Identification of a Novel Glucocorticoid Receptor Mutation in Budesonide-Resistant Human Bronchial Epithelial Cells
Susan Kunz,
Robert Sandoval,
Peter Carlsson,
Jan Carlstedt-Duke,
John W. Bloom and
Roger L. Miesfeld
From the Departments of Biochemistry and Molecular Biophysics (S.K., R.S., R.L.M.), Pharmacology (J.W.B.) and the Respiratory Sciences Center at the University of Arizona (J.W.B.), Tucson, Arizona 85721; Karo Bio AB (P.C.), S-141 57 Huddinge, Sweden; and Department of Medical Nutrition (P.C., J.C.-D.), Karolinska Institutet, Huddinge University Hospital, Novum, S-141 86 Huddinge, Sweden
Address all correspondence and requests for reprints to: Roger L. Miesfeld, Department of Biochemistry and Molecular Biophysics, 1041 East Lowell Street, University of Arizona, Tucson, Arizona 85721. E-mail: RLM{at}u.arizona.edu.
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ABSTRACT
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We developed a molecular genetic model to investigate glucocorticoid receptor (GR) signaling in human bronchial epithelial cells in response to the therapeutic steroid budesonide. Based on a genetic selection scheme using the human Chago K1 cell line and integrated copies of a glucocorticoid-responsive herpes simplex virus thymidine kinase gene and a green fluorescent protein gene, we isolated five Chago K1 variants that grew in media containing budesonide and ganciclovir. Three spontaneous budesonide-resistant subclones were found to express low levels of GR, whereas two mutants isolated from ethylmethane sulfonate-treated cultures contained normal levels of GR protein. Analysis of the GR coding sequence in the budesonide-resistant subclone Ch-BdE5 identified a novel Val to Met mutation at amino acid position 575 (GRV575M) which caused an 80% decrease in transcriptional regulatory functions with only a minimal effect on ligand binding activity. Homology modeling of the GR structure in this region of the hormone binding domain and molecular dynamic simulations suggested that the GRV575M mutation would have a decreased affinity for the LXXLL motif of p160 coactivators. To test this prediction, we performed transactivation and glutathione-S-transferase pull-down assays using the p160 coactivator glucocorticoid interacting protein 1 (GRIP1)/transcriptional intermediary factor 2 and found that GRV575M transcriptional activity was not enhanced by GRIP1 in transfected cells nor was it able to bind GRIP1 in vitro. Identification of the novel GRV575M variant in human bronchial epithelial cells using a molecular genetic selection scheme suggests that functional assays performed in relevant cell types could identify subtle defects in GR signaling that contribute to reduced steroid sensitivities in vivo.
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INTRODUCTION
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GLUCOCORTICOIDS ARE POTENT antiinflammatory agents that have been used to treat a variety of clinical symptoms including arthritis, respiratory disease, and hematopoietic cancers. Inhaled glucocorticoids such as budesonide (Bud) (1, 2) and fluticasone (3) have been shown to be effective in the treatment of asthma because of their high potency and reduced systemic effects compared with oral glucocorticoids (4). However, long-term steroid therapy for chronic diseases can sometimes lead to complications, and not all asthma patients respond similarly to the same dose of inhaled glucocorticoids (5). In the most extreme cases of steroid insensitivity, individuals are found to be functionally glucocorticoid-resistant (6). The molecular basis for steroid insensitivity in asthma treatment is poorly understood, partly due to the complexity of the disease and to the number cell types involved (7). It is known that inhaled glucocorticoids are able to mediate responses in bronchial epithelial cells (8), circulating thymocytes (9), and infiltrating eosinophils (10), all of which are present at high levels in asthmatic airways. Glucocorticoid action in each of these cell types is highly diverse, ranging from down-regulation of cytokine gene expression in bronchial epithelial cells (11) and T cells (12), to glucocorticoid receptor (GR)-mediated apoptosis in eosinophils (13).
The most abundant GR isoform in cells is the 90-kDa GR
protein (14). Two alternatively spliced forms of GR have also been described, the GRß isoform, which is defective in ligand binding due to a 50-amino acid deletion in the C terminus (15), and GR
, an exon 3 splice variant that contains an deleterious arginine insertion at position 452 (16, 17, 18). Other protein determinants required for glucocorticoid signaling include immunophilin proteins and chaperonins, which sequester unliganded GR in a large multisubunit complex in the cytoplasm (19). There is also evidence for membrane-bound steroid transport proteins that may play a role in modulating hormone bioavailability (20, 21). Upon ligand activation, GR is transported to the nuclear compartment where it regulates gene expression by direct interactions with specific DNA sequences called glucocorticoid response elements, or through DNA-independent protein-protein interactions (22). Two types of GR-interacting proteins (GRIPs) have been characterized, the p160 coreceptor proteins GRIP1/transcriptional intermediary factor 2 (TIF2), steroid receptor coactivator 1, and receptor coactivator 3/amplified in breast cancer-1 that contain LXXLL receptor binding motifs (23), and transcription factors such as p300/cAMP response element binding protein (CREB)-binding protein, CREB, activator protein 1, signal transducer and activator of transcription 5, and nuclear factor
B (NF
B), which have been shown to interact with GR based on coimmunoprecipitation assays (22). Other protein determinants that could affect GR function include a variety of cellular kinases and phosphatases that have been proposed to directly or indirectly modulate transcriptional regulatory activity (24, 25).
Alterations in the GR
coding sequence that affect ligand binding, DNA binding, and protein-protein interactions have been shown to cause glucocorticoid insensitivity (22). It has also been reported that altered cell-specific expression of the GRß (26, 27, 28, 29) or GR
(16, 17, 18) isoforms could contribute to steroid insensitivity, as well as elevated levels of immunophilin proteins such as FKBP51 (30). One way to investigate cell-specific signal transduction pathways is to use a molecular genetic approach to identify phenotypic variants that can be isolated and characterized. For example, mouse and human T cell lines have been used to select for resistance to the synthetic glucocorticoid hormone dexamethasone (Dex) on the basis of a failure to initiate the apoptotic pathway (31, 32). Yamamoto and colleagues (33) have exploited yeast as a model eukaryotic cell to develop powerful genetic strategies that have led to the isolation of yeast-encoded ligand-effect modulator genes such as LEM3 and LEM4, which control intracellular concentrations of steroid. The advantage of using yeast is the ability to combine genetic analysis with functional genomics. A potential drawback, however, is that important cell-specific hormone responses in humans may not be recapitulated in this single cell organism.
We are interested in cell-specific glucocorticoid signaling pathways that mediate the effects of steroid therapy, especially as they relate to the treatment of asthma (34). While complete glucocorticoid resistance is relatively rare in asthma patients (35, 36), it has been observed that there is a broad range of glucocorticoid sensitivity among asthmatics that respond to steroid therapy (37). The molecular basis for variable glucocorticoid responsiveness in these patients is unknown, but it could be due to altered expression of GR isoforms (16, 17, 18, 26, 27, 28, 29) or to the activity of nonreceptor determinants that control GR functions (38, 39). Since GR structure-function studies based on transient transfection assays using monkey kidney CV-1 cells may not be suitable for detecting subtle GR signaling defects, and high-throughput, whole-genome analysis is not yet feasible for large-scale studies of diverse populations, we constructed a model system using the human bronchial epithelial cell line Chago K1 that permits a genetic selection for Bud-resistant (BudR) mutants. Bud is a synthetic glucocorticoid that is commonly used as a therapeutic agent in asthma treatment (40, 41).
In this report we describe our initial findings using this molecular genetic system and its application to the isolation and characterization of five BudR cell lines. One BudR variant, called Ch-BdE5, was chosen for detailed molecular studies and found to contain a novel GR mutation (V575M) that disrupts binding of the p160 coactivator GRIP1/TIF2 without effecting receptor protein stability or ligand binding activities. The GRV575M mutation represents a GR signaling defect that would not be detected by conventional assays of human biopsy material, suggesting that this selection strategy could be a generalized approach for investigating additional cell type-selective steroid responses. Moreover, in conjunction with other BudR cell lines we isolated, the GRV575M receptor may prove to be a useful biological reagent to study coactivator functions specifically in human bronchial epithelial cells.
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RESULTS
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Generation of a Bud-Sensitive Human Bronchial Epithelial Cell Line
Chago K1 cells are a human bronchial epithelial cell line derived from the lung tissue biopsy of a 45-yr-old male diagnosed with bronchogenic carcinoma (42). The cells are hyperdiploid with a modal chromosome number of 52 and have been shown to express the MUC-1 and MUC-2 (mucin) genes commonly associated with cancer cells (43). The overall strategy we used to develop a Chago K1 cell line for the genetic selection of BudR variants is outlined in Fig. 1
.

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Fig. 1. Flow Scheme Illustrating the Strategy Used to Isolate BudR ChagoK1 Cell Variants
ChagoK1 cells were stably transfected with the reporter gene pMMTV-GFP-neo. The cell line Ch-GFP.9 was stably transfected with the reporter gene pMMTV-HSVtk-Zeo to generate the Bud-sensitive founder lines Ch-P10 and Ch-P8. Growth of Ch-P10 and Ch-P8 cells in media containing ganciclovir, Bud, and zeomycin (Zeo) led to the isolation of the BudR cell lines Ch-Bd1, Ch-Bd2, and Ch-Bd3. Two additional BudR variants, Ch-BdE4 and Ch-BdE5, were isolated after chemical mutagenesis of Ch-P8 cells with ethylmethane sulfonate (EMS). The relative proportion of GFP+ and GFP- BudR cell lines in each selection strategy is shown.
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The basis for the selection scheme was to isolate mutant cells that failed to activate two stably integrated glucocorticoid-responsive reporter genes: 1) pMMTV-HSVtk-Zeo, which contains the glucocorticoid-responsive mouse mammary tumor virus (MMTV) promoter controlling the expression of the herpes simplex virus (HSV) thymidine kinase (tk) gene, and 2) pMMTV-GFP-neo containing the MMTV promoter driving the expression of the Aequorea victoria green fluorescence protein (GFP) gene. Because the nucleotide analog ganciclovir (Gnc) is toxic to cells expressing the HSV tk protein (44), this strategy permits a genetic selection for BudR variants that grow in media containing Bud+Gnc. The purpose of stably integrating the pMMTV-GFP-neo reporter gene was to permit screening of BudR clones for independent loss of Bud-induced GFP expression. BudR clones with defects in general glucocorticoid signaling, rather than simply a defect in HSV tk expression or enzyme activity, would be expected to be GFP-negative in Bud-containing media.
Because it was important to have a reliable screen for Bud-induced GFP expression in BudR mutants, we first isolated a neomycin-resistant Chago cell line that displayed Bud-dependent green fluorescence as judged by fluorescent activated cell sorting (FACS). One such cell line, Ch-GFP.9, was shown to display a dose-dependent increase in percent GFP+ cells at both 24 and 48 h after treatment with Bud. As can be seen in Fig. 2A
, 10% of the Ch-GFP.9 cells were found to be GFP+ at 10-9 M Bud, with a maximal response of 85% GFP+ cells at 48 h in media containing 10-7 M Bud. The lack of fluorescence in approximately 15% of the Ch-GFP.9 cells at 48 h could be due to cell cycle effects on GR activity in asynchronous cultures (45). We next stably transfected Ch-GFP.9 cells with pMMTV-HSVtk-Zeo and screened zeomycin-resistant clonal isolates for growth in Bud+Gnc media. Two cell lines, Ch-P10 and Ch-P8, were found to be extremely Bud sensitive in Gnc-containing media and were chosen as founder cell lines for the genetic selection strategy (see Fig. 1
). A representative experiment measuring Ch-P8 cell viability in media containing 10-7 M Bud and 4 µM Gnc is shown in Fig. 2B
. It can be seen that after 10 d in Bud+Gnc media, the number of viable Ch-P8 cells was reduced more than 80% as compared with cells cultured in Gnc media lacking Bud.

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Fig. 2. Characterization of the Bud-Sensitive Phenotype in the Ch-P8 Cell Line
A, Bud induction of GFP expression in the Ch-P8 parental cell line Ch-GFP.9 measured by FACS. B, Ganciclovir killing of Ch-P8 cells in the presence of Bud. Data for cell viability are presented as mean ± SEM.
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Isolation of Bud-Resistant (BudR) Chago Cell Variants
Ch-P10 and Ch-P8 cells (2 x 106) were plated in selective media (100 nM Bud, 4 µM Gnc, and 50 µg/ml Zeocin) and 36 spontaneous BudR clones were isolated and expanded 14 d later. The subclones were screened for Bud-induced GFP expression by FACS analysis to identify BudR variants with defects in glucocorticoid responsiveness. Only three of the BudR cell lines, Ch-Bd1, Ch-Bd2 and Ch-Bd3, were found to be defective in GFP expression after 48 h of Bud treatment, suggesting that most of the spontaneous mutants were due to defects in HSV tk or Gnc metabolism. As depicted in Fig. 1
, we also isolated two additional BudR variants from a plating of 7 x 106 Ch-P8 cells that had been pretreated for 16 h with 400 µM ethylmethane sulfonate (EMS). In this case, 60 BudR clones were initially isolated of which only two, Ch-BdE4 and Ch-BdE5, were found to be GFP negative in Bud-containing media.
We reasoned that if the defect in Bud responsiveness was due to a mutation in general glucocorticoid signaling, then treatment of these cells with the glucocorticoid analog Dex should reveal a DexR phenotype. Figure 3A
shows the results of transient transfection assays in these cells using a glucocorticoid-responsive MMTV-luciferase (MM-Luc) reporter gene (46). All five of the BudR cell variants were found to be defective in mediating Dex-induced luciferase activity as compared with the parental cell lines Ch-P8 and Ch-P10. We also measured GR protein levels by Western blotting using whole-cell extracts prepared from the parental and mutant cell lines. As shown in Fig. 3B
, all five of the BudR variants were found to express detectable levels of GR
; however, the steady-state level of protein was variable. The Western blot was scanned and GR expression levels were quantitated relative to a loading control. As shown in Table 1
, Ch-Bd1 expressed the lowest amount of GR protein (26% of the parental line Ch-P10), whereas Ch-BdE4 and Ch-BdE5, the two EMS-treated cell lines, expressed near-normal levels of GR protein compared with the parental cell line Ch-P8.

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Fig. 3. Characterization of GR Signaling in BudR ChagoK1 Variant Cell Lines
A, GR-mediated transcriptional activation functions in BudR variants using a transient transfection assay including the reporter plasmid pMMTV-Fluc and control plasmid pTk-Rluc. Fold-induction values were determined using the relative fluorescence units obtained from extracts prepared from cells cultured in the absence or presence of 10-6 M Dex. Data are presented as mean ± SEM. B, Western blot analysis of GR protein expression in Chago cells using an anti-GR antibody.
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To determine whether the BudR phenotype in any of the mutants was due to decreased steroid binding activity, we prepared whole-cell extracts and measured the amount of [3H]Dex-specific binding activity per µg protein using a saturating concentration of Dex (10 nM). Table 1
lists the mean values obtained from triplicate assays and shows that all three of the spontaneous BudR mutants, Ch-Bd1, Ch-Bd2, and ChBd3, had a reduced level of [3H]Dex binding activity that was proportional to a decrease in GR protein expression. This association between protein levels and steroid binding activity was not the case for the two EMS-induced BudR mutants. Ch-BdE4 cells were found to have less than 40% the level of steroid binding activity relative to Ch-P8, even though the level of GR protein in these cells (based on Western blots) was higher than in the three spontaneous mutants. In contrast, [3H]Dex binding activity in Ch-BdE5 cells was found to be the same or higher than Ch-P8 cells, suggesting that the majority of GR protein expressed in this BudR cell line retains functional steroid binding activity (Table 1
).
Analysis of Bud-Regulated Transcription in Ch-Bd1 and Ch-Bd2
To directly determine whether increasing the level of GR protein expression in the spontaneous mutants Ch-Bd1 and Ch-Bd2 could complement the BudR phenotype, we transiently transfected GR
cDNA into these two cell lines and analyzed GR transcriptional transactivation and transrepression functions. Consistent with the observed defect in Dex-induced transcription of MM-Luc in Ch-Bd1 and Ch-Bd2 cells (Fig. 3
and Table 1
), we found that induction of this same reporter gene with Bud was reduced as much as 90% relative to the parental cell lines as shown in Fig. 4A
. More importantly, cotransfection of a GR
cDNA expression vector (CMX-GR
) with the MM-Luc reporter gene resulted in a 30-fold increase in Bud-dependent luciferase activity in both Ch-Bd1 and Ch-Bd2 cell lines. This result suggested that the BudR phenotype of Ch-Bd1 and Ch-Bd2 was not due to defects in steroid bioavailability or in expression of coreceptor proteins required for MM-Luc transactivation, but rather suboptimal levels of functional GR.
Defects in Bud-dependent transcriptional induction of the MM-Luc reporter gene were predicted based on the dependence of our genetic screen on activation of the MMTV promoter in the pMMTV-HSVtk-Zeo gene construct (Fig. 1
). However, if decreased levels of GR protein were the only defect in these two spontaneous Budr mutants, then GR-mediated transrepression of NF
B activity should also be compromised. Figure 4B
shows results from transrepression assays in which Ch-Bd1 and Ch-Bd2 cells were transfected with an NF
B-luciferase (NF
B-Luc) reporter gene and stimulated with TNF
in the presence or absence of Bud. Although Bud-dependent transrepression of NF
B activity in Ch-Bd2 cells was greatly reduced, inhibition of NF
B activity in Ch-Bd1 cells was normal. Cotransfecting Ch-Bd1 and Ch-Bd2 cells with the pCMX-GR
and pNF
B-Luc plasmids led to increased levels of transcriptional transrepression in both cell lines. Taken together, these data suggest that the GR signaling defects in Ch-Bd1 and Ch-Bd2 cells are not the same since the decreased activation function in Ch-Bd1 is not associated with alterations in NF
B transrepression.
Use of Denaturing HPLC to Screen for GR Sequence Mutations
Mutations in the GR gene coding sequence that do not affect protein expression levels can best be identified by direct sequencing of the GR gene. However, the gene is large, containing nine coding exons, and alternative splice variants have been reported that would not be detected by exonic sequencing. Therefore, we chose to screen for GR mutations using a combination of RT-PCR and denaturing HPLC (DHPLC). This strategy permitted us to efficiently identify base pair mismatches in DNA heteroduplexes formed between GR cDNA derived from parental cell line Ch-P8, and GR cDNA produced from the Ch-P8-related variant cell lines Ch-Bd2, Ch-BdE4, and Ch-BdE5. The basis of DHPLC is that under partially denaturing conditions, heteroduplexes containing single-base pair mismatches will be eluted ahead of homoduplexes that are fully double stranded under the chosen conditions (47).
Figure 5
shows the RT-PCR strategy that was used to cover a 740-amino acid region of the GR coding sequence with four overlapping DNA segments (G, H, B, and E segments). For these experiments, total RNA was isolated from each of the five cell lines, and RT-PCR products corresponding to the four regions were produced. Equal amounts of corresponding RT-PCR products from two cell sources were mixed and subjected to DHPLC analysis using the WAVE System from Transgenomics, Inc. (Omaha, NE). Figure 5
shows representative elution profiles of DNA duplexes formed between GR cDNA derived from Ch-P8 and from each of the three related BudR cell lines (Bd2/P8, BdE4/P8, BdE5/P8). Results from a control P8/P8 homoduplex reaction is also shown. By comparing the elution profiles of each GR segment between the various heteroduplex combinations, it can be seen that the Bd2/P8 hybridization reactions resulted in heteroduplex products that are indistinguishable from the homoduplex P8/P8 control. This result is consistent with our data indicating that the BudR phenotype in Ch-Bd2 cells is due to decreased expression of wild-type GR (Table 1
).

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Fig. 5. Detection of Sequence Variations in the GR Coding Sequence Using RT-PCR and Denaturing HPLC (DHPLC)
Location of RT-PCR primers used in the DHPLC analysis are shown relative to the location of GR activation function 1 sequence (AF-1), DNA binding domain (DBD), and ligand binding domain. Traces of the elution profile of partially denatured heteroduplexes are derived from equimolar mixtures of RT-PCR products from the indicated cell lines. P8/P8 is the homoduplex control. Samples were analyzed using the WAVE DNA fragment analysis system (Transgenomic, Inc., Omaha, NE).
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Results of the DHPLC analyses indicated that no unique sequence alterations were present in G, H, and E segments of the GR from any of the cell lines because the elution profiles from the mixed reactions were identical to that found in the P8/P8 homoduplex control (Fig. 5
). However, significant differences were found in the DHPLC elution profiles from the B segment region of GR present in Ch-BdE4 and Ch-BdE5 cells. This region spans amino acids 373584 and encodes the GR DNA-binding domain (DBD) and the amino-terminal end of the ligand binding domain. These data indicate that one or more nucleotides differ between the GR cDNA generated with RNA from Ch-P8 cells, and the GR cDNA derived from Ch-BdE4 and Ch-BdE5 RNA.
DNA Sequencing Reveals the Presence of GR
Transcripts and a Novel Mutation at V575M
Because results of ]3H]Dex binding assays (Table 1
) suggested that Ch-BdE4 cells express a GR protein with a defect in steroid binding activity, we focused our molecular analysis on the nature of the GR sequence alternation in Ch-BdE5 cells. As a control, we also characterized the B segment region of GR in the parental Ch-P8 cells and the spontaneous mutant Ch-Bd2. As shown in Fig. 6
, DNA sequence analysis of approximately 20 randomly selected B segment cDNA clones obtained from T:A cloning of the RT-PCR products from these three cell lines identified two deviations from the previously reported GR
coding sequence. First, approximately 10% of the cDNA inserts obtained from the three cell lines were found to encode the previously described GR
variant (16, 17, 18). This form of the receptor has been proposed to be the result of an alternative splicing event at the exon 3 boundary resulting in the insertion of an arginine codon between amino acids 451 and 452 (17). This single-amino acid insertion lies within the spacer region between the two zinc fingers. Second, we identified a novel point mutation that converts Val-575 to Met in GR cDNA clones derived from Ch-BdE5 RNA. Based on the chemical nature of the mutation (G to A transition) and its relative frequency in random plasmid clones (60%), it is most likely the result of an EMS-induced alteration in the GR exon 5 coding sequence. The position of the GRV575M mutation corresponds to a region of the ligand binding domain that is likely a p160 coactivator interaction site based on sequence homology to the human estrogen, thyroid, and peroxisome proliferator-activated receptors (see Discussion).
The GRV575M Receptor Is Defective in Transcriptional Regulatory Activities
To determine whether the transcriptional regulatory functions of the GRV575M mutant receptor could account for the BudR phenotype of Ch-BdE5 cells, we introduced the V575M mutation into the cloned GR
cDNA sequence to directly measure the ligand binding and transcriptional regulatory activity of GRV575M. We also inserted the Arg codon at position 452 of GR
to generate the GR
coding sequence. The GR
, GRV575M, and GR
receptor constructs were cloned into the pCMX expression vector (48) and transfected into COS-7 cells. Forty-eight hours later, cell extracts were prepared and analyzed for GR expression by Western blotting and by [3H]Dex binding assays. The results of these studies are shown in Fig. 7
. It can be seen that all three GR constructs produce high levels of full-length receptor. Consistent with the results reported by Ray et al. (16), we found that GR
and GR
bound [3H]Dex with similar affinities. In addition, these data confirm that the ligand binding activity of GRV575M is not significantly different than GR
, which explained why the Ch-BdE5 whole-cell binding data were comparable to that of the wild-type parental cell line Ch-P8 (see Table 1
).
Figure 8
shows the results from transient transfection assays in which the same receptor constructs were cotransfected into Ch-Bd2 cells with either the MM-Luc or NF
B-Luc reporter genes. Our analysis of the Ch-Bd2 phenotype indicated that this spontaneous BudR variant expressed significantly reduced levels of GR (Fig. 3
and Table 1
), and therefore could serve as a suitable genetic background to characterize GRV575M functions within the context of a human bronchial epithelial cell. Maximal transcriptional transactivation and transrepression activities of GR
, GRV575M, and GR
in transfected Ch-Bd2 cells were found to differ over a range of Bud concentrations from 10-10 M to 10-7 M. It was seen that whereas GR
is able to induce luciferase activity nearly 100-fold at 10-9 M Bud, maximal induction by the GRV575M mutant was only 15-fold at this same steroid concentration. In addition, we found that even at the highest Bud concentration (10-7 M), the MM-Luc reporter gene was only induced 40-fold by the GRV575M receptor. Note that the dose-response profile of GR
appeared to be similar to GR
; however, the maximal transactivation activity was greatly reduced. Figure 8B
shows the results of NF
B transrepression assays using these same receptor constructs in Ch-Bd2 cells. These data show that both GRV575M and GR
have reduced levels of transrepression activity (50% of GR
at 10-9 M) and that maximal transrepression function is achieved with 10-8 M Bud. Based on similar defects in Bud-regulated transcriptional activation observed in the Ch-BdE5 cell line (Fig. 3
) and the recombinant GRV575M receptor in transfected Ch-Bd2 cells (Fig. 8
), we propose that the GRV575M mutation is a primary determinant of the BudR phenotype in Ch-BdE5 cells.
GR Binding to the p160 Coactivator GRIP1 Is Defective in GRV575M
Numerous point mutations in the GR hormone-binding domain (HBD) have been characterized, most of which disrupt ligand binding activity (49). Recently however, Vottero et al. (50) described a human GR mutation at amino acid position 747 that was identified in a patient with familial glucocorticoid resistance. Biochemical characterization of the GRI747M mutation showed that the receptor had a 2-fold decrease in affinity for Dex but an approximately 25-fold reduction in transcriptional regulatory activity. Based on the location of residue 747 in the activation function 2 (AF-2) region of GR, they tested the ability of GRI747M to functionally interact with the p160 coactivator GRIP1. They found that reduced affinity of GRI747M for GRIP1 binding in vitro was associated with decreased GRIP1-mediated transactivation in vivo.
Because the GRV575M mutation we identified in Ch-BdE5 cells also maps to the AF-2 region of the GR HBD, we used molecular modeling and dynamic simulations to predict binding interactions with a p160 peptide from the coactivator TIF2 as shown in Fig. 9
. A ClustalW alignment of amino acid residues surrounding GRV575M with the analogous AF-2 region of 16 other nuclear receptors (Fig. 9A
) reveals that Val-575 is conserved in progesterone receptor (PR), mineralocorticoid receptor, retinoic acid receptor, and the retinoid X receptor. Moreover, based on recent data describing the predicted molecular structure of the human GR HBD using x-ray crystallography (51, 52), it can be seen that Val-575 lies within helix 3. This same residue corresponds to Thr in the peroxisome proliferator-activated receptor (PPAR), and to an Ile residue in the estrogen receptor (ER) and vitamin D receptor. Importantly, the Ile-358 residue in human ER
has been shown to be part of a shallow groove adjacent to helix 3 of the ligand binding domain that binds to the LXXLL motif of coactivator proteins through van der Waals contacts (53, 54). Moreover, Thr-297 of human PPAR
(55) and Val-284 of human TRß (56) have also been shown by x-ray crystallography to interact directly with LXXLL motifs in p160 coactivator peptides.
Using the published x-ray structures of the human PR (57) and ER (53), we generated a molecular model of the human GR HBD shown in Fig. 9B
. The structural features of this homology model agree very well with the recently published x-ray models of GR (51, 52) (data not shown). The homology model contains Bud in the ligand binding pocket and includes the TIF2 box 2 peptide with a LXXLL motif that was used in the molecular structure analysis of ER
as reported by Shiau et al. (53). This GR model predicts that Val-575 is oriented toward the surface of the receptor and is within the hydrophobic coactivator binding pocket associated with helix 3 of ER
(58). The primary effect of the larger and bulkier methionine side chain in GRV575M appears to be a constraint on the rotameric freedom of the Leu +1 side chain in the LXXLL peptide. Molecular dynamic simulations suggested that the relative free energy difference between the wild-type and the mutant would be about 0.7 kcal/mol, which translates to an approximately 10-fold reduction in TIF2 binding affinity. Note that the shortest distance from GR residue 575 to the Bud ligand is approximately 15 Å according to this model. Only electrostatic forces would have a significant and direct effect on other atoms at this long distance. Considering that both Val and Met are neutral side chains possessing weak partial charges, we would expect very little direct electrostatic influence of this mutation on the ligand binding. This prediction is consistent with our ligand binding data (Table 1
and Fig. 7
).
To directly test our prediction that the GRV575M mutation disrupts p160 coactivator interactions, we performed a transactivation assay in Ch-Bd2 cells using pCMX-GR
or pCMX-GRV575M and the GRIP1 expression plasmid pSG5.GRIP1 (59). The results shown in Fig. 10A
reveal that while Bud induction of the MMTV-Fluc reporter plasmid was enhanced more than 2-fold in Ch-Bd2 cells by the coexpression of GRIP1, the transactivation activity of GRV575M was unaffected by GRIP1 expression under these same conditions. A defect in GRIP1 binding by GRV575M was confirmed using a glutathione-S-transferase (GST) pull-down assay as shown in Fig. 9B
. These results demonstrate that GR
, but not GRV575M, displayed Bud-dependent GRIP1 binding to the nuclear receptor interaction domain that includes all three LXXLL motifs (59). Taken together, the results from molecular dynamic simulations using a TIF2 peptide (Fig. 9
), and the in vivo and in vitro functional assays using GRIP1 expression plasmids (Fig. 10
), suggest that the GRV575M defect in p160 coactivator interactions contributes to the BudR phenotype in Ch-BdE5 cells. In support of this conclusion, the Ch-P8, Ch-Bd2, and Ch-BdE5 subclones were found to contain a similar steady-state level of GRIP1/TIF2 protein as Jurkat cells based on Western blotting (data not shown).

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Fig. 10. Functional Interactions Between GRV575M and the p160 Coactivator GRIP1 Are Defective in Vivo and in Vitro
A, Results of transactivation assays in which Ch-Bd2 cells were transfected with pCMX-GR or pCMX-GRV575M, pMMTV-Fluc, pTk-Rluc, with or without pSG5.GRIP1 in the presence or absence of 10-7 M Bud. The relative luciferase units for these experiments were determined as described in Materials and Methods. B, GST pull-down assays using in vitro synthesized [35S] methionine-labeled GR , GRV575M, or luciferase proteins, incubated with glutathione-coupled sepharose beads bound with GST or GST-GRIP563-1121 protein produced in E. coli. Binding experiments were performed in the presence or absence of 10-7 M Bud as indicated, and eluted proteins were separated by SDS-PAGE and visualized by autoradiography. The input radiolabeled proteins present in 2 µl of reticulocyte lysate were loaded in lanes 1, 2, and 9, whereas all other lanes show the eluted proteins recovered from GST (lane 5) and GST-GRIP563-1121 (lanes 3, 4, and 68) binding reactions containing 10 µl reticulocyte lysate.
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DISCUSSION
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We have developed a molecular genetic model to investigate mechanisms of glucocorticoid insensitivity in a human bronchial epithelial cell line that represents a therapeutic target in asthma treatment. At least four types of BudR phenotypes were identified. The first class of mutants is represented by Ch-Bd2, which had a decreased amount of GR protein (36% of its wild-type parent Ch-P8) and contained defects in transcriptional transactivation and NF
B transrepression. Down-regulation of GR expression could account for the BudR phenotype and is consistent with earlier studies showing that GR content is rate-limiting for steroid responsiveness (60). Ch-Bd1 is a second type of BudR mutant in that it also contained a reduced level of GR protein; however, NF
B transrepression function was found to be normal (35% compared with 31% for the wild-type parent Ch-P10). Although we do not know what accounts for the difference in NF
B transrepression function between Ch-Bd1 and Ch-Bd2, we did find that ectopic expression of GR cDNA in Ch-Bd1 and Ch-Bd2 cells complemented the loss of function defects in transcriptional regulatory activity (Fig. 4
).
A third class of BudR phenotypes is represented by the EMS-induced mutant Ch-BdE4. This cell line expressed near wild-type levels of GR protein based on Western blotting (Fig. 3B
), suggesting that the BudR phenotype was not the result of reduced GR expression. However, Ch-BdE4 had the lowest level of [3H]Dex binding activity compared with all five Budr mutants (Table 1
), and GR cDNA produced an altered DHPLC elution profile in the B region (amino acids 373584). These data indicate that Ch-BdE4 cells express a GR variant with sequence alterations that effect ligand binding. Experiments are in progress to verify this prediction (Kunz, S., and R. Miesfeld, unpublished data).
The most unusual BudR cell line we isolated was Ch-BdE5, which is characterized by normal GR protein levels and [3H]Dex binding activity, but with defects in transcriptional regulatory functions. Sequence analysis of GR cDNA generated from Ch-BdE5 cell RNA revealed that 60% of the randomly isolated cDNA clones contained a point mutation at V575M (Fig. 6
). A comparison of GR with other nuclear receptors showed that V575 was highly conserved and corresponded to a region in helix 3 previously shown to be involved in coactivator binding (53, 54). Molecular dynamic simulations (Fig. 9
) and protein interaction assays using the p160 coactivator GRIP1/TIF2 (Fig. 10
) confirmed that GRV575M was a poor substrate for GRIP1, suggesting that this defect may be the molecular basis for the BudR in the Ch-BdE5 cell line. Interestingly, Rogatsky et al. (61) recently reported that GRIP1 can also function as a GR corepressor to inhibit NF
B signaling though the interleukin-8 gene regulatory region. Because we found that GRV575M was defective in mediating maximal repression of NF
B signaling in transfected Ch-Bd2 cells (Fig. 8
), it is likely that decreased transrepression functions of GRV575M are also due to altered GRIP1 binding properties.
Does expression of the GRV575M mutant receptor explain the Ch-BdE5 BudR phenotype? GR transactivation functions in the BudR Ch-BdE5 cell line were only approximately 20% that of the parental Ch-P8 cell line (Table 1
), yet about half (40%) of the GR transcripts analyzed from Ch-BdE5 cells encoded the wild-type GR
receptor based on cDNA sequence analysis (Fig. 6
). If this crude measure of GR
and GRV575M transcript ratios were correct, then one explanation for the BudR phenotype would be that the GRV575M mutation had a inhibitory effect on GR
activity. This type of dominant negative activity would be similar to what Vottero et al. (50) found when they cotransfected the GRI747M mutant with GR
at a 1:1 ratio in CV-1 cells. To test this idea, we recently used transient cotransfection assays of GRV575M and GR
into CV-1 or Ch-Bd2 cells at various molar ratios and measured Bud-dependent transactivation using the MMTV-Luc reporter (Kunz, S., and R. Miesfeld, unpublished data). Results from these cotransfection assays were inconclusive, however, because the inhibitory effects of GRV575M on GR
activity appeared to be additive, rather than synergistic, using molar ratios of up to 5:1 of pCMX-GRV575M relative to pCMX-GR
. An alternative explanation for the BudR phenotype would be that the steady-state level of GRV575M protein in Ch-BdE5 cells is much greater than that of GR
protein due to differences in protein stability. If this were the case, then the observed defect in Bud-regulated GR signaling in Ch-BdE5 cells would be due to elevated levels of GRV575M protein relative to GR
protein. For example, if coactivator binding destabilizes the GR
receptor complex as a mechanism of negative feedback signaling, then the level of GRV575M protein in the cell would accumulate relative to GR
because of differences in sensitivity to such feedback mechanisms. Interestingly, transfection of equal amounts of pCMX-GR
and pCMX-GRV575M plasmid DNA into COS-7 cells resulted in higher steady-state levels of GRV575M protein than GR
protein in cell extracts as determined by Western blotting (Fig. 7
). Therefore, it is possible that GRV575M protein is inherently more stable than GR
and constitutes a greater proportion of the total GR protein in the cell, which would be consistent with the additive inhibitory effects we observed in cotransfection assays (Kunz, S., and R. Miesfeld, unpublished data).
Reconstitution experiments using stable transfections of GR
and GRV575M into the GR-deficient Ch-Bd2 variant are underway to more directly determine the role of GRV575M in mediating the BudR phenotype.
In addition to identifying functional GR mutations such as GRV575M, the Chago cell system we developed could also be used to find non-GR defects that cause steroid insensitivity. Although we have focused this initial analysis on identifying GR mutations, it is likely that a larger screen for BudR cells would lead to the identification of additional GR signaling variants that are GR independent. This could be facilitated by integrating multiple copies of GR cDNA into the Ch-P8 founder cell line to minimize the chance of selecting for BudR cells with GR mutations. One type of mutation we could find using this type of strategy would be defects in the coactivator proteins themselves, e.g. mutations in GRIP1/TIF2, steroid receptor coactivator 1, and receptor coactivator 3/amplified in breast cancer-1. Another application of this molecular genetic model could be for high throughput screens to identify steroid analogs or other small molecules that reverse the BudR phenotype resulting from GR signaling defects. The sensitivity of such an assay could be increased by stably integrating the MMTV-Luc reporter gene into selected BudR variants. In the case of Ch-BdE5, it might be possible to screen small molecule libraries for compounds that stabilize coactivator binding to GRV575M in the presence of ligand and thus restore normal transcriptional regulatory activity. Finally, cell-specific, and perhaps even ligand-specific, GR target genes could be identified by analyzing the RNA expression profiles of BudR variants under various conditions. This approach would exploit isogenic cell line panels that have minimal differences due to the use of founder cell lines. Moreover, by comparing RNA expression profiles generated from treating the same steroid-insensitive cell line with different ligands, it should be possible to identify gene targets that track with specific hormonal responses.
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MATERIALS AND METHODS
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Cell Culture
The Chago K1 cell line was obtained from the American Type Culture Collection (ATCC, Manassas, VA) and cultured in RPMI 1640 media with L-glutamine (Irvine Scientific, Santa Ana, CA), plus 10% defined calf bovine serum (CBS, Hyclone Laboratories, Inc., Logan, UT), 100 U/ml penicillin, and 0.1 mg/ml streptomycin (Sigma Chemical Co., St. Louis, MO). Cultures were maintained in a 37 C incubator with 5% CO2, at 90% humidity. COS-7 cells were grown in DMEM low-glucose pyruvate medium (Irvine Scientific) containing 10% CBS.
Plasmids
The plasmid pMMTV-GFP-neo contains a 1.4-kb fragment MMTV long terminal repeat (LTR) from pMM-CAT (62) cloned into the XhoI/SalI-HindIII sites of pEGFP-1 reporter vector (CLONTECH, Palo Alto, CA). The plasmid pMMTV-HSVtk-Zeo was constructed by inserting the MMTV LTR (XhoI-HindIII) promoter region, and the HSVtk (XbaI-BamHI) coding region into the vector pBluescript SK (Stratagene, La Jolla, CA). The zeomycin resistance gene (Zeo) from pSV40-Zeo (Invitrogen, Carlsbad, CA) was excised with NotI and XbaI and inserted into the corresponding sites of pBluescript SK. The pMMTV-Rluc and pMMTV-Fluc plasmids used for Dual Luciferase Assay were constructed by inserting the MMTV LTR promoter into the pRL-null (Renilla) and pGL3-Basic (Firefly) vectors (Promega Corp., Madison, WI) using XhoI and HindIII (46). PNF
B-luc was obtained from Stratagene. The pCMX-hGR
expression vector (63) was used to construct the GR V575 M and GR
variants using QuikChange XL site-directed mutagenesis kit (Stratagene) and appropriate mutagenic primers. The GRIP1 bacterial expression vector pGEX2TK/GRIP563-1121 and eukaryotic expression vector pSG5.HA-GRIP were obtained from M. R. Stallcup and have been described previously (59). The GR templates for in vitro coupled transcription/translation for the GST pull-down assays were created by cloning the KpnI/XhoI fragments of pCMX-hGR
or pCMX-hGRV575M into the multiple cloning site of pBluescript II SK+ cloning vector (Stratagene).
Generation of Isogenic Chago K1 Cell Lines
Stable transfection of Chago K1 cells was done using 15-cm plates that were seeded to a density of 2 x 106 cells per plate and grown in RPMI media supplemented with 10% CBS and antibiotics for 24 h. The following day, each plate was rinsed with PBS, and 4 ml RPMI media (no serum or antibiotics) were added before transfection with 10 µg linearized pMM-GFP plasmid DNA in DOTAP:DOPE transfection reagent (Avanti Lipids, Alabaster, AL) Lipofectamine (Invitrogen) using a lipid to DNA ratio of 4:1 (wt/wt). After a 6-h incubation at 37 C in 5% CO2, the lipid mixture was aspirated and replaced with RPMI growth media. After an overnight recovery, media were aspirated and replaced with selection media containing 200 µg/ml G418 (Geneticin, Calbiochem). Selection media were changed every fourth day. After 15 d, 48 Neo-resistant colonies were picked and plated in fresh media without G418. Fifteen expanded colonies were split to 10-cm plates and treated with 10-7 M Bud (AstraZeneca, Lund, Sweden) for 48 h. One of these cell lines, Ch-GFP.9, was stably transfected as described above with pMM-HSVtk-Zeo construct. After transfection, cells were allowed to recover for 48 h before addition of RPMI selection media containing 50 µg/ml Zeocin (Invitrogen) and 10% CBS. Media were changed every 34 d and Zeo-resistant colonies were picked and expanded in 12-well plates. Ten Zeo-resistant cell lines were screened for sensitivity to Bud by treating with 1 µM ganciclovir sodium (Cytovene-IV, Hoffmann-LaRoche, Inc., Nutley, NJ) with or without steroid (10-7 M Bud). Two Bud-sensitive subclones (Ch-P8 and Ch-P10) were selected for mutational analysis.
Analysis of GFP by FACS
To screen for Bud-induced GFP expression by FACS analysis, cells were seeded at a density of 3 x 105 cells per well and allowed to attach overnight. After hormone treatment (10-7 M Bud), cells were harvested 24 or 48 h later with Trypsin-EDTA, washed once with PD buffer (137 mM NaCl, 2.7 mM KCl, 1.5 mM KH2PO4, 8.1 mM Na2HPO4, pH 7.2), and fixed for 30 min in 4% paraformaldehyde. After a final wash, cells were either stored at 4 C overnight or examined immediately by FACS (FACScan with Lysis II software, Becton Dickinson and Co., Franklin Lakes, NJ).
Quantitation of GR Levels by Immunoblotting
Whole-cell protein extracts were prepared from approximately 2 x 106 cells that had been harvested by trypsinization, washed with ice-cold PBS, and resuspended in 200 µl cold PBSTDS (1% Triton X-100, 0.5% sodium deoxycholate, 0.1% sodium dodecyl sulfate), with protease inhibitors (1 µg/ml leupeptin, 1 µg/ml aprotinin, 1 mM EDTA, and 0.5 mM phenylmethylsulfonylfluoride) in PBS. After 10 min on ice, the cell lysate was cleared by centrifugation at 14,000 x g, for 10 min at 4 C. Cell extracts (30 µm protein) were separated by SDS-PAGE on a 7.5% polyacrylamide gel and transferred to nitrocellulose by electroblotting in transfer buffer at 4 C for 1 h at 90 V. Nonspecific sites on the membrane were blocked for 1 h in 3% nonfat dry milk solution in TBST (100 mM Tris, pH 8.0, 0.9% NaCl, 0.05% Tween 20). GR was detected using anti-hGR polyclonal antibody PA1512 (Affinity BioReagents, Inc., Golden, CO) at a 1:1200 dilution for 1 h. After washes, peroxidase-labeled goat antirabbit IgG secondary antibody (Life Technologies, Gaithersburg, MD) was applied at a 1:20,000 dilution for 30 min. SuperSignal chemiluminescent substrate (Pierce Chemical Co., Rockford, IL) and Bio Max autoradiographic film (Eastman Kodak, Rochester, NY) were used to identify GR protein on the membrane.
Hormone Binding Assay
Cells were grown in RPMI media containing 10% charcoal-stripped CBS to approximately 60% confluence on 15-cm tissue culture plates. To harvest cells, plates were aspirated and washed once with PBS before addition of 10 ml PDTE (20 mM Tris-HCl, pH 7.5;10 mM EDTA in PBS). After 10 min at room temperature, cells were removed by repeated pipeting and centrifuged at 1500 rpm for 5 min at 4 C. Approximately 2 x 107 cells were resuspended in PBS, pelleted, quick frozen in liquid N2, and stored at -80 C. Pellets were thawed on ice in 250 µl TEGN50 [50 mM NaCl, 1 mM EDTA, 12% (vol/vol) glycerol, 1 mM 2-mercaptoethanol, 10 mM Na-molybdate, 1 mM phenylmethylsulfonylfluoride, and 10 mM Tris-HCl, pH 7.5, at 4 C). Cells were lysed by ultrasonic disruption using a Branson probe sonicator at setting 1, with a 50% cycle for 10 sec followed by centrifugation at 10,000 x g for 10 min at 4 C. Soluble protein concentration was determined by colorimetric assay (BCA, Pierce Chemical Co.). Binding assays were set up in triplicate, with each reaction containing 65 µl cell extract and saturating amounts of [3H]Dex (10 nM), specific activity 81 Ci/mmol (Amersham Pharmacia Biotech, Piscataway, NJ). Nonradioactive ligand was added at 1000-fold molar excess to one tube of each set. Samples were incubated on ice for 2 h. Unbound hormone was removed by addition of 100 µl of a charcoal-dextran suspension in TEGN50 (10 mg/ml activated charcoal and 1 mg/ml dextran) followed by passage through a 0.45 µm spin filter. Charcoal-free filtrate was added directly to scintillation cocktail and counted. Receptor-specific binding was calculated by subtracting the value of the sample containing excess cold ligand from those containing [3H]Dex-labeled ligand only.
Transient Transfection Assay
Cells were plated at a density of 2 x 105 cells per well in 12-well tissue culture plates in RPMI media supplemented with 10% charcoal-stripped CBS, and 100 U/ml each penicillin and streptomycin. After overnight recovery, media were aspirated and cells rinsed once with PBS. One milliliter of serum-free RPMI media was added to each well. Plasmid DNA (2 µg reporter gene; pMMTV-Fluc and 0.5 µg control gene; pTk-Rluc/well) was added to the cationic lipid DOTAP:DOPE (Avanti Polar Lipids) at a lipid to DNA ratio of 2:1 (wt/wt). Lipid-DNA complexes were allowed to form for 15 min at room temperature before incubation with cells for 6 h at 37 C. The lipid mixture was then replaced with growth media and cells were allowed to recover overnight. Cells were treated for 18 h with 10-6 M Dex or 10-7 M Bud for the transactivation assays, or with 10-6 M Dex plus 1 ng/ml TNF
(R&D Systems, Minneapolis, MN) for the NF
B transrepression studies. Forty-eight hours after transfection, cells were harvested for dual luciferase assay (Promega Corp.) using passive lysis buffer. Lysates were assayed for firefly and Renilla luciferase activity by the addition of appropriate substrate and measurement of fluorescence using a model TD-20/20 luminometer (Turner Designs, Sunnyvale, CA). Relative luciferase units (RLU) were normalized by dividing the reporter value by the control value. The COS-7 and Ch-Bd2 cell transfections were done in 10-cm dishes or six-well plates using Polyfect Transfection Reagent (QIAGEN, Valencia, CA) following manufacturers protocol. The Ch-Bd2 cell transfections using the GRIP1 eukaryotic expression plasmid contained 300 ng pSG5.HA-GRIP, 50 ng pCMX, pCMX-hGR
, or pCMX-hGRV575M, and the same amounts of luciferase reporter plasmids as described above. The relative luciferase units for pCMX-hGR
or pCMX-hGRV575M transfections shown in Fig. 10A
were determined by subtracting the amount of luciferase activity in cell extracts obtained from pCMX transfections to account for the low level of endogenous GR in the Ch-Bd2 cells.
Molecular Modeling
A preliminary homology model of the GR was constructed using the Swiss Protein Data Bank (PDB) Viewer version 3.7b (64) based on the amino acid sequence of the GR (accession code P04150) retrieved from the SwissProt database (65) and the molecular structure of the PR (accession code 1A28A) retrieved from the SwissProt ExPDB. The structures of initial GR homology model and the PR crystallographic structure were superimposed using the iterative magic fit option of the Swiss PDB Viewer. The structure of the progesterone ligand (Chemical Abstracts Services registry number [57-83-0]) and the crystallographic water molecules were copied from the PR structure (PDB accession code 1A28) to the GR homology model. The GR homology was then aligned to chain A of the crystallographic structure of ER
/raloxifene core/NR box 2 TIF2 peptide complex (PDB accession code 1GWQ) and the TIF2 peptide (chain C) was copied from the crystallographic structure to the homology model. Residues A688 to I689 (except for the backbone CA, C, and O atoms of I-689) and Q695 to D696 (except for the N and CA atoms of Q695) were deleted from the copied TIF2 peptide. Residues H691 and R692 were both mutated to alanine, resulting in the capped peptide Ac-LAALL-NHMe as a simplified mimic of the LXXLL NR box. SCWRL (side chain placement with a rotamer library; University of California San Francisco, San Francisco, CA) (66) version 2.9 was used to assign the side chain conformations of the amino acid residues in the GR/Bud/Ac-LAALL-NHMe model complex holding fixed (-s option) conserved amino acid residues lining the ligand binding cavity (L563, L566, Q570, W600, M601, M604, L608, R611, F623, M646, L732, Y735, C736, F740, F750). The structure of progesterone ligand was converted to Bud (Chemical Abstract Services registry number [51333-22-3]) using the structure builder of Maestro version 4.1.012 (Schrödinger, Inc., New York, NY) and minimized in the presence of the rigid receptor using the AMBER* force field. Molecular dynamics simulations with the CHARMM force field with a 600-psec equilibration phase and 1000 psec collection phase were performed on the GR/Bud/Ac-LAALL-NHMe model complex. An explicit water sphere with radius of 20 Å was centered on the peptide, and spherical boundary conditions were applied. All solvent exposed charged side chains (Asp, Glu, Arg, and Lys) outside this sphere were neutralized. Weak positional constraints were applied to all
carbons of the protein, whereas all peptide atoms were unconstrained. Estimates of the relative free energy of the LXXLL motif binding to the wild-type and mutant receptor, respectively, were calculated by the linear interaction energy method (67) using parameters a = 0.18 and b = 0.33.
GST Pull-down Assays
GST or GST-GRIP563-1121 proteins were isolated from E. coli BL21 (DE3) pLysS cells after induction with 0.2 mM isopropyl-ß-D-thiogalactopyranoside for 3 h. Bacterial cells were harvested, resuspended in NETN buffer (100 mM NaCl, 1 mM EDTA, 20 mM Tris-HCl, pH 8.0) containing 0.5% Nonidet P-40 (NP-40) detergent plus a protease inhibitor cocktail (Sigma Chemical Co.). Cells were then lysed using a probe sonicator (Branson) at 60% duty cycle twice for 15 sec while on ice. Triton X-100 was added to a final concentration of 1% before centrifugation at 10,000 x g in a Sorvall SA-600 rotor for 30 min at 4 C. Purification of GST proteins from these extracts was performed by incubation of the supernatant for 30 min at 4 C with gentle agitation using prewashed glutathione sepharose 4B (Amersham Pharmacia Biotech). GST-bound beads were washed by centrifugation (500 x g for 5 min at 4 C) once with NETN lysis buffer (NETN buffer with 0.5% NP-40 and protease inhibitors), and then twice with cold NETN binding buffer (NETN buffer with 0.1% NP-40 and protease inhibitors). The [35S] methionine-labeled GR
, GRV575M, and firefly luciferase proteins were synthesized in the presence or absence of 10-7 M Bud using the TNT-T7 coupled Reticulocyte Lysate System (Promega Corp.) according to the manufacturers instructions. The binding assay was conducted essentially as described previously (59). Briefly, 40 µl of bead slurry containing GST or GST-GRIP563-1121 fusion protein were incubated with 10 µl of the in vitro synthesis reaction and 50 µl NETN binding buffer. Tubes were rotated slowly at 4 C for 2 h, and then the beads were washed four times by centrifugation and resuspended in NETN binding buffer at 4 C. The Bud concentration was maintained at 10-7 M for all +Bud samples during the binding and washing steps. Finally, GST proteins were eluted from the beads using 25 µl of 10 mM reduced glutathione, and protein samples were analyzed by SDS-PAGE and autoradiography using Amplify fluorographic reagent (Amersham Pharmacia Biotech).
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ACKNOWLEDGMENTS
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We wish to thank Drs. Ross Rocklin and Ralph Brattsand, formerly of Astra Draco, for supporting our initial work on developing a molecular genetic model to investigate Bud resistance; Dr. Ron Evans for the gift of human GR
expression plasmid; Dr. Roger Askew for the HSV tk plasmid; Dr. Michael Stallcup for the GRIP1 expression plasmids; Dr. Konrad Koehler at Karo Bio for help with the molecular modeling; Dr. Kerr Whitfield for critical comments on the manuscript; and Felisa Blackmer of the Arizona Research Laboratories Division of Biotechnology for help with the Denaturing HPLC analysis.
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FOOTNOTES
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This work was supported by grants (to R.L.M.) from AstraZeneca, Inc., the NIH (HL-60201), and the Jack Findlay Doyle II Charitable Fund. P.C. was supported by the Foundation for Knowledge and Competence Development and Karo Bio AB, Huddinge, Sweden.
Abbreviations: AF-2, Activation function 2; Bud; budesonide; CBS, calf bovine serum; Dex; dexamethasone; DHPLC; denaturing HPLC; EMS, ethylmethane sulfonate; ER, estrogen receptor; FACS; fluorescent activated cell sorting; GFP, green fluorescent protein; Gnc; ganciclovir; GR; glucocorticoid receptor; GRIP1; glucocorticoid receptor interacting protein 1; GST; glutathione S-transferase; HBD, hormone binding domain; HSV, herpes simplex virus; LTR, long terminal repeat; MMTV; mouse mammary tumor virus; NF
B, nuclear factor
B; NP-40, Nonidet P-40; PDB, Protein Data Bank; PMSF; phenyl-methylsulfonyl fluoride; PPAR, peroxisome proliferator-activated receptor; PR, progesterone receptor; TIF2, transcriptional intermediary factor 2; tk, thymidine kinase.
Received for publication May 2, 2003.
Accepted for publication August 7, 2003.
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