Glucocorticoid-binding Proteins Research Articles

Differences between cytoplasmic glucocorticoid-binding proteins shown by heterogeneity of nuclear acceptor sites.

IT is now widely accepted that most, if not all, glucocorticoid responses in target tissue are mediated by specific steroid receptor molecules in the cytoplasm1,2. Following association between steroid and receptor, there is transport of the steroid to the nucleus where subsequent interaction with the genome presumably alters transcriptional processes3. This scheme does not yet satisfactorily explain the widely differing responses of various tissues to glucocorticoids. Specificity of the kind of response observed could lie in differences in the receptor molecule itself and/or in the ability of a common receptor molecule to interact only with certain ‘exposed’ parts of the nuclear material in a given cell. Some evidence has favoured the latter interpretation4, but there has not yet been support for the former contention. In this report, we present evidence that at least some degree of specificity of response may be mediated by subtle differences in the cytoplasmic corticosteroid-binding proteins between tissues with differing kinds of responses to glucocorticoids. Our data further suggest that there are intranuclear binding sites for cytoplasmic glucocorticoid receptors which are of high affinity and saturability and which can distinguish between glucocorticoid receptors of differing origin.

Isolation of cytoplasmic glucocorticoid-binding protein(s) from rat liver by means of affinity chromatography, and its partial characterization

Intracellular corticosteroid-binding protein(s) was isolated from rat liver cytosol by means of affinity chromatography, using amino-sepharose coupled to cortisol hemisuccinate. This protein material displayed apparent homogeneity on Sephadex G-100 and G-200 gel filtrations. Its molecular weight is 93 000 (average of two estimations), as calculated from sedimentation and diffusion constants. The amino acid composition of this protein closely resembles that of transcortin, although some quantitative differences between the two exist. The carbohydrate content of this protein was found to be appreciably smaller than that of transcortin. Calculation of the amount of possible contamination of the isolated protein(s) with residual blood present within the vascular bed of the liver tissue prior to homogenization indicates that at most one-fourth of the cytoplasmic corticosterone-binding protein(s) could be due to contamination with transcortin from plasma. On starch gel and polyacrylamide gel electrophoresis, the isolated protein material resolved into two major and one minor components, migrating in the α 1- and α 2- globulin region . Competitive binding studies with different steroids revealed that the isolated protein(s) has high affinity for progesterone, corticosterone, and cortisol. For all the three steroids, at least two binding components were present on the Scatchard plot: one possessing high affinity but low capacity ( K A = 10 −8 M, n = 15 · 10 −9 M ), the other, lower affinity but higher capacity ( K A = 10 −6 M , n = 160 · 10 −9 M .

Characterization of a distinct glucocorticoid-binding protein in the lactating mammary gland of the rat

Specific substances binding [ 3H]triamcinolane acetonide were detected in the cytosol fraction of the lactating mammary gland of the rat using sucrose gradient centrifugation. These receptors, which were protein in nature, exhibited sedimentation coefficients of 7–8 S and dissociated into lower molecular weight components sedimenting at 4–5 S when separated on sucrose gradients containing 0.4 M KCl. The cytoplasmic form of the binding protein was relatively specific for glucocorticoids although progesterone inhibited binding significantly. The dissociation constant ( K d) of the receptor-ligand complex was in the range of 10 −8 M. p-Chloromercuribenzoate diminished the ligand-binding capacity of the receptor suggesting a role for sulfhydryl groups in the binding reaction. Cytosols from mammary tissue obtained from virgin and pregnant rats revealed a paucity of binding sites as compared to those in the lactating gland. Examination of ligand-binding specificity indicates that these glucocorticoid-binding sites are distinct and easily discriminated from those of either the estrogen receptor of the mammary gland or the triamcinolone-binding component in plasma.

Glucocorticoid-binding proteins in human acute lymphoblastic leukemic blast cells.

The first known step in steroid hormone action is the association of the steroid with specific cytoplasmic steroid-binding proteins (SBP). Using a competitive binding assay, we detected, quantified, and partially characterized such a SBP in cytosol from glucocorticoid-sensitive human lymphoblastic leukemic blasts. The affinity of steroids for the SBP was directly related to their known killing potency. For example, steroids without glucocorticoid effect such as androstenedione, etiocholanolone, and tetrahydrocortisol were unable to displace radiolabeled dexamethasone from the SBP in the binding reaction. The dose-response curve for in vitro inhibition of [(3)H]thymidine uptake in leukemic blasts correlated closely with the binding affinity of glucocorticoids to the SBP, providing additional support for an essential physiologic role for SBP in steroid action. SBP activity was either greatly diminished or absent in glucocorticoid-resistant cells. Six patients who intially had SBP in their blasts and were responsive to combinations of drugs including glucocorticoids no longer had SBP activity detectable at a time when they no longer responded to combinations of drugs including glucocorticoids. In vitro [(3)H]thymidine uptake was not inhibited by steroids in leukemic blast cells lacking SBP activity. Other patients who had received some antileukemic therapy including glucocorticoids and who still had SBP in their leukemic blasts, were still responsive to drug combinations that included glucocorticoids. This appears to be the first study demonstrating glucocorticoid receptors in a human tissue.

Glucocorticoid binding proteins in human leukaemic lymphoblasts.

The binding of steroid hormones to highly specific cytoplasmic receptor proteins has been shown to be the first step in steroid hormone action in many systems1. These steroid binding proteins (SBP) seem to be found exclusively in target tissues and their kinetics of association and dissociation as well as their affinity for steroids correlate very well with biologic effects of the hormone2–6. SBP can be readily demonstrated in cells such as rat thymocytes and mouse lymphosarcoma cells in culture and these cells are lysed by glucocorticoids7,8. Conversely, when steroid resistant cell lines are cloned from mouse lymphoma cells, a marked decrease in SBP has been demonstrated9. In addition, loss of the biologic effect of glucocorticoids has been correlated with a decrease in SBP in cultured hepatoma and fibroblast cells10,11. As yet it has not been determined whether glucocorticoid receptors are of similar importance in human disease and so we examined human leukaemic lymphoblasts to see if a specific SBP was detectable and whether or not its presence or absence would correlate with glucocorticoid response in clinical situations and intact cells in culture. Very significant morbidity and mortality are associated with glucocorticoid therapy, particularly in the treatment of human malignancy12,13. The eventual goal would be the development of a rapid and reliable in vitro test to predict steroid responsiveness in human leukaemic cells and thus avoid the hazards of unnecessary steroid therapy.

Glucocorticoid-binding Proteins of Rat Liver Cytosol: II. PHYSICAL CHARACTERIZATION AND PROPERTIES OF THE BINDING PROTEINS

Abstract The three glucocorticoid-binding proteins present in rat liver cytosol, A, B, and G, can be separated upon chromatography on Sephadex gels or DEAE-cellulose. All three proteins are thermolabile, but the A and B proteins resist heating for 10 min at 40°, whereas the G protein is inactivated by this treatment. The binding of glucocorticoids to the G protein is inhibited by sulfhydryl-blocking reagents, whereas the binding to the A and B proteins does not require free sulfhydryl groups. Glucocorticoid binding to the G protein is optimal at physiological pH and ionic strength and is markedly affected by deviations from these conditions. The data from Sephadex gel filtration, sucrose gradient centrifugation, and equilibrium centrifugation in CsCl, allow the calculation of the Stokes radius, sedimentation coefficient, and buoyant density of the binding proteins, which yielded molecular weights of 51,000 and 64,000 for the A and B proteins, respectively. In buffer of low ionic strength the G protein forms heavy complexes (7 S, molecular weight over 200,000) which readily further aggregate under our experimental conditions. At higher ionic strength the G protein behaves as a rather assymetrical molecule (4 S, frictional ratio 1.35) with a molecular weight of about 66,000. Many of these values were obtained with crude cytosol and await confirmation by comparable studies upon homogeneous preparations. The A and B proteins bind natural glucocorticoids with high affinity and specificity but show no detectable affinity for the biologically very potent 9α-fluorinated glucocorticoids. The G protein binds all the tested physiologically active natural and synthetic glucocorticoids with high affinity and specificity. The apparent association constant, Ka, of the three proteins for glucocorticoids decreases with increasing temperatures from 0–37°, but the steroid binding to the A and B proteins is much more affected by the higher temperature. At physiological temperatures the Ka of the G protein for dexamethasone is about one order of magnitude higher than the Ka of the A and B proteins for cortisol. Thermodynamic calculations showed that the binding of cortisol to the A and B proteins is accompanied by a negative change of entropy, but the binding of dexamethasone of the G protein involves a positive ΔS°.

Glucocorticoid-binding Proteins of Rat Liver Cytosol: I. SEPARATION AND IDENTIFICATION OF THE BINDING PROTEINS

Abstract The specific interaction of glucocorticoid hormones with liver cytosol proteins of adrenalectomized rats was studied by an adsorbent technique for the separation of free and protein-bound steroid. By this rapid procedure a high affinity binding site for glucocorticoids can be detected which is usually overlooked when the slower technique of equilibrium dialysis is used. This metastable binding protein can be quantitatively estimated in whole cytosol preparations because of its high affinity not only for natural glucocorticoids but also for [3H]dexamethasone, which does not bind to the other two more stable glucocorticoid-binding proteins previously reported in rat liver cytosol, the A and B proteins. It can also be distinguished from these two binding proteins for natural glucocorticoids on the basis of its differential fractionation and physical properties. Unlike the A and B proteins, which in sucrose gradients sediment at 4 S independently of ionic strength, the dexamethasone-binding protein sediments in low salt sucrose gradients largely as a heavy complex, 7 S, which reverts to a lighter form, 4 S, in the presence of 0.3 m KCl. A similar salt-dependent behavior is observed upon chromatography of the dexamethasone-binding protein through Sephadex G-200 columns. The fact that specific dexamethasone binding can only be detected in the cytosol of glucocorticoid target cells, in conjunction with previously presented evidence on its relation to hormonal enzyme induction in vivo, makes it reasonable to assume that this binding protein, which we will call the G protein, may indeed represent the physiologically significant hepatic glucocorticoid receptor.

Correlation between glucocorticoid binding to specific liver cytosol receptors and enzyme induction in vivo

The degree of saturation in , vivo of a soluble hepatic glucocorticoid binding protein as a function of time and dosage of cortisol administration to adrenalectomized rats was found to correlate closely with the extent of hormonal induction of the hepatic enzymes tryptophan dioxygenase and tyrosine amino-transferase. These findings in conjunction with the high affinity and specificity for glucocorticoids manifested by this binding protein indicate that it may be the functionally significant glucocorticoid “receptor” of rat liver cytosol.

Studies of the glucocorticoid-binding protein from thymocytes: III. pH dependence of the binding and density-gradient centrifugation of the protein

1. 1. The binding of corticosterone to the glucocorticoid-binding protein from thymocytes was maximal at pH 7–8 and decreased rapidly below pH 7. 2. 2. Glucocorticoid-binding protein was precipitated at pH 5–6 together with most of the protein in crude extracts from thymocytes. 3. 3. After density gradient centrifugation of glucocorticoid-binding protein at a low salt concentration (0.1 M KCl) 2 peaks of binding activity were found. The sedimentation constant of the major peak was 10.0 ± 0.5 S and that of the minor peak 4.5 ± 0.2 S. At higher salt concentrations (0.25–1.0 M KCl) only the 4.5-S peak was observed. 4. 4. The complex with corticosterone and with triamcinolone acetonide showed the same sedimentation behaviour, although a partial dissociation of the corticosterone complex occurred during the run. 5. 5. Binding of triamicinolone acetonide in intact cells at 37 °C followed by extraction with a buffer containing 0.6 M KCl caused the appearance of a 3.5-S form of bound hormone, the sedimentation constant of which was uninfluenced by the KCl concentration in the density gradient.

Investigations on the glucocorticoid-binding protein from rat thymocytes: II. Stability, kinetics and specificity of binding of steroids

1. 1. The glucocorticoid-binding protein from rat thymocytes was shown to be stabilized by 40% (v/v) glycerol at −5°. 2. 2. The temperature dependence of the stability of rat thymocyte glucocorticoid-binding protein in the presence and absence of glycerol was investigated at temperatures up to 30°. Glycerol stabilized only at temperatures below approximately 8°. The inactivation of the binding activity seemed, in all cases, to be a simple first order process. 3. 3. The dissociation of bound steroid from the protein at −5° seemed to be a simple first order process. The half-lives of dissociation were determined to be approximately 800 min for corticosterone and cortisol, 450 min for 11-deoxycorticosterone, 1850 min for dexamethasone and at least several days for triamcinolone acetonide. 4. 4. The dissociation constants for the binding of the steroids mentioned above were determined, as well as the inhibition constants for different non-radioactive steroids inhibiting the binding of tritiated corticosterone. 5. 5. The rate constants for the binding of tritiated corticosterone, cortisol, 11-deoxycorticosterone, dexamethasone, triamcinolone acetonide and aldosterone to glucocorticoid-binding protein were determined at −5°. Furthermore, the dissociation constants were measured and compared with the dissociation constants calculated from the rates of binding and dissociation. 6. 6. Rat thymocyte glucocorticoid-binding protein was shown not to be identical with rat transcortin by measuring its temperature lability and sensitivity to SH reagents.

Studies of the glucocorticoid-binding protein from thymocytes: I. Localization in the cell and some properties of the protein

1. 1.Suspensions of rat thymocytes were incubated at 4° or 37°, rapidly washed at 4° and a nuclear fraction prepared by density gradient centrifugation. At 37° nearly all of the corticosterone-binding protein was present in the nucleus, while at 4° approx. 1 3 was found in the cytoplasm. 2. 2.By Sephadex filtration or coal adsorption, corticosterone binding to high-molecular-weight compounds could be shown in a 100 000 × g supernatant from sonicated cells. 3. 3.This binding was abolished by incubation with proteases or thiol reagents. 4. 4.The binding was unstable. The apparent dissociation constant of the corticosterone binding was 2–5 times greater than the dissociation constant of the binding to whole cells. 5. 5.The molecular weight of the corticosterone-binding compound was at least 100 000, as determined by Sephadex filtration. 6. 6.The affinity for corticosterone was highest between pH 7 and 8.