Evidence that the 90-kDa phosphoprotein associated with the untransformed L-cell glucocorticoid receptor is a murine heat shock protein

Two phosphoproteins are adsorbed to protein-A-Sepharose when cytosol from 32P-labeled L-cells is incubated with a monoclonal antibody against the glucocorticoid receptor: one is a 98-100-kDa phosphoprotein that contains the steroid-binding site and the other is a 90-kDa nonsteroid-binding phosphoprotein that is associated with the untransformed, molybdate-stabilized receptor (Housley, P. R., Sanchez, E. R., Westphal, H.M., Beato, M., and Pratt, W.B. (1985) J. Biol. Chem. 260, in press). In this paper we show that the 90-kDa receptor-associated phosphoprotein is an abundant cytosolic protein that reacts with a monoclonal antibody that recognizes the 90-kDa phosphoprotein that binds steroid receptors in the chicken oviduct. The 90-kDa protein immunoadsorbed from L-cell cytosol with this antibody reacts on Western blots with rabbit antiserum prepared against the 89-kDa chicken heat shock protein. Immunoadsorption of molybdate-stabilized cytosol by antibodies against the glucocorticoid receptor results in the presence of a 90-kDa protein that interacts on Western blots with the antiserum against the chicken heat shock protein. The association between the 90-kDa protein and the receptor is only seen by this technique when molybdate is present to stabilize the complex; and when steroid-bound receptors are incubated at 25 degrees C to transform them to the DNA-binding state, the 90-kDa protein dissociates. These observations are consistent with the proposal that the untransformed glucocorticoid receptor in L-cells exists in a complex with the murine 90-kDa heat shock protein.     

The molybdate-stabilized glucocorticoid binding complex of L-cells contains a 98-100 kdalton steroid binding phosphoprotein and a 90 kdalton nonsteroid-binding phosphoprotein that is part of the murine heat-shock complex

This paper summarizes our work performed with glucocorticoid-binding complexes in molybdate-stabilized cytosol prepared from 32P-labeled L-cells. In our early work, we showed that cytosol prepared from 32P-labeled L-cells contains two phosphoproteins (a 90 and a 98-100 kdalton protein) that elute from an affinity resin of deoxycorticosterone agarose in a manner consistent with the predicted behavior of the glucocorticoid receptor. Both phosphoproteins are immunoadsorbed onto protein-A-Sepharose from molybdate-stabilized cytosol incubated with a monoclonal antibody against the receptor. The 98-100 kdalton phosphoprotein binds steroid and the 90 kdalton phosphoprotein is a structurally different, nonsteroid-binding protein that is bound to the untransformed, molybdate-stabilized glucocorticoid receptor. The 90 kdalton protein reacts on Western blots with a monoclonal antibody raised against a 90 kdalton protein from the water mold Achlya ambisexualis. This antibody recognizes an epitope that is conserved in 90 kdalton phosphoproteins from rodent and human cells, and it reacts with the 90 kdalton phosphoprotein that copurifies with the molybdate-stabilized, untransformed chick oviduct progesterone receptor. The 90 kdalton nonsteroid-binding phosphoprotein is an abundant cytosolic protein that dissociates from the glucocorticoid receptor when it is transformed, and unlike the steroid-binding protein, it does not bind to DNA. The 90 kdalton phosphoprotein determines the acidic behavior of the untransformed glucocorticoid receptor on DEAE-cellulose. This abundant cytosolic 90 kdalton phosphoprotein reacts with rabbit antiserum raised against the gel purified 89 kdalton chicken heat-shock protein (hsp89). This antiserum recognizes 90 kdalton heat-shock proteins in human, rodent, frog and Drosophila cells. Immunoadsorption of molybdate-stabilized cytosol with antibody directed against the 98-100 kdalton steroid receptor results in the immune-specific adsorption of a 90 kdalton phosphoprotein that reacts with anti-hsp89 antibody on Western blots. These observations suggest that, like the transforming proteins from several avian sarcoma viruses, the untransformed glucocorticoid receptor exists in a complex with the 90 kdalton heat-shock protein.     

Transformation of glucocorticoid and progesterone receptors to the DNA-binding state

This brief review explores some recent observations relating to the structure of untransformed glucocorticoid and progesterone receptors and the mechanism by which the receptors are transformed to the DNA-binding state. In their molybdatestabilized, untransformed state, progesterone and glucocorticoid receptors exist as a heteromeric 8-9S complex containing one unit of steroid binding phosphoprotein and one or two units of the 90 kD heat shock protein hsp90. When the receptors are transformed, the steroid-binding protein dissociates from hsp90. In cytosol preparations, temperature-mediated dissociation proceeds much more rapidly in the presence of hormone. The dissociated receptor binds to DNA with high affinity, regardless of whether it is in the hormone-bound or the hormone-free state. These observations raise the possibility that the primary, and perhaps the only, role for the hormone is to promote dissociation of the receptor-hsp90 complex. Molybdate, vanadate, and tungstate inhibit receptor transformation to the DNA-binding form, an effect that appears to reflect the ability of these transition metal oxyanions to stabilize the complex between the steroid receptor and hsp90. By promoting the formation of disulfide bonds, hydrogen peroxide also stabilizes the glucocorticoid receptor-hsp90 complex and prevents receptor transformation. A small, heat-stable factor present in all cytosol preparations inhibits receptor transformation, and, when the factor is removed, glucocorticoid receptors are rapidly transformed. This ubiquitous factor has the physical properties of a metal anion, and it is proposed that molybdate and vanadate affect steroid receptor complexes by interacting with a metal anion-binding site that is normally occupied by this endogenous receptor-stabilizing factor.     

The molybdate-stabilized L-cell glucocorticoid receptor isolated by affinity chromatography or with a monoclonal antibody is associated with a 90-92-kDa nonsteroid-binding phosphoprotein

We have previously reported that molybdate-stabilized cytosol prepared from 32P-labeled L-cells contains two phosphoproteins (a 90-92- and a 98-100-kDa protein) that elute from an affinity resin of deoxycorticosterone-derivatized agarose in a manner consistent with the predicted behavior of the glucocorticoid receptor (Housley, P. R., and Pratt, W. B. (1983) J. Biol. Chem. 258, 4630-4635). In the present work we report that both the 90-92- and 98-100-kDa 32P-labeled proteins are also extracted from molybdate-stabilized cytosol by incubation with a monoclonal antibody and protein A-Sepharose. Only the 98-100-kDa protein is specifically labeled when either L-cell cytosol or L-cell cytosol proteins bound to the affinity resin are labeled with the glucocorticoid binding site-specific affinity ligand [3H]dexamethasone 21-mesylate. The 98-100-kDa protein labeled with [3H]dexamethasone mesylate is adsorbed to protein A-Sepharose in an immune-specific manner after reaction with the monoclonal antibody. Sodium dodecyl sulfate-polyacrylamide gel analysis of the protein A-Sepharose-bound material resulting from incubating the monoclonal antibody with a mixture of 32P-labeled cytosol and [3H]dexamethasone mesylate-labeled cytosol demonstrates identity of the 98-100-kDa [3H]dexamethasone mesylate-labeled band with the 98-100-kDa 32P-labeled band and clear separation from the nonsteroid-binding 90-92-kDa phosphoprotein. The results of immunoblot experiments demonstrate that the 90-92-kDa protein is structurally distinct from the 98-100-kDa steroid-binding protein. As the 90-92-kDa nonsteroid-binding phosphoprotein co-purified with the 98-100-kDa uncleaved form of the glucocorticoid receptor by two independent methods, one of which is based on recognizing a steroid-binding site and the other of which is based on recognizing an antibody binding site, we propose that the 90-92-kDa phosphoprotein is a component of the molybdate-stabilized, untransformed glucocorticoid-receptor complex in L-cell cytosol.     

Relationship of the 90-kDa murine heat shock protein to the untransformed and transformed states of the L cell glucocorticoid receptor

Incubation of molybdate-stabilized L cell cytosol with a monoclonal antibody directed against the 100-kDa glucocorticoid-binding protein causes the immune-specific adsorption to protein A-Sepharose of both the 100-kDa glucocorticoid receptor and the 90-kDa murine heat shock protein (hsp90) (Sanchez, E. R., Toft, D. O., Schlesinger, M. J., and Pratt, W. B. (1985) J. Biol. Chem. 260, 12398-12401). When the glucocorticoid receptor in cytosol is transformed to the DNA-binding state, hsp90 dissociates. In this paper, we show that temperature-mediated dissociation of hsp90 from the receptor is a hormone-dependent event in the same manner as temperature-mediated transformation to the DNA-binding state. In contrast to temperature-mediated transformation, ammonium sulfate causes both dissociation of hsp90 from the receptor and conversion of the receptor to the DNA-binding form in a manner that does not require the presence of steroid. The untransformed form of the glucocorticoid receptor and the strongly negatively charged hsp90 protein behave similarly on DEAE-cellulose chromatography, suggesting that the hsp90 component may contribute significantly to the net negative charge behavior of the non-DNA-binding form of the receptor complex.     

Phosphorylation of L-cell glucocorticoid receptors in immune complexes: evidence that the receptor is not a protein kinase

Two phosphoproteins are absorbed to protein A-Sepharose when cytosol from 32P-labeled L-cells is incubated with a monoclonal antibody against the glucocorticoid receptor: one is a 98K phosphoprotein that contains the steroid binding site, and the other is a 90K non-steroid-binding phosphoprotein that is associated with the molybdate-stabilized receptor [Housley, P. R., Sanchez, E. R., Westphal, H. M., Beato, M., & Pratt, W. B. (1985) J. Biol. Chem. 260, 13810-13817]. In this paper we have incubated L-cell cytosol with rabbit antiserum against the mouse glucocorticoid receptor and show that incubation of protein A-Sepharose-bound immune complexes with [gamma-32P]ATP and Mg2+ results in phosphorylation of the 98K steroid-binding protein but not of the 90K receptor-associated protein. Phosphorylation occurs regardless of whether the receptor is unoccupied or is present as the untransformed or transformed steroid-receptor complex. No phosphorylation occurs in the presence of Ca2+ instead of Mg2+. If protein A-Sepharose-bound immune complexes prepared with a monoclonal antibody against the receptor are incubated with [gamma-32P]ATP and Mg2+, neither protein is phosphorylated. If the protein A-Sepharose pellet is obtained from molybdate-stabilized cytosol that has been incubated both with monoclonal antibody to provide the 98K receptor and its 90K associated protein and with preimmune rabbit serum, which causes the nonspecific adsorption of an L-cell protein kinase, then incubation with [gamma-32P]ATP and Mg2+ causes receptor phosphorylation.(ABSTRACT TRUNCATED AT 250 WORDS)     

Phosphotryptic peptide analysis of human progesterone receptor. New phosphorylated sites formed in nuclei after hormone treatment

Human progesterone receptors (PR) exist as two independent naturally occurring steroid-binding forms of approximately 120 kDa (B-receptors) and 94 kDa (A-receptors). Both are phosphorylated in hormone-untreated T47Dco breast cancer cells. Hormone treatment leads to receptor transformation and an increased phosphorylation state: the 32P-labeling intensity is 3-5 times higher after progestin treatment and 8-10 times higher after RU 486 treatment. Only serine residues are phosphorylated. To determine whether there are unique phosphorylation sites in transformed nuclear PR, we analyzed the phosphopeptides of untransformed and transformed A- and B-receptors by tryptic cleavage and reverse-phase high pressure liquid chromatography. Untransformed A- and B-receptors share at least five common phosphopeptides, and a sixth is unique to B. Following transformation by either R5020 or RU 486, A-receptors generate at least six and B-receptors seven phosphopeptides. Compared with untransformed PR, there are at least two different phosphopeptides in transformed nuclear PR. Cyanogen bromide cleavage of transformed nuclear A-receptors, which lack the proximal 165 amino-terminal residues of the 933 amino acid B-receptors, produces two large fragments of approximately 43 and 19 kDa. These fragments contain all of the 32P label and comprise amino acids 165-595. Cleavage of transformed B-receptors also produces peptides of 43 and 19 kDa plus an additional 36-kDa fragment corresponding to residues 1-165. No 32P-labeled low molecular mass peptides are detected. Thus, all the hormone-dependent phosphoserine residues produced in nuclei are located in the first 595 amino acids of human PR, representing the amino terminus and 28 residues of the DNA-binding domain.     

Localization of phosphorylation sites with respect to the functional domains of the mouse L cell glucocorticoid receptor

Digestion of the rat liver glucocorticoid receptor with chymotrypsin results in the generation of a 42-kDa fragment which contains the steroid-binding and DNA-binding domains and the antigenic site for the BuGR anti-glucocorticoid receptor monoclonal antibody, while digestion with trypsin generates a 15-kDa receptor fragment containing only the DNA-binding function and the BuGR epitope (Eisen, L.P., Reichman, M.E., Thompson, E.B., Gametchu, B., Harrison, R. W., and Eisen, H.J. (1985) J. Biol. Chem. 260, 11805-11810). In this paper, glucocorticoid receptor of mouse L cells that were grown in the presence of [32P]orthophosphate was digested with trypsin or chymotrypsin (either before or after immune purification with BuGR antibody) and analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, autoradiography, and Western blotting. The receptor is endogenously phosphorylated only on serine residues. Chymotrypsin digestion results in a 32P-labeled 42-kDa receptor fragment which contains steroid-binding, DNA-binding, and BuGR-reactive sites. Trypsin digestion generates a 27-kDa steroid-bound fragment (meroreceptor) which is not labeled with 32P and a 32P-labeled 15-kDa fragment which contains both the DNA-binding domain and the BuGR epitope. We have calculated that there are 4 times as many phosphate residues in the intact receptor than in the 42-kDa chymotrypsin fragment. From examination of 32P-labeled receptor fragments, we have deduced that one phosphate is located between amino acids 398 and 447, a region containing the BuGR epitope and about one-third of the DNA-binding domain, and the remaining three phosphates appear to be clustered just to the amino-terminal side of the BuGR epitope in a region defined by amino acids 313 to 369. Treatment of intact 32P-labeled receptor in cytosol with alkaline phosphatase removes these three phosphates, but it does not remove the phosphate from the DNA-binding-BuGR-reactive fragment and it does not affect the ability of the transformed receptor to bind to DNA-cellulose.     

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)     

Evidence that the 90-kDa heat shock protein is necessary for the steroid binding conformation of the L cell glucocorticoid receptor

Using L cell glucocorticoid receptors that have been immunopurified by adsorption to protein A Sepharose with a monoclonal antireceptor antibody, we have developed an assay to study the requirements for maintenance of steroid-binding capacity. After rapid purification by immunoadsorption, heteromeric receptor complexes retain the ability to bind glucocorticoid hormone. When the receptor complexes are warmed at 20 degrees C, steroid-binding capacity is lost, and the 90-kDa heat shock protein (hsp90) dissociates from the receptor. The rates of both temperature- and salt-dependent dissociation of hsp90 parallel the rates of loss of hormone-binding activity. Molybdate and hydrogen peroxide stabilize the hsp90-receptor complex against temperature-dependent dissociation. Molybdate, however, is much more effective in stabilizing steroid-binding capacity than peroxide. Receptors that have been inactivated in the absence of molybdate or peroxide cannot be reactivated. Inactivation of steroid-binding capacity occurs in the presence or absence of reducing agent, and inactivation is not accompanied by receptor cleavage or dephosphorylation. Under no conditions does an hsp90-free receptor bind steroid. Receptor bound to hsp90 can be cleaved to the 27-kDa meroreceptor in the presence of molybdate with retention of both hsp90 and steroid-binding activity. These observations lead us to propose that hsp90 is necessary but not sufficient for maintaining a competent high affinity glucocorticoid-binding site. Although the 27-kDa meroreceptor fragment is not itself sufficient for a competent binding site, it is sufficient when it is associated with hsp90.     

Glucocorticoid receptor phosphorylation, transformation, and DNA binding

Glucocorticoid receptors were isolated by immunoadsorption from cytosol of L cells that were cultured for 18 h in the presence of [32P]orthophosphate, and the phosphorylation state of the receptor was examined before and after transformation to the DNA-binding state. Temperature-mediated transformation of the glucocorticoid receptor under cell-free conditions results in no change in receptor size or degree of phosphorylation. When cytosol containing transformed receptors is incubated with DNA-cellulose, 30-50% of the receptors are able to bind to DNA and the remainder do not bind to DNA. Both the heated receptors that bind to DNA and the receptors that do not bind to DNA are phosphorylated to the same degree. When intact cells containing 32P-labeled receptors are incubated for 2 h at 0 degree C with triamcinolone acetonide and then for 20 min at 37 degrees C in the presence of the hormone, 80% of the receptor becomes tightly associated with the nucleus in a manner that is both temperature-dependent and ligand-dependent. Approximately 80% of the nuclear-bound receptor is extracted with 0.4 M NaCl. Both the cytosolic receptor from cells incubated at 0 degree C and the salt-extracted nuclear receptor from cells incubated at 37 degrees C have been resolved by immunoadsorption to protein A-Sepharose with the BuGR1 monoclonal antibody and sodium dodecyl sulfate-polyacrylamide gel electrophoresis, followed by immunoblotting and autoradiography of the immunoblots. In addition, direct measurements of the amounts of 32P contained per unit of receptor protein were performed for receptors transformed both in the intact cell and in cell-free lysates. The results demonstrate that the untransformed receptor and the nuclear-bound transformed receptor are labeled with 32P to the same extent.     

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.     

The Mr 90,000 heat shock protein-free androgen receptor has a high affinity for steroid, in contrast to the glucocorticoid receptor

Transformed and bacterially expressed glucocorticoid receptors free from Mr 90,000 heat shock protein (hsp90) have a 100-fold lower steroid-binding affinity than the hsp90-bound nontransformed receptor, suggesting that hsp90 is needed for high-affinity steroid binding [Nemoto, T., Ohara-Nemoto, Y., Denis, M., & Gustafsson, J.-A. (1990) Biochemistry 29, 1880-1886]. To investigate whether or not this phenomenon is common to all steroid receptors, we investigated the steroid-binding affinities of bacterially expressed and transformed androgen receptors. The C-terminal portion of the rat androgen receptor containing the putative steroid-binding domain was expressed as a fusion protein of protein A in Escherichia coli. The recombinant protein bound a synthetic androgen, [3H]R1881, with high affinity (Kd = 0.8 +/- 0.3 nM). Glycerol gradient analysis revealed that the recombinant protein sedimented at around the 3S region irrespective of the presence of molybdate, indicating that the receptor is present in monomeric form. The steroid-free transformed androgen receptor was obtained by exposure of rat submandibular gland cytosol to 0.4 M NaCl in the absence of steroid. High-performance ion-exchange liquid chromatography analysis showed that the transformed androgen receptor bound to [3H]R1881 with high affinity. Thus these observations indicate that, in contrast to the glucocorticoid receptor, hsp90 is not required for the high-affinity steroid binding of the androgen receptor. In addition, the hsp90-free androgen receptor prebound with radioinert R1881 was efficiently relabeled with [3H]R1881, while the triamcinolone acetonide-bound, transformed glucocorticoid receptor failed in ligand exchange. The inability to achieve ligand exchange probably reflects the low steroid-binding affinity of this entity.     

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)     

Evidence that the hormone-binding domain of the mouse glucocorticoid receptor directly represses DNA binding activity in a major portion of receptors that are "misfolded" after removal of hsp90.

After dissociation of cytosolic heteromeric glucocorticoid receptor complexes by steroid, salt, and other methods, only 35-60% of the dissociated receptors can bind to DNA-cellulose. The DNA-binding and non-DNA-binding forms of the dissociated receptors have the same Mr and are phosphorylated to the same extent (Tienrungroj, W., Sanchez, E. R., Housley, P. R., Harrison, R. W., and Pratt, W. B. (1987) J. Biol. Chem. 262, 17347-17349). The basis for the different DNA-binding activities is unknown, but the DNA-binding fraction of the receptor has a more basic pI than the non-DNA-binding fraction (Smith, A. C., Elsasser, M. S., and Harmon, J. M. (1986) J. Biol. Chem. 261, 13285-13292). We have separated the non-DNA-binding state of the receptor from the DNA-binding state and then cleaved it with trypsin and chymotrypsin. We find that the 15-kDa tryptic fragment derived from the non-DNA-binding state of the dissociated receptor is fully competent in binding DNA, whereas the 42-kDa chymotryptic fragment containing both the hormone-binding and DNA-binding domains does not bind DNA. Trypsin cleavage of the molybdate-stabilized untransformed receptor also yields a 15-kDa fragment that is fully competent in binding DNA. Reducing agents do not restore DNA-binding to the non-DNA-binding fraction of the receptor and the hormone-binding domain can be separated from the DNA-binding domain on nonreducing gel electrophoresis. These results argue that the two domains are not linked by disulfide bridges, and they are consistent with the proposal that there are two least energy states of folding after dissociation of hsp90. A significant portion of the receptors is "misfolded" in such a manner that the steroid binding domain is directly preventing DNA-binding activity.     

Cell-free synthesis of rat glucocorticoid receptor in rabbit reticulocyte lysate. In vitro synthesis of receptor in Mr 90,000 heat shock protein-depleted lysate

The glucocorticoid receptor is present in the cytosol of cell extracts as a large nonactivated (i.e. non-DNA-binding) approximately 9 S (Mr 300,000) complex. Experimental evidence indicates that the purified nonactivated glucocorticoid receptor contains a single steroid-binding protein and two approximately 90-kDa nonsteroid-binding subunits identified as heat shock protein (hsp) 90. Translation of the glucocorticoid receptor mRNA in vitro in reticulocyte lysates produces a large nonactivated glucocorticoid receptor complex similar to that found in cytosols. The cell-free synthesized glucocorticoid receptor is able to bind steroid and can be activated further to the DNA-binding form. To test the hypothesis of an active role played by hsp90 in the stabilization of a competent steroid-binding conformation of the glucocorticoid receptor, we have synthesized the receptor in a reticulocyte lysate that has been depleted of hsp90 by immunoadsorption with AC88 anti-hsp90. Although the translation capacity of the reticulocyte system was reduced considerably upon hsp90 removal, the glucocorticoid receptor was synthesized, and a significant number of molecules were found to bind [3H]triamcinolone acetonide. Chromatography on DEAE-cellulose showed that most of the receptor molecules synthesized in hsp90-depleted lysate had lost the capacity to form an oligomeric receptor complex. Addition of purified rat liver hsp90 to the hsp90-depleted lysate before translation did not increase steroid binding nor did it restore formation of the heteromeric receptor complex. Analysis of [35S] methionine-labeled glucocorticoid receptor molecules synthesized in the hsp90-depleted lysate showed the production of polypeptides differing from the expected chromatographic pattern on DEAE-cellulose. Upon addition of purified hsp90 to the hsp90-depleted lysate, before translation, the 35S-labeled synthesized receptor fractionated on DEAE-cellulose as an intermediate peak between activated and nonactivated receptor forms. The data suggest that hsp90 alone may not be sufficient for the formation of the nonactivated steroid receptor complex.     

Elimination and reconstitution of the requirement for hormone in promoting temperature-dependent transformation of cytosolic glucocorticoid receptors to the DNA-binding state

Cytosols contain a heat-stable, chelatable, anionic, molybdate-like factor that stabilizes glucocorticoid receptors in a heteromeric complex with hsp90 (refers to the 90-kDa heat shock protein) and inhibits their transformation to the DNA-binding state (Meshinchi, S., Grippo, J.F., Sanchez, E.R., Bresnick, E.H., and Pratt, W.B. (1988) J. Biol. Chem. 263, 16809-16817). In this work, we demonstrate that removal of this factor by passage of L cell cytosol through the metal-chelating resin Chelex-100 makes the glucocorticoid receptor unstable, thus markedly facilitating both its dissociation from hsp90 and its transformation to the DNA-binding state. In normal cytosol, both temperature-mediated dissociation of hsp90 and temperature-mediated receptor transformation are hormone-dependent events. In the Chelex-treated, metal-depleted cytosol, however, temperature-mediated dissociation of hsp90 and receptor transformation occur very rapidly in a manner that is no longer hormone-dependent. When boiled L cell cytosol is added to the metal-depleted receptor system, the hormone dependence of both temperature-mediated dissociation of receptor from hsp90 and receptor transformation to the DNA-binding state is reconstituted. Like boiled cytosol, molybdate stabilizes the receptor complex and inhibits its transformation in metal-depleted cytosol, but it does not reconstitute the hormone dependence of the system. These results support the proposal that an endogenous metal anion interacts with the glucocorticoid receptor to stabilize it in the heteromeric, inactive, non-DNA-binding state in cytosol and that binding of the hormone promotes conversion of the receptor to the DNA-binding state through an effect on this metal anion center.     

The non-activated glucocorticoid receptor: structure and activation

Glucocorticoid hormone receptors are present in the soluble fraction of target cell homogenates as large entities (Mr approximately 300,000) that are unable to interact with DNA. These large complexes contain an Mr approximately 94,000 steroid- and DNA-binding polypeptide, in association with an Mr approximately 90,000 non-ligand-binding entity, which has been identified as a heat shock protein, hsp90. This protein has been purified to near homogeneity as a component of the non-activated receptor complex. Characterization of the purified protein revealed its presence as a dimer in the large receptor form. Dissociation of the receptor-hsp90 complex can be induced by heat treatment only when ligand is bound to the receptor, as demonstrated by specific DNA-binding assay and sucrose gradient ultracentrifugation, hsp90 represents ca 1% of total proteins in rat liver cytosol, and milligram amounts were purified using a combination of high performance ion exchange and gel permeation chromatography. Monospecific antibodies were raised in rabbits. They were found to precipitate the intact non-activated glucocorticoid receptor, as well as the Mr approximately 27,000 steroid-binding fragment of the receptor generated by trypsin treatment, indicating that hsp90 interacts with the steroid-binding domain of the glucocorticoid receptor. Finally, translation of glucocorticoid receptor mRNA in reticulocyte lysate yields a protein which also interacts with hsp90 and binds to DNA only after ligand-binding and heat treatment. Thus, the glucocorticoid receptor is synthesized in a non-activated form also in vitro.     

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.