A specific glucocorticoid binding macromolecule of rabbit uterine cytosol

A high affinity (K_d=2.7 × 10^?10M at 0°) dexamethasone binding macro-molecule has been identified in the cytosol fraction of rabbit uteri. Competition studies show high specificity for glucocorticoids since binding of labeled dexamethasone is inhibited by cortisol and corticosterone but not by progesterone, testosterone, or estradiol 17β. The binding component has a sedimentation coefficient of 8S and its concentration in uterine cytosol is about 0.2 pmoles per mg protein. Uptake of labeled dexamethasone by isolated uterine nuclei requires the presence of cytosol and is temperature dependent. The KCl-extractable nuclear complex sediments at 4S. Thus the dexamethasone binding components of the rabbit uterus have properties similar to those described for steroid hormone receptors present in target tissues. Specific dexamethasone binding could not be demonstrated in rat uterine cytosol.     

ATP-dependent activation of glucocorticoid receptor from rat liver cytosol.

The glucocorticoid--receptor complex from freshly prepared rat liver cytosol is in a non-activated form, with very little affinity to bind to isolated nuclei. When such preparations were incubated with 5--10 mM-ATP at 4 degrees C, the receptor complex acquired the properties of an 'activated' transformed form, which readily bound to nuclei, ATP--Sepharose, phosphocellulose and DNA--cellulose. This transformation was comparable with the activation achieved by warming the steroid--receptor complex at 23 degrees C. The effect of ATP was specific, as it was more effective than ADP, whereas AMP had no such effect on activation. The process of receptor activation was sensitive to the presence of 10 mM-sodium molybdate; the latter blocked activation by both ATP and heat. Bivalent cations had no observable effect on the receptor activation at low temperature, but they decreased the extent of activation by ATP. The steroid-binding properties of glucocorticoid receptor remained intact under the above conditions. However, a significant increase in steroid binding occurred when ATP was preincubated with cytosol receptor before the addition of [3H]triamcinolone acetonide. ATP also stabilized the glucocorticoid--receptor complexes at 23 degrees C. These results suggest a role for ATP in receptor function and offer a convenient method of studying the activation process of glucocorticoid receptor under mild assay conditions.     

Purification of the glucocorticoid receptor from rat liver cytosol.

The [3H]-triamcinolone acetonide-labeled glucocorticoid receptor from rat liver cytosol was purified to 85% homogeneity according to sodium dodecyl sulfate gel electrophoresis. It consisted of one subunit with a molecular weight of 89,000 and had one ligand-binding site per molecule. The purification involved sequential chromatography on phosphocellulose, DNA-cellulose twice, and Sephadex G-200. Between the two chromatography steps on DNA-cellulose, the receptor was heat activated. The receptor was affinity eluted from the second DNA-cellulose column with pyrodixal 5'-phosphate. The purification achieved in the first three chromatographic steps varied between 60 and 95% homogeneity in different experiments. After chromatography on the second DNA-cellulose column, the steroid.receptor complex had a Stokes radius of 6.0 nm and a sedimentation coefficient of 3.4 S in 0.15 M KCl. In the absence of KCl, the sedimentation coefficient was 3.6 S. After concentration on hydroxylapatite, the steroid.receptor complex was analyzed by isoelectric focusing in polyacrylamide gel. The radioactivity was shown to focus together with the major protein band with pI 5.8. Following limited proteolysis with trypsin, the radioactivity, together with the major protein band, focused at pI 6.2 as previously described for the unpurified steroid.receptor complex.     

Characterization of a progestin-binding macromolecule in the amphibian oocyte cytosol.

A [³H]-progestin-binding macromolecule has been isolated from $\text{R}\text{.}\text{pipiens}$ oocyte cytosol and characterized using binding assays, gel filtration, DEAE-cellulose chromatography and ultracentrifugal techniques. Macromolecules present in the prophase oocyte cytosol have a high affinity and specificity for the synthetic progestin R5020 and the intact oocyte will concentrate both R5020 and progesterone 20–40 fold from the medium. This may be the first published case of a cytosol steroid binding macromolecule in a cell system in which the steroid appears to act at an extranuclear level.     

Subunit composition of the untransformed glucocorticoid receptor in the cytosol and in the cell.

We have used bifunctional reagents to examine the subunit composition of the non-DNA-binding form of the rat and human glucocorticoid receptor. Treatment of intact cells and cell extracts with a reversible cross-linker, followed by electrophoretic analysis of immunoadsorbed receptor revealed that three proteins of apparent approximate molecular masses, 90, 53 and 14 kDa are associated with the receptor. The first of these was identified immunochemically as a 90-kDa heat-shock protein (hsp90). The complex isolated from HeLa cells contained 2.2 mol hsp90/mol steroid-binding subunit. Cross-linking of the receptor complex in the cytosol completely prevented salt-induced dissociation of the subunits. The cross-linked receptor was electrophoretically resolved into two oligomeric complexes of apparent molecular mass 288 kDa and 347 kDa, reflecting the association of the 53-kDa protein with a fraction of the receptor. Since no higher oligomeric complexes could be generated by cross-linking cell extracts under different conditions, we conclude that most of the untransformed cytosolic receptor is devoid of additional components.     

Unsaturated fatty acids inhibit glucocorticoid receptor binding of trout hepatic cytosol.

1. The effect of free fatty acids [FFAs (saturated (S) and unsaturated (U))] on dexamethasone binding in vitro using liver cytosol from rainbow trout was examined. 2. All UFFAs but none of the SFFAs tested suppressed binding. This suppression is dose-dependent and correlated roughly with the degree of unsaturation of the FFAs. 3. Scatchard analysis indicated that the addition of linoleic C18:2 (150 microM) increased the dissociation constant (Kd = 5.1 +/- 0.4 x 10(-8) M vs control of 1.7 +/- 0.3 x 10(-8) M) but minimally affected the binding capacity (Bmax = 68 +/- 6.2 vs control of 88 +/- 15.2 fmol/mg protein) suggesting C18:2 caused a conformational change of the receptor. 4. Lineweaver-Burk plot revealed a mixed non-competitive type of inhibition by C18:2. 5. Free acid appeared to be required for inhibition as esterification or derivatization of the acid greatly diminished its potency. 6.C18:2 also promotes the dissociation of bound [3H]-dexamethasone from the steroid-receptor complex but slower in rate and lesser in magnitude compared to that caused by dexamethasone or the glucocorticoid antagonist RU 38486. 7. UFFAs and some of their derivatives can thus modulate glucocorticoid receptor function in vitro and might play essential roles in regulating glucocorticoid action in fish as well. 8. These fatty acids presumably acts at a site different from that of the glucocorticoid binding site.     

Characterization of the purified activated glucocorticoid receptor from rat liver cytosol

The activated glucocorticoid receptor (GR) from rat liver cytosol was purified by sequential chromatography on DNA-cellulose and DEAE-Sepharose. Analysis by sodium dodecyl sulfate-gel electrophoresis demonstrated a main band with Mr = 94,000 (94K band). Two minor bands with Mr = 79,000 (79K band) and 72,000 (72K band) were also seen in this preparation. Photoaffinity labeling showed that the hormone is bound to the 94K and 79K components but not to the 72K component. Immunoblotting using antibodies raised against the 94K protein demonstrated cross-reactivity between the 94K and 79K components but not with the 72K species. The 72K species could be partially separated from the 94K and 79K components by density gradient centrifugation. Limited proteolysis of the purified GR with trypsin or alpha-chymotrypsin led to degradation of the 94K and 79K components and appearance of a 39K fragment which still retained the hormone and could be bound to DNA-cellulose. The 72K component was not affected by digestion with trypsin or alpha-chymotrypsin. However, chromatography on DNA-cellulose of the alpha-chymotrypsin-treated GR resulted in elution of the 72K component in the flow-through of the column while the 39K fragment was retained on the column and eluted with 0.18 M NaCl. In the control experiment where no alpha-chymotrypsin treatment was performed, the 72K component could not be detected in the flow-through fraction but was eluted together with the 94K and 79K components at 0.18 M NaCl. These results suggest that the 72K protein might be bound to the 94K and/or 79K component. The 39K fragment did not bind antibodies raised against the 94K protein. The 39K fragment was further degraded by trypsin but not by alpha-chymotrypsin to a 27K and a 25K fragment while both still retained the ligand. These data obtained with limited proteolysis of the purified GR are in agreement with previous findings on proteolysis of the GR in crude cytosol (Wrange, O., and Gustafsson, J.-A. (1978) J. Biol. Chem. 253, 856-865; Carlstedt-Duke, J., Okret, S., Wrange, O., and Gustafsson, J.-A. (1982) Proc. Natl. Acad. Sci. U.S.A. 79, 4260-4264).     

The pituitary function of androgen receptor constitutes a glucocorticoid production circuit

Androgen receptor (AR) mediates diverse androgen actions, particularly reproductive processes in males and females. AR-mediated androgen signaling is considered to also control metabolic processes; however, the molecular basis remains elusive. In the present study, we explored the molecular mechanism of late-onset obesity in male AR null mutant (ARKO) mice. We determined that the obesity was caused by a hypercorticoid state. The negative feedback system regulating glucocorticoid production was impaired in ARKO mice. Male and female ARKO mice exhibited hypertrophic adrenal glands and glucocorticoid overproduction, presumably due to high levels of adrenal corticotropic hormone. The pituitary glands of the ARKO males had increased expression of proopiomelanocortin and decreased expression of the glucocorticoid receptor (GR). There were no overt structural abnormalities and no alteration in the distribution of cell types in the pituitaries of male ARKO mice. Additionally, there was normal production of the other hormones within the glucocorticoid feedback system in both the pituitary and hypothalamus. In a cell line derived from pituitary glands, GR expression was under the positive control of the activated AR. Thus, this study suggests that the activated AR supports the negative feedback regulation of glucocorticoid production via up-regulation of GR expression in the pituitary gland.     

The principal glucocorticoid binding macromolecule in hepatoma cells in culture is similar to corticosteroid Binder II of rat liver cytosol

Reuber H-35 hepatoma (H4-II-E-C3) and HTC cells are known to retain differentiated corticosteroid induced functions in cell culture and to bind corticosteroids to macromolecules in cytosol which subsequently enter the cell nuclear fraction. Using both cell types we have demonstrated the major macromolecular fraction in cytosol to have properties (elution position from DEAE columns, pI, ³H-dexamethasone binding), very similar to those of rat liver corticosteroid Binder II which may be the hormone receptor.     

Cellular localization and distribution of glucocorticoid receptor immunoreactivity and the expression of glucocorticoid receptor messenger RNA in rat pituitary gland

By means of double immunohistochemical techniques and a nonradioisotopic in situ hybridization method, we determined the colocalization pattern of glucocorticoid receptor (GR) and pituitary hormones and the GR messenger RNA (mRNA) expression in the pituitaries of Wistar adult male rats. Immunoreactivity for GR was detected in the nuclei of cells in the anterior and posterior pituitary. Double immunohistochemistry revealed that the colocaliza- tion of GR and anterior pituitary hormones occurred in almost 99% of the growth hormone (GH)-producing cells and adrenocorticotropic hormone (ACTH)-producing cells, and in 67% of the thyroid stimulating hormone (TSH)-producing cells. Almost all of the folliculostellate cells (93%), marginal layer cells (94%) in the anterior pituitary, and pituicytes (96%) in the posterior pituitary immunostained for S100 protein antibody were also immunostained with GR. GR mRNA was abundant in the cytoplasm of anterior and intermediate pituitary cells but scattered sparsely in that of the posterior pituitary. These results suggest that glucocorticoids directly influence certain pituitary cells in order to regulate cell function, including the synthesis and/or secretion of hormones.     

Inclusion of the glucocorticoid receptor in a hypothalamic pituitary adrenal axis model reveals bistability

Background

This article deals with the theoretical size distribution (of number of sub-taxa) of a fossil taxon arising from a simple null model of macroevolution.

Model

New species arise through speciations occurring independently and at random at a fixed probability rate, while extinctions either occur independently and at random (background extinctions) or cataclysmically. In addition new genera are assumed to arise through speciations of a very radical nature, again assumed to occur independently and at random at a fixed probability rate.

Conclusion

The size distributions of the pioneering genus (following a cataclysm) and of derived genera are determined. Also the distribution of the number of genera is considered along with a comparison of the probability of a monospecific genus with that of a monogeneric family.     

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 approximately 6 nm, a sedimentation coefficient of 4S and a molecular mass of approximately 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, 9S 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.     

Formation of a fluorescent glucocorticoid receptor-steroid complex in HTC cell cytosol

An intensely fluorescent rhodamine derivative of dexamethasone (i.e. Dex-C2-Rho) was synthesized. Dex-C2-Rho possessed high affinity for HTC cell glucocorticoid receptors in cell-free systems. Whole cell activity and receptor affinity of Dex-C2-Rho were both much lower, apparently due to problems with cell permeability and/or metabolism. A specific, fluorescent receptor-steroid complex at concentrations as low as 1 X 10(-9) M could readily be observed with crude HTC cell receptors after removal of the free Dex-C2-Rho. This appears to be the first report of a fluorescent glucocorticoid receptor-steroid complex.     

Stabilization of glucocorticoid receptor association with RNA by a low molecular weight factor from rat liver cytosol

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.     

Evidence for a factor in liver cytosol which controls the competition between progesterone and triamcinolone acetonide for the glucocorticoid receptor

There is an inhibitory factor in rat liver which prevents progesterone and certain of its analogues from competing with triamcinolone acetonide for binding to the blucocorticoid receptor. The effects of this inhibitor can be eliminated by dilution of the cytosol or by gel filtration. Neither rat thymus nor serum contain this factor.     

Decreased liver cytosol glucocorticoid receptors in protein-deficient rats

A low protein diet (5% as compared to a control 21% protein diet, $\text{ad libitum}$) caused a significant decrease in the concentration of liver cytoplasmic glucocorticoid receptors; the equilibrium dissociation constant (Kd) did not change. The maximum decrease occurred in two weeks and was reversible upon substitution of the low protein by a control diet. This influence of protein deficiency could not be attributed to elevated plasma corticosterone levels since a comparable increase in plasma corticosterone of calorie-deficient rats (21% protein diet in restricted quantity) did not decrease glucocorticoid receptors and the difference in receptor levels of control and protein deficient animals persisted following adrenalectomy. These results suggest that glucocorticoids might not exert their usual biologic effects in the presence of protein malnutrition.     

Localization of putative gonadotrophin releasing hormone receptor protein in the anterior pituitary

Specific binding of a fully biologically active 125I-gonadotrophin releasing hormone (GnRH) to isolated anterior pituitary cells is time dependent, saturable and the concentration dependent binding curves exhibit positive cooperativity. Binding to intact or solubilized plasma membranes and an affinity purified GnRH receptor protein reveals in all instances multiple high affinity binding sites. Thus, GnRH receptor protein appears to be an intrinsic constituent of the cell membrane, and perhaps, other membranous organelles. To investigate the latter, the binding of 125I-GnRH to various subcellular fractions was studied and its affinity and time requirements determined. GnRH binding to plasma membranes and secretory granules was to multiple high affinity sites, while that to nuclei and microsomes was to a single high affinity site. Binding was 1.83 +/- 0.07, 0.78 +/- 0.04, 0.31 +/- 0.03 and 0.27 +/- 0.03 fmol micrograms-1 protein for isolated plasma membranes, secretory granules, microsomes and nuclei, respectively, after 30 min incubation with 10(-9) M GnRH. The magnitude of binding to microsomes did not change during the incubation period. It did not show any decrease (p greater than 0.05) in isolated nuclei and plasma membranes, except for the 24 h time period, when a significant drop (p less than 0.001) was seen. Binding to the secretory granule fraction culminated at 15 min and then decreased (p less than 0.001) steadily to a non-detectable level at 24 h. Thus GnRH receptor protein or its portion may be an integral part of some membranous particles in the anterior pituitary cells.(ABSTRACT TRUNCATED AT 250 WORDS)