Repression of glucocorticoid receptor gene transcription by c-Jun.

The regulation of glucocorticoid receptor gene expression by members of the AP-1 family was examined in glucocorticoid-free NIH3T3 cells transfected with the human glucocorticoid receptor gene promoter driving expression of a CAT reporter gene. c-Jun inhibited the promoter activity by 80% and JunB by 30%, whereas c-Fos and JunD had no inhibitory effect. Electrophoretic mobility shift assays showed that c-Jun is unable to efficiently interact with the AP-1-like site present in the human glucocorticoid receptor promoter. Moreover, c-Jun was still able to repress promoter mutants in which the region containing the AP-1-like site was deleted. NIH3T3 cell clones overexpressing c-Jun exhibited lower glucocorticoid receptor mRNA levels, which suggests that the murine glucocorticoid receptor gene can also be regulated by AP-1. These results provide a new mechanism for cross-talk between the glucocorticoid receptor and the AP-1 family of transcription factors in the absence of glucocorticoid ligands.

Evidence that the peptidylprolyl isomerase domain of the hsp90-binding immunophilin FKBP52 is involved in both dynein interaction and glucocorticoid receptor movement to the nucleus.

We have previously shown that immunoadsorption of the FKBP52 immunophilin component of steroid receptor.hsp90 heterocomplexes is accompanied by coadsorption of cytoplasmic dynein, a motor protein involved in retrograde transport of vesicles toward the nucleus. Coimmunoadsorption of dynein is competed by an expressed fragment of FKBP52 comprising its peptidylprolyl isomerase (PPIase) domain (Silverstein, A. M., Galigniana, M. D., Kanelakis, K. C., Radanyi, C., Renoir, J.-M., and Pratt, W. B. (1999) J. Biol. Chem. 52, 36980-36986). Here we show that cotransfection of 3T3 cells with the FKBP52 PPIase domain and a green fluorescent protein (GFP) glucocorticoid receptor (GR) chimera inhibits dexamethasone-dependent movement of the GFP-GR from the cytoplasm to the nucleus. Cotransfection with FKBP12 does not affect GFP-GR movement. Inhibition of movement by the FKBP52 PPIase domain is abrogated in cells treated with colcemid to eliminate microtubules prior to steroid addition. After withdrawal of colcemid, microtubules reform, and PPIase inhibition of GFP-GR movement is restored. These observations are consistent with the notion that FKBP52 targets retrograde movement of the GFP-GR along microtubules by linking the receptor to the dynein motor. Here, we also show that native GR.hsp90 heterocomplexes immunoadsorbed from L cell cytosol contain dynein and that GR.hsp90 heterocomplexes assembled in reticulocyte lysate contain cytoplasmic dynein in a manner that is competed by the PPIase domain of FKBP52.

Separable features of the ligand-binding domain determine the differential subcellular localization and ligand-binding specificity of glucocorticoid receptor and progesterone receptor.

Glucocorticoid receptor (GR) and progesterone receptor (PR) are closely related members of the steroid receptor family of transcription factors. The two receptors share a similar domain structure, substantial sequence identity, DNA binding specificity, and the ability to induce many of the same genes. Despite these similarities, the unliganded GR is localized predominantly in the cytoplasm, while unliganded PR is found predominantly in the nucleus. By expressing green fluorescent protein (GFP)-tagged receptors and assessing subcellular localization in living cells by confocal microscopy, we have investigated the structural basis for the differential localization of GR and PR. By constructing a series of GFP-tagged receptor chimeras between GR and PR, we have shown that multiple features in the N-terminal half of the ligand-binding domain (LBD) are the critical determinants that mandate the differential localization of GR and PR. Replacement of residues encompassing helices 1-5 of GR with those of PR yields a receptor that is nuclear. However, this domain is unable to mediate nuclear import by itself when removed from the context of the receptor. The chimeric receptors also indicate that regions encompassing helices 6 and 7 are key determinants of the ligand binding potential and the transactivation potential of receptors. Thus, the determinants specifying localization of hormone-free receptors are separable from those governing ligand binding character.

Inhibition of glucocorticoid receptor nucleocytoplasmic shuttling by okadaic acid requires intact cytoskeleton.

It has been shown previously that glucocorticoid receptors (GRs) that have undergone hormone-dependent translocation to the nucleus and have subsequently exited the nucleus upon hormone withdrawal are unable to recycle into the nucleus if cells are treated during hormone withdrawal with okadaic acid, a cell-permeable inhibitor of certain serine/threonine protein phosphatases. Using a green fluorescent protein (GFP) GR chimera (GFP-GR), we report here that okadaic acid inhibition of steroid-dependent receptor recycling to the nucleus is abrogated in cells treated for 1 h with colcemid to eliminate microtubule networks prior to steroid addition. After withdrawal of colcemid, normal cytoskeletal architecture is restored and okadaic acid inhibition of steroid-dependent GFP-GR nuclear recycling is restored. When okadaic acid is present during hormone withdrawal, GR that is recycled to the cytoplasm becomes complexed with hsp90 and binds steroid, but it does not undergo the normal agonist-dependent dissociation from hsp90 upon retreatment with steroid. However, when the cytoskeleton is disrupted by colcemid, the GR in okadaic acid-treated cells recycles from the cytoplasm to the nucleus in an agonist-dependent manner without dissociating from hsp90. This suggests that under physiological conditions where the cytoskeleton is intact, a dephosphorylation event is required for loss of high affinity binding to hsp90 that is required for receptor translocation through the cytoplasm to the nucleus along cytoskeletal tracts.

Heat shock protein 90-dependent (geldanamycin-inhibited) movement of the glucocorticoid receptor through the cytoplasm to the nucleus requires intact cytoskeleton.

We use here a chimera of the green fluorescent protein (GFP) and the glucocorticoid receptor (GR) to test the notion that the protein chaperone heat shock protein-90 (hsp90) is required for steroid-dependent translocation of the receptor through the cytoplasm along cytoskeletal tracks. The GFP-GR fusion protein undergoes steroid-mediated translocation from the cytoplasm to the nucleus, where it is transcriptionally active. Treatment of 3T3 cells containing steroid-bound GFP-GR with geldanamycin, a benzoquinone ansamycin that binds to hsp90 and disrupts its function, inhibits dexamethasone-dependent translocation from the cytoplasm to the nucleus. The t1/2 for translocation in the absence of geldanamycin is approximately 5 min, and the t1/2 in the presence of geldanamycin is approximately 45 min. In cells treated for 1 h with the cytoskeletal disrupting agents colcemid, cytochalasin D, and beta,beta'-iminodipropionitrile to completely disrupt the microtubule, microfilament, and intermediate filament networks, respectively, the GFP-GR still translocates rapidly to the nucleus in a strictly dexamethasone-dependent manner but translocation is no longer affected by geldanamycin. After withdrawal of the cytoskeletal disrupting agents for 3 h, normal cytoskeletal architecture is restored, and geldanamycin inhibition of dexamethasone-dependent GFP-GR translocation is restored. We suggest that in cells without an intact cytoskeletal system, the GFP-GR moves through the cytoplasm by diffusion. However, under physiological conditions in which the cytoskeleton is intact, diffusion is limited, and the GFP-GR utilizes a movement machinery that is dependent upon hsp90 chaperone activity. In contrast to the GR, GFP-STAT5B, a signaling protein that is not complexed with hsp90, undergoes GH-dependent translocation to the nucleus in a manner that is not dependent upon hsp90 chaperone activity.

Modulation of hormone-dependent glucocorticoid receptor function using a tetracycline-regulated expression system.

The glucocorticoid receptor (GR) is a ligand-dependent transcription factor capable of stimulating and inhibiting the expression of target genes. To better understand the biological action of glucocorticoids and the function of GR, we have utilized the tetracycline (Tc)-regulated mammalian expression system to develop a novel cell line, E8.2/GR3, derived from GR null mouse L929 fibroblasts, that exhibits conditional expression of rat GR. The intracellular concentration of rGR in E8.2/GR3 cells--from undetectable levels to levels more than 10-fold greater than that observed in wild-type L929 cells--could be manipulated by varying the Tc concentration in the culture media. Similarly, dexamethasone (DEX)-dependent transactivation of the mouse mammary tumor virus long terminal repeat and transrepression of the cadmium-induced activity of the mouse heme oxygenase-1 gene enhancer, SX2, were strictly dependent on the presence of rGR, and the levels of these activities could be modulated by Tc. Similar levels of Tc, and thus rGR, were required for half-maximal transactivation and transrepression whereas a 6-fold lower concentration of DEX was required for half-maximal transrepression than for transactivation. RU486 inhibited both DEX-dependent transactivation and transrepression. DEX decreased the steady-state level of rGR mRNA and protein in a Tc dependent manner. DEX also induced morphological changes in E8.2/GR3 cells that were dependent on rGR as no alterations were observed in the presence of Tc. These cells provide a powerful system for examining the various activities of GR, particularly as a function of different intracellular receptor concentrations.

Glucocorticoids stimulate growth of human papillomavirus type 16 (HPV16)-immortalized human keratinocytes and support HPV16-mediated immortalization without affecting the levels of HPV16 E6/E7 mRNA.

We investigated the effects of the glucocorticoids hydrocortisone and dexamethasone on human papillomavirus type 16 (HPV16)-mediated human cell carcinogenesis using normal human keratinocytes (HKc) and HKc immortalized by transfection with HPV16 DNA (HKc/HPV16). Normal HKc did not require glucocorticoids for proliferation. In contrast, growth of early passage HKc/HPV16 strictly required these hormones, although glucocorticoid dependence became less stringent during in vitro progression. Glucocorticoid dependence was acquired by HKc early after immortalization with HPV16 DNA, and glucocorticoids were required for efficient HKc immortalization. However, treatment of HKc/HPV16 with hydrocortisone or dexamethasone did not increase the steady-state levels of HPV16 E6/E7 mRNA or protein. Firefly luciferase activity expressed under the control of the HPV16 upstream regulatory region and P97 promoter increased by about fourfold following dexamethasone treatment of HeLa, but only twofold in HKc/HPV16, and less than twofold in SiHa. However, all of these cell lines expressed sufficient endogenous glucocorticoid receptors to allow for a dexamethasone response of the mouse mammary tumor virus promoter. These results indicate that mechanisms other than a direct influence by glucocorticoids on HPV16 early gene expression may contribute to the striking biological effects of these steroids on HPV16-mediated human cell carcinogenesis.

Initial processing of human proenkephalin in bovine chromaffin cells.

The opioid peptide precursor preproenkephalin (PPE) contains seven enkephalin sequences and is synthesized by epinephrine-producing adrenal chromaffin cells and various peripheral and central neurons. After removal of its signal peptide, PPE undergoes processing at dibasic amino acid sites to yield its final opioid products-Met-enkephalin, Leu-enkephalin, and various larger, enkephalin-containing peptides. Processing of PPE was examined in bovine chromaffin cells using a plasmid containing the human PPE (hPPE) cDNA under the control of the cytomegalovirus immediate early enhancer/promoter. Following transfection of this hPPE-containing plasmid into bovine chromaffin cells, several proenkephalin-immunoreactive bands were observed on western blots with monoclonal antibodies that recognize human, but not bovine, proenkephalin sequences. The pattern of hPPE-derived peptides observed was similar to that of bovine PPE processing products. A series of recombinant plasmids containing mutations in the hPPE sequence at putative processing sites was then constructed. Conversion of Lys-Lys and Lys-Arg sequences to Lys-Gln and of Arg-Arg to Arg-Gln altered initial hPPE processing at only three of the putative processing sites. When hPPE cDNA containing mutations at all of these initially processed sites was expressed, one or more alternative processing sites were revealed. These data suggest the importance of structural features in addition to the dibasic sequences that limit the processing of proenkephalin.

Optimization of calcium phosphate transfection for bovine chromaffin cells: relationship to calcium phosphate precipitate formation.

Optimal conditions for formation of calcium phosphate-DNA precipitates and for chromaffin cell transfection by the calcium phosphate method were examined. A relationship was observed between turbidity of calcium phosphate solutions and the ability of calcium phosphate-DNA mixtures to give efficient transfection of bovine chromaffin cells. Under optimal conditions up to 35% of chromaffin cells in cultures transfected with plasmid DNA encoding human proenkephalin or Escherichia coli beta-galactosidase expressed the respective proteins. Important factors for transfection were the pH (6.95) and buffer employed for calcium phosphate-DNA precipitate formation, the amount and type of DNA, and the absence of serum in the cultures. Additionally, phosphate and calcium concentrations in the culture medium during incubation of cells with DNA are critical. Optimal conditions for transfection of chromaffin cells were also useful for transfection of clonal BSC-40 cells, an African green monkey kidney cell line. These results suggest that the optimal conditions described here for chromaffin cells may have broad applicability to other cell types. In addition, the results suggest that it is possible to optimize the solutions used for transfection conditions by monitoring calcium phosphate formation.

Attenuation of glucocorticoid receptor levels by the H-ras oncogene.

Certain oncogene products are known to affect the cellular response to glucocorticoids. In particular, glucocorticoid-induced transcription is impaired in H-ras-transformed cells. In this study, we examine the mechanism for this effect in NIH3T3 cells containing stably integrated H-ras genomic sequences. NIH3T3ras cells transfected with the MMTV-CAT reporter exhibit a pronounced reduction in the level of glucocorticoid-induced CAT activity, compared to normal NIH3T3 cells. As the response to glucocorticoids depends on the amount of glucocorticoid receptor protein, we have examined the cellular receptor content in both cell lines. The cytosolic and total cellular GR protein are both markedly lower in NIH3T3ras cells, suggesting that the reduced response is directly due to an attenuation of receptor levels. The steady-state level of glucocorticoid receptor mRNA is appreciably reduced in NIH3T3ras cells, which accounts for the attenuated level of glucocorticoid receptor protein. The rate of glucocorticoid receptor gene transcription is concomitantly decreased in NIH3T3ras cells. Theras effect maps to the proximal promoter of the glucocorticoid receptor gene. These results suggest that a target for activated H-Ras protein may be a transcription factor which partially represses transcription of the glucocorticoid receptor gene.

Potentiation of glucocorticoid receptor-mediated gene expression by heat and chemical shock.

We have examined the effects of heat shock on glucocorticoid receptor (GR)-mediated gene transcription in an L929 cell line derivative (LMCAT2) stably transfected with the mouse mammary tumor virus-chloramphenicol acetyltransferase (MMTV-CAT) reporter plasmid. Exposure of the LMCAT2 cells to heat or chemical shock resulted in a large increase in dexamethasone (Dex)-induced expression of CAT enzyme activity. This potentiation of hormone-induced MMTV-CAT expression was dependent on the magnitude of the stress event and on the Dex concentration, with maximal increases observed for 1 microM Dex after 2 h at 43 C or 2 h at 200 microM sodium arsenite. Heat shock potentiation of MMTV-CAT expression was not seen in an L929 cell derivative devoid of GR or in LMCAT2 cells treated with RU486 antagonist, suggesting that this effect of stress on CAT gene expression was mediated by the GR. Using a quantitative Western blot procedure, the amount of GR protein in the nucleus of cells subjected to combined heat shock and Dex treatment was no greater than the amount of nuclear GR in cells treated with hormone alone, indicating that the stress potentiation effect was not the result of increased nuclear translocation or retention by the GR. In addition, equally strong potentiations of MMTV-CAT expression were observed for cells subjected to heat shock either before or after Dex-mediated translocation of the GR to the nucleus. Thus, the major effect of stress on GR transcription enhancement activity appears to occur after the GR is bound to its high affinity nuclear acceptor sites. We have used a series of MMTV-CAT reporter constructs containing varying portions of the long terminal repeat regulatory region to show that a putative heat shock transcription factor-binding sequence at position -437 of the long terminal repeat is not required for this effect of heat shock on MMTV-CAT expression. A stress-induced increase in hormone-mediated CAT gene expression was observed for a minimal CAT reporter controlled by two synthetic glucocorticoid response elements and a TATA box sequence. Thus, it is unlikely that any DNA-binding transcription factor, other than GR, is required for this effect of stress on transcription by the hormone-bound GR. Based on these results, a model of heat shock enhancement of GR-mediated gene expression is developed in which stress acts on the DNA-bound GR, on a putative heat shock-activated adaptor, or on components of the RNA-polymerase-II complex.

Site-directed mutagenesis of the phosphorylation sites in the mouse glucocorticoid receptor.

The functional significance of receptor phosphorylation in mediating the actions of glucocorticoids remains undefined. The identification of seven phosphorylation sites in the mouse glucocorticoid receptor (Bodwell, J. E., Orti, E., Coull, J. M., Pappin, D. J. C., Smith, L. I., and Swift, F. (1991) J. Biol. Chem. 266, 7549-7555) permits a direct examination of the potential regulatory role of glucocorticoid receptor phosphorylation in transactivation. Using oligonucleotide-directed mutagenesis of the mouse glucocorticoid receptor cDNA, we have substituted alanine or aspartate for the residues phosphorylated in this ligand-dependent transcription factor. COS-1 cells were cotransfected with mutant receptor cDNA expression vectors and a reporter plasmid containing the glucocorticoid-inducible mouse mammary tumor virus promoter linked to chloramphenicol acetyltransferase in order to characterize the effect of these substitutions on receptor-mediated gene expression. Substitution of alanine or aspartate at single phosphorylation sites does not prevent receptor transactivation. Receptors containing multiple substitutions of alanine or aspartate at the major phosphorylation sites in the acidic domain elicit levels of hormone-induced reporter gene expression that are comparable to wild-type receptors. Mutant receptors substituted with alanine at the five phosphorylation sites conserved among the rat, human, and mouse receptors exhibit a 22% decrease in transcriptional activity. Receptors mutated at all seven sites display a similar modest reduction. These results demonstrate that receptor phosphorylation at these seven identified residues is not a major determinant in glucocorticoid receptor transcriptional activity at the mouse mammary tumor virus promoter.

Demonstration by confocal microscopy that unliganded overexpressed glucocorticoid receptors are distributed in a nonrandom manner throughout all planes of the nucleus.

Mouse glucocorticoid receptors (GR) that are over-expressed in Chinese hamster ovary (CHO) cells behave like progesterone receptors, in that the unliganded receptor localizes to the nucleus where it resides in a loosely bound docking complex, probably in association with the 90-kDa heat shock protein (hsp90) and hsp70. In this paper we examine the localization of the overexpressed GR within the CHO cell nucleus by confocal microscopy. In hormone-free cells the receptor distributes in a mottled pattern throughout all planes of the nucleus. The receptor is not present in nucleoli and shows no preferential localization in the periphery vs. the center of the nucleus. The mottled distribution in each plane of the nucleus demonstrates clearly that there are regions that do not contain receptor; thus, the distribution of the GR is not random. When triamcinolone acetonide is added to the CHO cells, there is no detectable change in receptor distribution. Overexpressed receptors that have either no hormone-binding activity or no DNA-binding activity because of point mutations localize in the same mottled pattern as the wild-type receptor. These observations are consistent with the proposal that the overexpressed GR can enter the nucleus in its unliganded state and proceed to loci distributed throughout the nucleus, where it is retained in an inactive docking complex until the binding of hormone triggers its progression to high affinity sites where the primary events in transcriptional activation occur. As there is no detectable change in localization with the addition of ligand, we suggest that the docking complex may be located very near or possibly at the site where the primary events in transcriptional activation occur.

Regulation of insulin-like growth factor-I messenger ribonucleic acid expression in Leydig cells.

In the present study, we evaluated insulin-like growth factor-I (IGF-I) messenger RNA expression in the rat testis. Crude interstitial cells were separated into three distinct bands on 15-60% Percoll density gradients. IGF-I mRNA was mainly localized in the Leydig cell-enriched fraction (band 3), while band 1 and band 2 cells did not contain significant amounts of IGF-I mRNA. Leydig cell IGF-I mRNA consisted of multiple species varying from 0.8 to 7.5 kb and was present in rat Leydig cells all ages examined, from 25 to 55 days old. To further document that IGF-I mRNAs are present in Leydig cells, the method of Klinefelter et al. (Biol. Reprod. (1987) 36, 769-783) was used to isolate highly purified (greater than 98% pure) Leydig cells. Most of the IGF-I mRNA was localized in these Leydig cells, while there was no detectable IGF-I mRNA in the whole testis or other interstitial cells. Furthermore, IGF-I mRNA in Leydig cells was increased more than 2-fold by growth hormone (GH) administration in vivo. This suggests that IGF-I mRNA in Leydig cells is also GH dependent. Interstitial IGF-I produced in Leydig cells may have both autocrine and paracrine effects in the testis.

Evidence that the conserved region in the steroid binding domain of the glucocorticoid receptor is required for both optimal binding of hsp90 and protection from proteolytic cleavage. A two-site model for hsp90 binding to the steroid binding domain.

Steroid hormone receptors contain a conserved sequence of amino acids within the steroid binding domain, and we have previously speculated that this conserved region is the site of interaction of the glucocorticoid receptor with hsp90 (Danielsen, M., Northrop, J. P., and Ringold, G. M. (1986) EMBO J. 5, 2513-2522; Pratt, W. B., Jolly, D. J., Pratt, D. V., Hollenberg, S. M., Giguere, V., Cadepond, F. M., Schweizer-Groyer, G., Catelli, M.-G., Evans, R. M., and Baulieu, E.-E. (1988) J. Biol. Chem. 263, 267-273). In this work, we transfect COS-7 cells with three mutants of the mouse glucocorticoid receptor deleted for all or part of this conserved region. The mutant receptor missing the entire conserved region is very unstable and is found predominantly as cleavage products. Approximately one-third of the cleavage products have lost most or all of the steroid binding domain. This mutant receptor has a constitutive activity that is about one-third that of the steroid-bound wild type receptor in stimulating transcription from a reporter gene. We propose that the partial constitutive activity results from proteolytic cleavage of the steroid binding domain from the rest of the receptor, thus removing the functional repression determined by this domain. This mutant receptor is associated with hsp90 in cytosols prepared in the presence of molybdate but, when molybdate is not present, the receptor is unstable and there is very little receptor-associated hsp90. This observation is consistent with the proposal that binding of hsp90 helps to stabilize the glucocorticoid receptor against proteolysis, and it demonstrates that the site of molybdate interaction with the receptor lies outside of the conserved sequence. Our data are interpreted according to a two-site model in which hsp90 interacts with the steroid binding domain at two sites. One site is in the conserved sequence, and the other is at a transition metal oxyanion binding site, located between the conserved sequence and the COOH terminus.

Aluminum fluoride inhibition of glucocorticoid receptor inactivation and transformation.

Fluoride, in the presence of aluminum ions, reversibly inhibits the temperature-mediated inactivation of unoccupied glucocorticoid receptors in cytosol preparations from mouse L cells. The effect is concentration-dependent, with virtually complete stabilization of specific glucocorticoid-binding capacity at 2 mM fluoride and 100 microM aluminum. These concentrations of aluminum and fluoride are ineffective when used separately. Aluminum fluoride also stabilizes receptors toward inactivation by gel filtration and ammonium sulfate precipitation. Aluminum fluoride prevents temperature-dependent transformation of steroid-receptor complexes to the DNA-binding state. Aluminum fluoride does not inhibit calf intestine alkaline phosphatase, and unoccupied receptors inactivated by this enzyme in the presence of aluminum fluoride can be completely reactivated by dithiothreitol. The effects of aluminum fluoride are due to stabilization of the complex between the glucocorticoid receptor and the 90-kDa mammalian heat-shock protein hsp90, which suggests that aluminum fluoride interacts directly with the receptor. Endogenous thermal inactivation of receptors in cytosol is not accompanied by receptor dephosphorylation. However, inactivation is correlated with dissociation of hsp90 from the unoccupied receptor. These results support the proposal that hsp90 is required for the receptor to bind steroid and dissociation of hsp90 is sufficient to inactivate the unoccupied receptor.

Isolation and characterization of a mouse L cell variant deficient in glucocorticoid receptors.

The growth of mouse L cell fibroblasts is inhibited by glucocorticoids, and we have selected spontaneous glucocorticoid-resistant L cells in culture. One cloned variant exhibits a stable phenotype in the absence of selective conditions. This variant contains no specific glucocorticoid-binding capacity, no immunoreactive glucocorticoid receptor protein, and no detectable glucocorticoid receptor messenger RNA. A glucocorticoid-dependent reporter gene requires exogenous glucocorticoid receptor cDNA and steroid in order to be expressed in this variant. Genomic DNA analysis of the variant cell line indicates that there has been no gross alteration in receptor gene structure. These results suggest that the variant may be deficient in transcription of the glucocorticoid receptor gene.

Biochemical and molecular characterization of the glucocorticoid receptor of lymphosarcoma P1798 variants.

Three phenotypically distinct isolates from lymphosarcoma P1798 have been compared with respect to properties of the glucocorticoid receptor. Wild type P1798 cells express functional receptors and glucocorticoid treatment of such cells causes cytolysis in vivo. Wild type cells do not undergo cytolysis in culture. Rather, such cells exhibit reversible inhibition of proliferation in the presence of dexamethasone. Two variant populations were selected from this background. One was selected for the ability to form tumors in mice receiving pharmacological doses of glucocorticoids. Cells from such tumors are resistant to the cytolytic effects of glucocorticoids in vivo, but are sensitive to the antiproliferative effects of the hormone in culture. Variants were also selected based upon their ability to proliferate in the presence of dexamethasone in culture. These variants were resistant to glucocorticoid-mediated cytolysis in vivo. Wild type P1798 cells express approximately 20,000 high affinity dexamethasone-binding sites per cell. Dexamethasone-mesylate labeling and immunoblotting experiments indicate that hormone binding is due to a polypeptide of Mr 90-100 K. This polypeptide is encoded in an mRNA species that resolved as a single entity of approximately 7000 nucleotides. Variants selected for resistance to cytolysis in vivo are indistinguishable in any of these respects from wild type cells. The receptors are fully functional, as evidenced by their ability to precipitate growth arrest of dexamethasone-treated cultures. Variants selected for resistance in culture harbor a receptor mutation. They express fewer than 500 dexamethasone-binding sites per cell. Such variants contain neither detectable dexamethasone-mesylate-binding protein nor any protein that is recognized by a receptor antibody.(ABSTRACT TRUNCATED AT 250 WORDS)

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.