -164) results in coordinated loss of progesterone and glucocorticoid-dependent gene expression. We show here that purified glucocorticoid receptor from rat liver and progesterone receptor from rabbit uterus yield similar or overlapping exonuclease III footprints in the promoter regions of MMTV and chicken lysozyme. Thus, the regulatory elements for different steroid hormones may be similar or at least share structural features.

Cloning of the human glucocorticoid receptor cDNA

We show that the human glucocorticoid receptor (GR), isolated from the breast cancer cell line MCF-7, has an apparent molecular weight identical to that of rat liver GR (94 kDa) and reacts with antibodies raised against the latter. These antibodies were used to clone cDNA sequences corresponding to the human GR from a lambda gt11 expression library constructed using MCF-7 poly(A)+ RNA. Three non-homologous cDNA clones with inserts of 125, 220 and 350 bp, which express epitopes recognised by the rat liver GR antibodies, were isolated. Rat liver GR antibodies, immunopurified using the immobilised purified beta-galactosidase fusion proteins, detect partially purified rat liver and human GRs on Western blots. In addition, these antibodies immuno-adsorb rat liver and human GRs affinity-labelled with [3H] triamcinolone acetonide. Northern blot analysis, using all three cDNA probes, reveals the presence of a major MCF-7 poly(A)+ RNA species of approximately 7 kb.

Effect of colchicine on the depletion and replenishment of cytoplasmic glucocorticoid receptor in rat liver after administration of glucocorticoid.

Pretreatment of rats with colchicine (3 mg/kg body weight) modified the time course of depletion of the cytoplasmic binding sites for 3H-dexamethasone after administration of prednisolone (0.5 or 1.5 mg/kg body weight). Colchicine also decreased the rate of the cytoplasmic receptor replenishment which was confirmed by application of this drug after completion of the cytoplasmic receptor translocation to nuclei (30 min after prednisolone injection). Addition of colchicine to the incubation mixture for in vitro binding of 3H-dexamethasone-labelled liver cytosol to isolated liver nuclei suspended in TKMS buffer (50 mM Tris-HCl, pH 7.5, 50 mM KCl, 5 mM MgCl2 and 250 mM sucrose) evoked no measurable changes in the rate of the nuclear binding.

Protein kinase activity of purified rat liver glucocorticoid receptor

Molybdate-stabilized nonactivated rat liver glucocorticoid receptor (GR) was purified to near homogeneity using a biospecific affinity adsorbent, Bio Gel A 0.5m and DEAE-Sephacel. The purified GR sedimented in the 9–10S region in 5–20% sucrose gradients containing 0.10M KCl and 20mM Na 2MoO 4. SDS-polyacrylamide gel electrophoresis revealed a major single band with an apparent molecular weight of 90,000 ± 2,000. Affinity labeling of GR with [ 3H]-dexamethasone mesylate showed association of the radioactivity with a peptide of 90,000 molecular weight. Purified receptor preparation was dialyzed to remove molybdate and was incubated with different protein substrates in the presence of 50μM [λ- 32P]-ATP and divalent cations. Radioactive phosphate from [λ- 32P]-ATP was seen to be incorporated into calf thymus histones, turkey gizzard myosin light chain kinase and rabbit skeletal muscle kinase in the presence of Mg 2+ and Ca 2+ ions. Addition of steroid ligand exogenously to the reaction mixture appeared to increase the extent of protein phosphorylation. No autophosphorylation of GR was evident under the above conditions. The data suggest that purified rat liver GR displays protein kinase activity.

Ontogeny of rat brain and liver glucocorticoid receptors.

Glucocorticoid hormone action occurs via a two-step process: Glucocorticoid receptor (GR) forms a cytoplasmic complex with the steroid (G - R complex) which is readily transformed to at least two more basic nucleophilic forms capable of rapid translocation into the nucleus, thereby eliciting a biological response (The transformation - translocation mechanism). Although age dependent differences in the concentration and the properties of rat liver G-R complexes have been reported (Grote et al. 1977), information concerning developing rat brain is limited. A detailed comparative study of GR concentration and G-R complex transformation - translocation effectiveness in the two tissues during development was therefore carried out. Preparation of cytoplasmic GR or G-R complex with [3H]dexamethasone, Scatchard analysis, and column chromatography were carried out as previously described (Alexis et al. 1983). For in vivo nuclear uptake of [3H]dexamethasone, 5 μg per 100 g body weight were injected ip one hour before sacrifice. Labelled

Characterization of a steroid hormone receptor gene and mRNA in wild-type and mutant cells.

The effects of steroid hormones are mediated by intracellular hormone-specific receptor proteins; the interaction between the hormone and its receptor increases the affinity of the receptor for nuclear binding sites, thereby modulating the expression of specific genes. The glucocorticoid receptor is a soluble protein of relative molecular mass (Mr) 94,000 (94K), present at a low relative abundance (less than or equal to 0.01%); it has been purified to near-homogeneity, and specific antisera and monoclonal antibodies have been produced. Purified glucocorticoid receptor binds in vitro with high affinity to defined regions of DNA near regulated promoters, and sequences essential for these interactions are functional in vivo as hormone-dependent transcriptional enhancer elements. We have now cloned complementary DNA (cDNA) for the rat liver glucocorticoid receptor and we describe here a 2.6-kilobase (kb) receptor cDNA isolated following polysome immuno-enrichment of receptor messenger RNA with glucocorticoid receptor-specific antibodies. The receptor appears to be encoded by a single-copy gene which specifies a approximately 6-kb transcript in rat and mouse cells; this mRNA is altered quantitatively and qualitatively in several mutant cell lines with specific defects in receptor function.

Inactivation of unbound rat liver glucocorticoid receptor by N-alkylmaleimides at sub-zero temperatures.

A series of N-alkylmaleimides was shown to inactivate effectively the rat liver glucocorticoid receptor at neutral pH. A partial purification of the unbound cytosolic receptor by protamine sulfate precipitation and a careful stabilization of the essential thiol by dithiothreitol and sodium molybdate before the alkylation step appeared essential to obtain pseudo-first-order kinetics. Moreover, performing the experiment at -12 degrees C in buffer containing 40% glycerol as antifreeze agent resulted in increased receptor stabilization and a slowing-down of the inactivation process, which could then be more accurately studied. This process was demonstrated to be dose- and pH-dependent in the case of N-ethyl- and N-nonylmaleimides. Furthermore, comparison of the various N-alkylmaleimides revealed a striking increase of receptor inactivation with increasing chain length of the maleimide derivative. Full protection against inactivation was afforded by previous [3H]dexamethasone binding on the receptor. Long-chain N-alkylmaleimides inactivated by beta-mercaptoethanol were still able to inhibit the [3H]-dexamethasone binding noncovalently. Likewise N-nonylsuccinimide was shown to compete with [3H]dexamethasone for receptor binding. It is suggested that the chain length effect observed in the inactivation process is related to nonpolar interactions in the binding of maleimides to the receptor prior to the irreversible alkylation of sulfhydryl groups. These groups lie in a hydrophobic environment, probably in the steroid binding site itself.