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 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.     

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)     

Glucocorticoid receptor phosphorylation in mouse L-cells

This paper summarizes our observations on the phosphorylation state of untransformed and transformed glucocorticoid receptors isolated from 32P-labeled L-cells. The 300-350-kDa 9S untransformed murine glucocorticoid receptor complex is composed of a 100-kDa steroid-binding phosphoprotein and one or possibly two units of the 90-kDa heat shock protein (hsp90), which is also a phosphoprotein. Transformation of this complex to the 4S DNA-binding state is accompanied by dissociation of hsp90. When receptors in cytosol are transformed by heating at 25 degrees C, there is no gross change in the degree of phosphorylation of the steroid-binding protein. Both receptors that are bound to DNA after transformation under cell-free conditions and receptors that are located in the nucleus of cells incubated at 37 degrees C in the presence of glucocorticoid are labeled with 32P. The results of experiments in which the 32P-labeled receptor was submitted to limited proteolysis suggest that the 16-kDa DNA-binding domain is phosphorylated and that the 28-kDa steroid-binding domain is not.     

The 56-59-kilodalton protein identified in untransformed steroid receptor complexes is a unique protein that exists in cytosol in a complex with both the 70- and 90-kilodalton heat shock proteins

It has previously been shown that 9S, untransformed progestin, estrogen, androgen, and glucocorticoid receptor complexes in rabbit uterine and liver cytosols contain a 59-kDa protein [Tai, P. K., Maeda, Y., Nakao, K., Wakim, N. G., Duhring, J. L., & Faber, L. E. (1986) Biochemistry 25, 5269-5275]. In this work we show that the monoclonal antibody KN 382/EC1 raised against the rabbit 59-kDa protein reacts with 9S, untransformed glucocorticoid receptor complexes in cytosol prepared from human IM-9 lymphocytes but not with 4S salt-transformed receptors. The human protein recognized by the EC1 antibody is a 56-kDa protein (p56) of moderate abundance located predominantly in the cytoplasm by indirect immunofluorescence. There are at least six isomorphs of p56 by two-dimensional gel analysis. N-Terminal sequencing (20 amino acids) shows that p56 is a unique human protein. When p56 is immunoadsorbed from IM-9 cell cytosol, both the 70- and 90-kDa heat shock proteins are coadsorbed in an immune-specific manner. Neither heat shock protein reacts directly with the EC1 antibody. We conclude that p56 exists in cytosol in a higher order complex containing hsp70 and hsp90, both of which in turn have been found to be associated with untransformed steroid receptors.     

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.     

Demonstration that the 90-kilodalton heat shock protein is bound to the glucocorticoid receptor in its 9S nondeoxynucleic acid binding form

The 9S molybdate-stabilized form of the glucocorticoid receptor of mouse L cell lysates was immunoadsorbed to protein-A-Sepharose with antiserum directed against the 89-kilodalton chicken heat shock protein (anti-hsp89). In order to achieve this, "free" (nonreceptor associated) hsp90 was first separated from the molybdate-stabilized 9S receptor by sucrose gradient sedimentation. Incubation of the 9S [3H]triamcinolone acetonide-labeled receptor peak with anti-hsp89 results in the immune-specific adsorption of 20% of the specifically bound radioactivity and adsorption of the 100-kilodalton receptor protein, as detected by Western-blotting, using the GR49 antireceptor monoclonal antibody as probe. These observations provide the only direct proof that hsp90 is a component of the 9S form of a steroid receptor.     

Direct demonstration of glucocorticoid receptor phosphorylation by intact L-cells

Glucocorticoid-sensitive L-cells were cultured for 18 h in the presence of [32P]orthophosphate and steroid-binding proteins of cytosol were separated by affinity chromatography and analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and isoelectric focusing. Cytosol contains a major phosphoprotein of Mr = 92,000 and a minor phosphoprotein of Mr = 100,000, both of which bind glucocorticoids in a stereospecific, high affinity manner and have the same Mr as glucocorticoid receptor species that have been covalently labeled with the site-specific affinity ligand [3H] 9 alpha-fluoro-16-methyl-11 beta,17 alpha,21-trihydroxypregna-1, 4-diene-3,20-dione 21-mesylate. Cytosol from 32P-labeled, glucocorticoid-resistant L-cells possessing 5% of the steroid-binding capacity of sensitive cells contains very little of the Mr = 92,000 phosphoprotein and none of the Mr = 100,000 phosphoprotein. These observations provide strong evidence that the glucocorticoid receptor is phosphorylated by intact L-cells. The Mr = 92,000 protein is phosphorylated on serine and it can be resolved into two species using isoelectric focusing, consistent with the proposal that there is more than 1 phosphorylated serine/steroid-binding unit. The glucocorticoid-resistant L-cell line produces a unique phosphoprotein of Mr = 104,000 that is recovered in variable amounts after affinity chromatography. It is not known whether this phosphoprotein is a separate gene product or whether it represents a precursor with weak steroid-binding activity that is not cleaved in the resistant cell to the high affinity, Mr = 92,000 mature receptor form.     

Direct stoichiometric evidence that the untransformed Mr 300,000, 9S, glucocorticoid receptor is a core unit derived from a larger heteromeric complex

We have used three methods to measure the stoichiometry of the glucocorticoid receptor and the 90-kDa heat shock protein (hsp90) in L-cell glucocorticoid receptor complexes that were purified by immunoadsorption to protein A-Sepharose with an anti-receptor monoclonal antibody, followed by a minimal washing procedure that permits retention of receptor-associated protein. In two of the methods, receptor was quantitated by radioligand binding, and receptor-specific hsp90 was quantitated against a standard curve of purified hsp90, either on Coomassie blue stained SDS gels by laser densitometry or on Western blots by quantitative immunoblotting with 125I-labeled counterantibody. The stoichiometry values obtained by densitometry and immunoblotting are 7 and 6 mol of hsp90/mol of receptor, respectively. In a third method, which detects total receptor protein rather than just steroid-bound receptor, the ratio of hsp90 to receptor was determined by immunopurifying receptor complexes from [35S]methionine-labeled L cells, and the amount of 35S incorporated into receptor and hsp90 was corrected for the established methionine content of the respective proteins. In complexes from L cells which are labeled to steady state (48 h), the ratio of hsp90 to GR is 4:1. When immunoadsorbed receptor complexes are washed extensively with 0.5 M NaCl and 0.4% Triton X-100 in the presence of molybdate, the ratio of hsp90 to GR is 2:1. In addition to hsp90, preparations of [35S]methionine-labeled untransformed receptor complex also contain a 55-kDa protein that the conclusion that the untransformed L-cell glucocorticoid receptor exists in cytosol in a much larger heteromeric complex than considered to date.(ABSTRACT TRUNCATED AT 250 WORDS)     

Immunological evidence that the nonhormone binding component of avian steroid receptors exists in a wide range of tissues and species

A monoclonal antibody to a fungal protein has been used to demonstrate the presence of the nonhormone binding component of molybdate-stabilized steroid receptors in a variety of vertebrate tissues. We recently identified a steroid receptor in the aquatic fungus Achlya ambisexualis where sexual morphogenesis of the male is directed by the steroid antheridiol. This receptor resembles receptors of higher organisms in exhibiting an 8S, molybdate-stabilized form. In the chick oviduct, a 90 000 molecular weight protein has previously been shown to be associated with the molybdate-stabilized complex of the progesterone receptor. We have isolated a similar protein of molecular weight about 88 000 from A. ambisexualis and have obtained a hybridomal-derived monoclonal antibody directed against it. This mouse anti-Achlya immunoglobulin G1 (IgG1) cross-reacts with the 90 000 molecular weight protein in chick oviduct cytosol and was used to detect analogous 90 000 molecular weight proteins in mammalian tissues. Tissue cytosols were incubated with antibody, and the complexes were isolated onto protein A-Sepharose. The resin-bound proteins were then analyzed by gel electrophoresis. This procedure revealed the presence of 90 000 molecular weight proteins in several mammalian tissues including rat liver, mouse liver and uterus, pig ovarian granulosa cells, human endometrium, and HeLa cells. These results demonstrate that the 90 000 molecular weight protein is not peculiar to the chick oviduct but is present in several different tissues from a variety of animals. This antibody should be a useful probe for further studies on the biological role of these proteins.     

Study of the heteromeric structure of the untransformed glucocorticoid receptor using chemical cross-linking and monoclonal antibodies against the 90K heat-shock protein

The untransformed rat glucocorticoid receptor is assumed to be a hetero-oligomeric complex, containing a non-steroid binding component, the 90K heat-shock protein (HSP 90). Direct measurement of its molecular weight by chemical cross-linking provides new evidence for a trimeric structure with a Mr of ca. 270,000. Resorting to an anti HSP 90 probe (AC 88), we show that the native dimeric HSP 90 possess two accessible epitopes for this monoclonal antibody, while when bound to the steroid-binding subunit, only one epitope remains accessible. These data clearly suggest that the untransformed rat glucocorticoid receptor is an asymmetrical hetero-oligomeric complex.     

Isolation of steroid receptor binding protein from chicken oviduct and production of monoclonal antibodies

Previous studies have shown that the molybdate-stabilized progesterone receptor from the chick oviduct contains a nonhormone binding component with a molecular weight of 90 000. This protein has also been shown to be associated with some other molybdate-stabilized steroid receptors of the oviduct. In order to access this larger pool of the receptor binding protein, we have developed an isolation procedure based on the observation that the protein is selectively shed from proteins adsorbed to heparin-agarose when molybdate is removed. The protein obtained by this procedure is shown to be the same as that isolated from affinity-purified progesterone receptor as compared by protease digestion and one-dimensional peptide mapping. Four immunoglobulin G secreting hybridoma cell lines were generated against the 90 000-dalton antigen. All of the antibodies recognize the 90 000-dalton protein obtained by electrophoretic transfer from sodium dodecyl sulfate-polyacrylamide gels. In addition, two of the antibodies complex the molybdate-stabilized progesterone receptor as demonstrated by sedimentation analysis on sucrose gradients. One of these antibodies was used to show the presence of the 90 000-dalton component in molybdate-stabilized glucocorticoid and androgen receptors and also to show its presence in brain, liver, and skeletal muscle, but not in serum.     

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.     

Association of the heat shock protein hsp90 with steroid hormone receptors and tyrosine kinase oncogene products

Monospecific, polyclonal rabbit antibody raised against the 90-kd non-hormone binding component of molybdate-stabilized steroid hormone receptor specifically recognises the 90-kd molecular weight heat shock protein (hsp 90) in mink cell extracts. Partial proteolytic digestion experiments indicate that this protein is identical to the 90-kd phosphoprotein found in a highly stable complex with the protein products of at least three members of the tyrosine kinase family of oncogenes (src, fes, fgr).     

A 50-kDa cytosolic protein complexed with the 90-kDa heat shock protein (hsp90) is the same protein complexed with pp60v-src hsp90 in cells transformed by the Rous sarcoma virus.

We have previously shown that a 50-kDa protein is one component of a heteromeric complex immunoprecipitated by the 90-kDa heat shock protein (hsp90) monoclonal antibodies 8D3 and 3G3 (Perdew, G. H., and Whitelaw, M. L. (1991) J. Biol. Chem. 266, 6708-6713). In this report, we compare the 50-kDa protein with that found in pp60v-src-hsp90-p50 complexes immunoprecipitated from Rous sarcoma virus-transformed cells with antibodies to pp60v-src. 35S- and 32P-labeled p50 proteins from each system were identical in their mobilities by sodium dodecyl sulfate-polyacryl-amide gel electrophoresis. The profile of N-chlorosuccinimide cleavage products derived from each 32P-labeled p50 protein were also identical when resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. We have developed a mouse monoclonal antibody, 3M/1B5p50, capable of detecting p50 on Western blots. This antibody detected the 50-kDa protein which co-purified with the pa104 pp60v-src mutant of the avian sarcoma virus oncoprotein in 44A rat fibroblasts. We did not detect p50 in association with native glucocorticoid receptor in L cells or with the overexpressed glucocorticoid receptor in Chinese hamster ovary cells. Two experiments utilizing immunochemical staining implied that essentially all cytosolic p50 is associated with hsp90. Firstly, immunoprecipitating hsp90 from Hepa 1 cytosol with monoclonal antibody 3G3 left the cytosol depleted of p50. Secondly, cytosol fractionated by sucrose gradient revealed that p50 cosedimented with hsp90, confirming the existence of p50 only in association with hsp90.     

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.     

Hormone-free mouse glucocorticoid receptors overexpressed in Chinese hamster ovary cells are localized to the nucleus and are associated with both hsp70 and hsp90

In this work, we examine the cellular localization and protein interactions of mouse glucocorticoid receptors that have been overexpressed in Chinese hamster ovary (CHO) cells (Hirst, M. A., Northrop, J. P., Danielsen, M., and Ringold, G. M. (1990) Mol. Endocrinol. 4, 162-170). We demonstrate that wild-type unliganded mouse glucocorticoid receptor, which is expressed in CHO cells to a level approximately 10 times that of L cells, is localized entirely to the nucleus by indirect immunofluorescence with the BuGR antireceptor monoclonal antibody. Overexpressed receptors that have either no hormone binding activity or no DNA binding activity because of point mutations also localize to the nucleus, providing genetic proof that the nuclear localization cannot reflect a steroid-mediated shift of the receptor from the cytoplasm to the nucleus and that DNA binding activity is not required for nuclear localization. Like unliganded progesterone receptors, which also associate in a loosely bound "docking" complex with the nucleus, the mouse glucocorticoid receptor overexpressed in CHO cells is associated with both hsp90 and hsp70. This is in contrast to the untransformed mouse glucocorticoid receptor in L cell cytosol, which is associated with hsp90 but not hsp70. The difference in hsp70 association between cell types could reflect overexpression of the receptor in CHO cells. However, like receptors in CHO cells selected for very high levels of overexpression, receptors in CHO cells selected for an intermediate level of receptor expression that is comparable to that of L cells are also bound to hsp70. This observation argues against an explanation of hsp70 association based purely on receptor overexpression, and we speculate that association of the unliganded glucocorticoid receptor with hsp70 might be a consequence of its nuclear localization in the CHO cells. Although there are differences between the mouse receptor in CHO cells and L cells, the nuclear localization signal of the untransformed mouse receptor reacts equivalently with the AP64 antibody against NL1 in cytosols prepared from both cell types.     

Evidence that the 90-kilodalton heat shock protein is associated with tubulin-containing complexes in L cell cytosol and in intact PtK cells

It has been established that the 90-kilodalton murine heat shock protein, hsp90, is associated with the untransformed, non-DNA-binding form of the glucocorticoid receptor in L cell cytosol. In this work, we show that incubation of L cell cytosol with Affi-Gel-coupled monoclonal antibodies directed against either alpha-tubulin alone or both alpha- and beta-tubulin results in the immune-specific adsorption of hsp90 identified by Western blotting with the AC88 monoclonal antibody. Similarly, the AC88 antibody, which is specific for hsp90, causes the immune-specific isolation of both alpha- and beta-tubulin from hypotonic cytosol. The distribution of hsp90 in cultured Potorous tridactylis kidney cells was examined by indirect immunofluorescence using the AC88 monoclonal as primary antibody. In interphase cells, AC88-dependent fluorescence was distributed like antitubulin antibody-dependent fluorescence in a fibrillar array located in the cytoplasm and around the periphery of the nucleus. In cells undergoing mitosis, AC88 fluorescence was located in the mitotic spindle. These observations suggest that a significant portion of hsp90 is associated with a tubulin-containing complex both in a hypotonic cytosol preparation from mouse fibroblasts and in intact marsupial kidney epithelial cells. The distribution of AC88 fluorescence in interphase Potorous tridactylis kidney cells is similar to the distribution of glucocorticoid receptor demonstrated by Wikstrom, A. C., Bakke, O., Okret, S., Bronnegard, M., and Gustafsson, J. A in rat hepatoma and human uterine cells.     

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.     

Subunit composition of the molybdate-stabilized non-activated glucocorticoid receptor from rat liver

A monoclonal IgG 2a antibody directed against the activated rat liver glucocorticoid receptor (GR) was used to prepare an immunoaffinity matrix of high capacity. The molybdate-stabilized GR from rat liver cytosol was immunoadsorbed on this gel. A non-hormone-binding protein of Mr approximately 90,000, as determined after denaturing gel electrophoresis, was eluted from this matrix following removal of molybdate and exposure to heat (25 degrees C) and salt (0.15 M NaCl). Subsequently, the Mr approximately 90,000 protein was purified to homogeneity using high-performance ion-exchange chromatography, covalently radiolabelled, and analyzed by high-performance size-exclusion chromatography and sucrose gradient ultracentrifugation. Hydrodynamic characterization indicates that, under our experimental conditions, the molybdate-stabilized rat liver GR (Rs approximately 7.4 nm, s20,w approximately 9.1 S, calculated mol. wt Mr approximately 285,000) includes one steroid-binding unit (Rs approximately 5.5 nm, S20,w approximately 4.3 S, calculated Mr approximately 100,000) and a dimer of Mr approximately 90,000 non-hormone-binding protein (Rs approximately 6.9 nm, S20,w approximately 6.1 S, calculated native Mr approximately 180,000).