Phosphorylation and dephosphorylation has been suggested to influence the function of glucocorticoid receptors, but evidence for hormone-dependent changes in the phosphorylation state under physiological conditions is lacking. Here we show that in intact WEHI-7 mouse thymoma cells, labeled for 18-20 h with [32P]orthophosphate and [35S]methionine, glucocorticoids rapidly increase the average number of phosphates on the steroid-binding protein approximately from three to five. This stimulation is agonist-dependent since the antiglucocorticoid RU 486 (17 beta-hydroxy-11 beta,4-dimethylaminophenyl-17 alpha-propynyl estra-4,9-diene-3-one) has no effect by itself and blocks the cortisol-induced phosphorylation. Furthermore, the salt-unextractable nuclear bound receptors lose at least two phosphates compared to cytosolic and nuclear extractable forms. These results show for the first time that these hormone-dependent transcription regulators undergo agonist-induced phosphorylation and dephosphorylation which may affect their activity.

Animals↗

PubMed1989

Phosphorylated sites within the functional domains of the approximately 100-kDa steroid-binding subunit of glucocorticoid receptors.

The steroid-binding subunit of the glucocorticoid receptor is known to be a approximately 100-kDa phosphoprotein composed of an immunogenic, DNA-binding, and steroid-binding domain. When isolated from WEHI-7 cells, this protein contains between two and three phosphoryl groups per steroid-binding site (Mendel WEHI-7 cells, this protein contains between two and three phosphoryl groups per steroid-binding site (Mendel et al., 1987). To identify the domains that contain these phosphorylated sites, we have analyzed the phosphate content of selected proteolytic fragments of the approximately 100-kDa steroid-binding protein from nonactivated and activated receptors. The approximately 100-kDa steroid-binding protein from WEHI-7 cells grown in the presence of [32P]orthophosphate was covalently labeled with [3H]dexamethasone 21-mesylate, purified with the BuGR2 monoclonal antibody, digested with chymotrypsin or trypsin, and analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Chymotrypsin digestion of this protein yields a approximately 45-kDa fragment containing both the steroid-binding and DNA-binding domains, which contained both 32P and 3H. Trypsin digestion of the protein yields a approximately 29-kDa fragment encompassing the steroid-binding domain but not the DNA-binding domain of the approximately 100-kDa protein, which also contained both 32P and 3H. The 32P/3H ratio of each fragment provides a measure of phosphate content per steroid-binding site and indicated that each fragment has approximately 30% of the phosphate content of the intact protein. This is sufficient to account for one of the three receptor phosphoryl groups.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1989

Phosphorylation of glucocorticoid receptor-associated and free forms of the approximately 90-kDa heat shock protein before and after receptor activation.

Several lines of evidence have suggested that glucocorticoid receptor function may be regulated by phosphorylation-dephosphorylation reactions, and it has been proposed that dephosphorylation accompanies activation to the DNA-binding form. The phosphate content of the approximately 100-kDa steroid-binding protein has been determined directly and was found not to change during activation in intact cells (Mendel, D.B., Bodwell, J.E., and Munck, A. (1987) J. Biol. Chem. 262, 5644-5648). We have now determined the effect of interaction with the receptor and of activation on the phosphate content of the approximately 90-kDa heat shock protein (Hsp 90), which is thought to be a non-steroid-binding subunit of nonactivated glucocorticoid receptors that dissociates on activation. Monoclonal antibodies AC88 and BuGR2 were used to purify free Hsp 90 and cytosolic nonactivated glucocorticoid-receptor complexes, respectively, from WEHI-7 cells grown in the presence of 32Pi and [35S] methionine. Cell-free activation of the nonactivated receptor-antibody complexes immobilized on protein A-Sepharose minicolumns allowed the recovery of the Hsp 90 dissociated from the complexes during activation. Proteins were separated by denaturing polyacrylamide gel electrophoresis, and the 32P/35S ratio, which was used as a measure of the phosphate content relative to protein, was determined for the free, receptor-associated, and dissociated forms of the Hsp 90, as well as for the approximately 100-kDa steroid-binding protein of non-activated and activated receptors. The three forms of the Hsp 90 had the same phosphate contents, as did the approximately 100-kDa steroid-binding protein before and after activation. Based upon these results, we conclude that no net change in the phosphorylation occurs when the Hsp 90 associates with the approximately 100-kDa steroid-binding protein to form nonactivated receptors and that neither protein component of nonactivated complexes is dephosphorylated when they dissociate during thermal activation under cell-free conditions.

Animals↗

PubMed1989

A dynamic model of glucocorticoid receptor phosphorylation and cycling in intact cells.

Glucocorticoid receptors have been proposed to undergo an ATP-dependent recycling process in intact cells, and a functional role for receptor phosphorylation has been suggested. To further investigate this possibility we have examined the phosphate content of the steroid-binding protein of all glucocorticoid receptor forms which have been isolated from WEHI-7 mouse thymoma cells. By labeling of intact cells with 32Pi for 18-20 h in the absence of hormone, covalent binding of [3H]dexamethasone 21-mesylate, immunopurification and SDS-PAGE analysis, the steroid binding protein was found to contain, on average, 2-3 phosphates as phosphoserine. One third of the phosphates were associated with proteolytic fragments encompassing the C-terminal steroid-binding domain. The central DNA-binding domain was not phosphorylated, leaving the other two thirds of the phosphates localized in the N-terminal domain. The phosphate content of various receptor forms from cells incubated with 32Pi and [35S]methionine was compared using 35S to normalize for quantity of protein. In ATP-depleted cells a non-steroid-binding form of the receptor (the "null" receptor) is found tightly bound to the nucleus, even without steroid. The phosphate content of null receptors was two thirds that of cytosolic receptors from normal cells, suggesting phosphorylation-dependent cycling in the absence of hormone. Addition of glucocorticoid agonists, but not antagonist, to 32P- and 35S-labeled cells increased the phosphate content of the cytosolic steroid-binding protein up to 170%, indicating an average increase in the phosphates from about 3 to 5. After 30 min of hormone treatment the phosphate content of the steroid-binding protein of cytosolic activated (DNA-binding) and nonactivated receptors, and that of nuclear receptors extractable with high salt concentrations and/or DNase I digestion, was the same. No change in the phosphate content of the 90-kDa heat shock protein associated with unliganded and nonactivated receptors was detected following association of the free protein with the receptor and following hormone binding of the receptor. Analysis of the unextractable nuclear receptors indicated that they contained less phosphate (60% of that of cytosolic receptors), similarly to null receptors, indicating that dephosphorylation is associated with the unextractable nuclear fraction. The rate of hormone-dependent phosphorylation appeared to be much faster than the rate of dephosphorylation in the presence of hormone, the latter determined by a chase of the 32P label with unlabeled phosphate. Our results show that phosphorylation and dephosphorylation are involved in the mechanism of action of glucocorticoid receptors.(ABSTRACT TRUNCATED AT 400 WORDS)

Adenosine Triphosphate↗

PubMed1988

Identification of cysteine-644 as the covalent site of attachment of dexamethasone 21-mesylate to murine glucocorticoid receptors in WEHI-7 cells.

Dexamethasone 21-mesylate is a highly specific synthetic glucocorticoid derivative that binds covalently to glucocorticoid receptors via sulfhydryl groups. We have identified the amino acid that reacts with the dexamethasone 21-mesylate by using enzymatic digestion and microsequencing for radiolabel. Nonactivated glucocorticoid receptors obtained from labeling intact WEHI-7 mouse thymoma cells with [3H]dexamethasone 21-mesylate were immunopurified and analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The purified approximately 100-kDa steroid-binding subunit was eluted from gel slices and subjected to enzymatic digestion. Trypsin digestion followed by reversed-phase high-performance liquid chromatography (reversed-phase HPLC) produced a single [3H]dexamethasone 21-mesylate labeled peptide. Automated Edman degradation of this peptide revealed that the [3H]dexamethasone 21-mesylate was located at position 5 from the amino terminus. Dual-isotope labeling studies with [3H]dexamethasone 21-mesylate and [35S]methionine demonstrated that this peptide contained methionine. Staphylococcus aureus V8 protease digestion of [3H]dexamethasone 21-mesylate labeled steroid-binding subunits generated a different radiolabeled peptide containing label at position 7 from the amino terminus. On the basis of the published amino acid sequence of the murine glucocorticoid receptor, our data clearly identify cysteine-644 as the single residue in the steroid-binding domain that covalently binds dexamethasone 21-mesylate.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1988

Cyclic, nonequilibrium models of glucocorticoid antagonism: role of activation, nuclear binding and receptor recycling.

Quantitative models that have been proposed to date to explain mechanisms of glucocorticoid antagonism have generally been of the equilibrium type, involving hypothetical allosteric equilibria between active and inactive states of the receptor or the steroid-receptor complex. We describe here the agonist-antagonist relationships predicted by a nonequilibrium cyclic model that we have recently devised to account for the kinetic behavior of glucocorticoid-receptor complexes in intact rat thymus cells. This model simulates quantitatively most kinetic and steady state results that have been obtained so far. It postulates the existence of only well-established receptor species, and its kinetic parameters can in principle be determined by receptor measurements with intact cells. To calculate the steady state agonist-antagonist properties it is assumed that biological activity is proportional to the total amount of nuclear-bound complex, whether formed by agonist or antagonist. The agonist activity of a steroid is determined by the steady state ratio of nuclear-bound to total complexes it forms. This ratio varies from 0 for a pure antagonist to 1 for a pure agonist. It turns out to be independent of agonist and antagonist concentrations, and a function only of the rate constants for the reactions of the complexes formed by a steroid. Analysis of the dependence of the ratio on each rate constant shows quantitatively how each reaction in the cyclic model--activation of the nonactivated complex, nuclear binding of the activated complexes, and dissociation and recycling of activated and nuclear-bound complexes--affects antagonist properties. Steady state interactions of agonists with antagonists are found to be determined by equations that are identical to those for competition in simple equilibrium systems. Predicted dose-response relations agree qualitatively with experimentally observed relations. They are similar to those predicted by two-state allosteric models, although the cyclic model has no allosteric mechanisms and is based on quite different assumptions. Present limitations of the model arise particularly from lack of information about the mechanisms by which nuclear-bound complexes generate biological activity; for lack of such information the model includes no steps to account for substances that have low agonist activity despite forming nuclear-bound complexes.

Allosteric Regulation↗

PubMed1988

Cell proliferation and early differentiation during embryonic development and metamorphosis of Hydractinia echinata.

The early embryonic development of Hydractinia lasts about 2.5 days until the developing planula larva acquires competence for metamorphosis. Most embryonic cells stop cycling on reaching the larval stage. In older larvae of Hydractinia, cells that are still proliferating occur exclusively in the endoderm in a typical distribution along the longitudinal axis. During metamorphosis, proliferation activity begins again. The number of S-phase cells has increased by the 9th hour after induction of metamorphosis. Proliferative activity starts in the middle gastric region and in basal parts of primary polyps. Tentacles and stolon tips are always free of replicating cells.

Animals↗

PubMed1988

[Gastro-esophageal reflux in pediatrics, an ever-present problem].

Explore the source record for details and available documents.

Gastroesophageal Reflux↗

PubMed1987

Dexamethasone causes translocation of glucose transporters from the plasma membrane to an intracellular site in human fibroblasts.

To investigate the mechanism by which glucocorticoids inhibit glucose transport in peripheral tissues, we have used a monoclonal antibody directed against the human glucose transporter to measure the relative amounts of glucose transporter polypeptide in various cell fractions of human foreskin fibroblasts after treatment with and without dexamethasone. In cells treated for 4 h with 100 nM dexamethasone, a decrease of 48% in glucose transport was accompanied by a decrease of 40% in the amount of glucose transporter polypeptide in a plasma membrane fraction enriched 10-fold in 5'-nucleotidase activity and a 78% increase in the amount of transporter polypeptide in a fraction of putative intracellular membranes, designated P2. There was no significant change in the amount of transporter polypeptide in whole cell lysates. Insulin (200 nM) stimulated glucose transport in basal fibroblasts by only 9%. However, addition of insulin for 30 min to cells that had been treated for 4 h with dexamethasone completely reversed the dexamethasone-induced decrease in glucose transport and also reversed the dexamethasone-induced changes in glucose transporter polypeptide content of the plasma membrane and P2 fractions. From these observations we conclude that dexamethasone decreases glucose transport by causing translocation of glucose transporters from the plasma membrane to an internal location and that insulin reverses the dexamethasone effect by reversing the translocation.

5'-Nucleotidase↗

PubMed1987

Activation of cytosolic glucocorticoid-receptor complexes in intact WEHI-7 cells does not dephosphorylate the steroid-binding protein.

In order to determine the ratio of phosphates to hormone-binding sites on nonactivated (non-DNA-binding) glucocorticoid receptors in WEHI-7 mouse thymoma cells, we have extracted these receptors from cells grown to a steady state with 32P, labeled them with a saturating concentration of [3H]dexamethasone 21-mesylate, purified them using a monoclonal antibody, and analyzed them by polyacrylamide gel electrophoresis under denaturing and reducing conditions. The complexes contained approximately 5 mol of phosphate/mol of bound steroid. Only half of the phosphates were associated with the approximately 100-kDa protein which is labeled with [3H]dexamethasone 21-mesylate. The remaining phosphates were associated with the approximately 90-kDa non-steroid-binding component of the nonactivated complex. Dual label studies, using [35S]methionine to measure receptor protein and 32P to measure receptor phosphates, have enabled us to determine the phosphate content, relative to receptor protein, of both nonactivated and activated cytosolic complexes generated in intact WEHI-7 cells exposed to triamcinolone acetonide at 37 degrees C. The total amount of phosphate associated with the activated complex is roughly half of that associated with the nonactivated complex, the decrease being accounted for by dissociation of the approximately 90-kDa phosphoprotein which accompanies activation. However, the ratio of 32P to 35S counts associated with the approximately 100-kDa steroid-binding protein is the same for the activated and nonactivated complexes. These results indicate that there is no net change in the phosphorylation of the approximately 100-kDa steroid-binding component of the cytosolic glucocorticoid-receptor complex upon activation in the intact cell.

Animals↗

PubMed1987

Steroid hormone antagonism and a cyclic model of receptor kinetics.

Steady state agonist-antagonist relations have been derived for a general version of a cyclic model of glucocorticoid-receptor kinetics. The model was previously shown to account quantitatively for the transient and steady state distribution of hormone-receptor complexes formed in thymus cells by several glucocorticoids. Agonist-antagonist properties of a steroid in the model are expressed quantitatively by its "agonist activity" A, the steady state ratio of nuclear-bound to total complexes it forms. For a pure agonist A = 1, for a pure antagonist A = 0. This ratio is found to be independent of steroid concentration and a function only of the rate constants of reactions involving complexes formed by the steroid. Analysis of the dependence of A on each rate constant reveals how each reaction in the cyclic model--activation, nuclear binding, dissociation of activated and nuclear-bound complexes--influences antagonist properties. The steady state interaction of an antagonist with an agonist is shown to be governed by relations that are indistinguishable from competition relations for the simplest equilibrium system, and to yield dose-response curves that are very similar to those produced by two-state allosteric models of steroid hormone antagonism, despite the fact that the cyclic model includes no allosteric mechanisms. With steroids for which relevant rate constants can be measured, the model is directly testable. Limitations of the model arise from lack of information about the nuclear events that lead to biological activity following binding of activated complexes to the nucleus.

Animals↗

PubMed1986

Molybdate-stabilized nonactivated glucocorticoid-receptor complexes contain a 90-kDa non-steroid-binding phosphoprotein that is lost on activation.

We have used a monoclonal antibody to purify glucocorticoid-receptor complexes from WEHI-7 mouse thymoma cells. Molybdate-stabilized, nonactivated complexes were found to contain two distinct proteins which could be separated by polyacrylamide gel electrophoresis under denaturing and reducing conditions. One of the proteins, 100 kDa, was labeled when cytosol was incubated with the affinity ligand [3H]dexamethasone 21-mesylate. The second protein, 90 kDa, was not labeled. Several lines of evidence, including Western blot analysis of purified nonactivated complexes, indicate that only the 100-kDa protein is directly recognized by the antibody. The 90-kDa protein appears to be purified as a component of the nonactivated complex due to noncovalent association with the 100-kDa protein. Both the 100-kDa and 90-kDa components of the nonactivated complex become labeled with 35S when cells are grown in medium containing [35S]methionine. Using cells labeled in this manner, we have shown that activated (i.e. DNA-binding) cytosolic complexes, formed by warming either in intact cells or under cell-free conditions, contain only the 100-kDa protein. Complexes extracted from nuclei of warmed cells similarly contain only the 100-kDa protein. These results indicate that the 100-kDa and 90-kDa components of nonactivated complexes separate upon activation. Purification of nonactivated complexes from cells grown in medium containing [32P]orthophosphoric acid indicates that both the 100-kDa and 90-kDa components are phosphoproteins which can be labeled with 32P. Therefore, resolution of the two proteins will be essential in order to determine whether the receptor is dephosphorylated on activation.

Animals↗

PubMed1986

Glucocorticoid physiology, pharmacology and stress.

Basal levels of glucocorticoids maintained by negative feedback regulation are known to modulate a wide range of physiological processes, through a variety of effects such as those on carbohydrate metabolism and "permissive" actions on effects of other hormones. Glucocorticoid levels increase sharply in response to the stress of any kind of threat to homeostasis. The increased levels have traditionally been ascribed the function of enhancing the organism's resistance to stress. How known physiological and pharmacological effects of high levels of glucocorticoids might accomplish this function, however, has been a mystery. A generalization that is beginning to emerge is that many of these effects may be secondary to modulation by glucocorticoids of the actions of numerous intercellular mediators, including established hormones, prostanoids, neutral proteinases, and cytokines such as interferon. These mediators participate in physiological mechanisms--endocrine, renal, immune, neural, etc.--that mount a first line of defense against such challenges to homeostasis as hemorrhage, metabolic disturbances, infection, anxiety, and others. Contrary to the traditional view that the role of glucocorticoids in stress is to enhance these defense mechanisms, it has become increasingly clear that glucocorticoids at moderate to high levels generally suppress them. This paradox first emerged when glucocorticoids were discovered to be antiinflammatory agents, and had remained a major obstacle to a unified picture of glucocorticoid function. We have suggested that stress-induced increases in glucocorticoid levels protect not against the source of stress itself but rather against the body's normal reactions to stress, preventing those reactions from overshooting and themselves threatening homeostasis. This hypothesis, the seeds of which are to be found in many earlier discussions of glucocorticoid effects, immediately accounts for the paradox noted above, and provides glucocorticoid physiology with a unified conceptual framework that can accommodate such apparently unrelated physiological and pharmacological effects as those on carbohydrate metabolism, inflammatory processes, shock and water balance. It also leads us to propose that some enzymes rapidly induced by glucocorticoids detoxify mediators released during stress-induced activation of primary defense mechanisms; those mediators could themselves cause damage if left unchecked.

Adrenocorticotropic Hormone↗

PubMed1986

[Primary intestinal lymphangiectasis with antenatal manifestation].

Antenatal ascites diagnosed by ultrasound examination was punctured and shunted in utero. Radiologic, endoscopic and histologic data led to diagnosis of primitive intestinal lymphangiectasia during the second year of life. Antenatal revelation of Waldmann's disease is extremely rare in pediatric literature.

Ascites↗