Molecular Endocrinology, in press (December 2003 ...

Molecular Endocrinology, in press (December 2003).

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 ¥, †

Roger L. Miesfeld §, ¶

From the Departments of §Biochemistry and Molecular Biophysics, ¥Pharmacologyand the †Respiratory Sciences Center at the University of Arizona, Tucson, AZ 85721,*Karo Bio AB, S-141 57 Huddinge, Sweden, #Department of Medical Nutrition,Karolinska Institutet, Huddinge University Hospital, Novum, S-141 86 Huddinge,Sweden.

running title: Molecular Genetics of Glucocorticoid-Resistancekey words: asthma, ganciclovir, dexamethasone, mutation, GRIP1, GRg

¶Address correspondences to:Roger L. Miesfeld

Department of Biochemistry and Molecular Biophysics1041 E. Lowell StreetUniversity of Arizona

Tucson, AZ 85721, USAtel. (520) 626-2343

FAX (520) 621-9288e-mail: [email protected]

Kunz et al. - page 2

ABSTRACT

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 GST pull down assays using the

p160 coactivator GRIP1/TIF2 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.


ABBREVIATIONS

Bud; budesonide

Dex; dexamethasone

DHPLC; denaturing high performance liquid chromatography

FACS; fluorescent activated cell sorting

Gnc; ganciclovir

GR; glucocorticoid receptor

GRIP1; glucocorticoid receptor interacting protein 1

GST; glutathione S-transferase

MMTV; mouse mammary tumor virus

NR; nuclear receptor

PMSF; phenyl-methylsulfonyl fluoride

RT-PCR; reverse transcriptase polymerase chain reaction

SEM; standard error of the mean


INTRODUCTION

Glucocorticoids are potent anti-inflammatory agents that have been used to treat a

variety of clinical symptoms including arthritis, respiratory disease and hemaetopoietic

cancers. Inhaled glucocorticoids such as budesonide (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 to 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 GR-mediated apoptosis in eosinophils (13).

The most abundant GR isoform in cells is the 90 kDa GRa protein (14). Two

alternatively spliced forms of GR have also been described, the GRb isoform which is

defective in ligand binding due to a 50 amino acid deletion in the C-terminus (15) , and

GRg, an exon 3 splice variant that contains an deleterious arginine insertion at position

452 (16-18). Other protein determinants required for glucocorticoid signaling include

immunophilin proteins and chaperonins which sequester unliganded GR in a large

multi-subunit 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 (GREs), or through DNA-

independent protein-protein interactions (22). Two types of GR-interacting proteins

have been characterized, the p160 coreceptor proteins GRIP1/TIF2, SRC1 and

RAC3/AIB1 that contain LXXLL receptor binding motifs (23), and transcription factors

such as p300/CBP, CREB, AP-1, STAT-5 and NFkB which have been shown to interact

with GR based on co-immunoprecipitation 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 GRa 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 GRb (26-29) or

GRg (16-18) 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 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, that control

intracellular concentrations of steroid (33). 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 amongst

asthmatics that respond to steroid therapy (37). The molecular basis for variable GC

responsiveness in these patients is unknown but it could be due to altered expression of

GR isoforms (16-18, 26-29), or to the activity of non-receptor 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 budesonide resistant (BudR) mutants. Budesonide 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.

RESULTS

Generation of a budesonide-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 year 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 figure 1.

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.

Since 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.


Since it was important to have a reliable screen for Bud-induced GFP expression

in BudR mutants, we first isolated neomycin-resistant Chago cell line that displayed Bud-

dependent green fluorescence as judged by 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

hours after treatment with Bud. As can be seen in figure 2A, 10% of the Ch-GFP.9 cells

were found to be GFP+ at 10-9M Bud, with a maximal response of 85% GFP+ cells at 48

hours in media containing 10-7M Bud. The lack of fluorescence in ~15% of the Ch-GFP.9

cells at 48 hours 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 figure

1). A representative experiment measuring Ch-P8 cell viability in media containing 10-7

M Bud and 4 mM Gnc is shown in figure 2B. It can be seen that after 10 days in Bud+Gnc

media, the number of viable Ch-P8 cells was reduced over 80% as compared to cells

cultured in Gnc media lacking Bud.

Isolation of budesonide-resistant (Budr) Chago cell variants

Ch-P10 and Ch-P8 cells (2 x 106) were plated in selective media (100 nM Bud, 4 mM Gnc

and 50 mg/ml Zeocin) and 36 spontaneous BudR clones were isolated and expanded 14

days 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 hrs of Bud treatment, suggesting that most of the spontaneous

mutants were due to defects in HSV tk or Gnc metabolism. As depicted in figure 1, we


also isolated two additional BudR variants from a plating of 7 x 106 Ch-P8 cells that had

been pre-treated for 16 hrs with 400mM 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 dexamethasone (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 to 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 figure 3B, all five of the BudR variants were found to express detectable levels

of GRa, 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 to the parental cell line Ch-P8.

To determine if the BudR phenotype in any of the mutants was due to decreased

steroid binding activity, we prepared whole cell extracts and measured the amount of3H-Dex-specific binding activity per mg 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-dexamethasone 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 <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 if increasing the level of GR protein expression in the

spontaneous mutants Ch-Bd1 and Ch-Bd2 could complement the BudR phenotype, we

transiently transfected GRa 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

(figure 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 figure 4A. More

importantly, co-transfection of a GRa cDNA expression vector (CMX-GRa) 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 co-receptor 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 (figure 1). However, if

decreased levels of GR protein were the only defect in these two spontaneous Budr


mutants, then GR-mediated transrepression of NFkB activity should also be

compromised. Figure 4B shows results from transrepression assays in which Ch-Bd1

and Ch-Bd2 cells were transfected with an NFkB-luciferase (NFkB-Luc) reporter gene

and stimulated with tumor necrosis factor alpha (TNFa) in the presence or absence of

Bud. Although Bud-dependent transrepression of NFkB activity in Ch-Bd2 cells was

greatly reduced, inhibition of NFkB activity in Ch-Bd1 cells was normal. Co-

transfecting Ch-Bd1 and Ch-Bd2 cells with the pCMX-GRa and pNFkB-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 NFkB transrepression.

Use of denaturing HPLC to screen for GR sequence mutations

Mutations in the GR gene coding sequence that do not effect 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 which

would not be detected by exonic sequencing. Therefore, we chose to screen for GR

mutations using a combination of reverse transcriptase-mediated PCR (RT-PCR) and

denaturing high-performance liquid chromatography (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 four related Budr cell lines

(Bd2/P8, BdE4/P8, BdE5/P8) cell lines. 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).

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 since the

elution profiles from the mixed reactions were identical to that found in the P8/P8

homoduplex control (figure 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 373-584 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 presence of GRg transcripts and a novel mutation at V575M

Since 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 figure 6, DNA sequence analysis of ~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 GRa coding sequence. First, approximately 10% of the cDNA inserts obtained

from the three cell lines were found to encode the previously described GRg variant (16-

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 if 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 GRa cDNA sequence in order to directly measure the ligand

binding and transcriptional regulatory activity of GRV575M. We also inserted the Arg

codon at position 452 of GRa to generate the GRg coding sequence. The GRa, GRV575M

and GRg 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 figure 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 GRa and GRg bound 3H-Dex with similar affinities. In

addition, these data confirm that the ligand binding activity of GRV575M is not

significantly different than GRa, 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 co-transfected into Ch-Bd2 cells with either the MM-Luc or

NFkB-Luc reporter genes. Our analysis of the Ch-Bd2 phenotype indicated that this

spontaneous BudR variant expressed significantly reduced levels of GR (figure 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 GRa, GRV575M and GRg 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 while GRa 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 GRg appeared to be similar to


GRa, however, the maximal transactivation activity was greatly reduced. Figure 8B

shows the results of NFkB transrepression assays using these same receptor constructs

in Ch-Bd2 cells. These data show that both GRV575M and GRg have reduced levels of

transrepression activity (50% of GRa 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 (figure 3) and the

recombinant GRV575M receptor in transfected Ch-Bd2 cells (figure 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 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 a ~25-fold

reduction in transcriptional regulatory activity. Based on the location of residue 747 in

the 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.

Since 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 figure 9.

A ClustalW alignment of amino acid residues surrounding GRV575M with the analogous

AF-2 region of 16 other nuclear receptors (figure 9A), reveals that Val-575 is conserved

in progesterone receptor (PR), mineralocorticoid receptor (MR), retinoic acid receptor


(RAR) and the retinoid X receptor RXR. 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 (VDR). Importantly, the Ile-358 residue

in human ERa 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 PPARg (55) and Val-284

of human TRb (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 figure 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 budesonide 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 ERa 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 ERa

(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 a ~10-fold reduction in TIF2 binding affinity. Note that the shortest

distance from GR residue 575 to the budesonide 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 figure 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-GRa or pCMX-GRV575M and the GRIP1 expression plasmid pSG5.GRIP1 (59). The

results shown in figure 10A reveal that while Bud-induction of the MMTV-Fluc reporter

plasmid was enhanced over 2-fold in Ch-Bd2 cells by the co-expression 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 GST

pull-down assay as shown in figure 9B. These results demonstrate that GRa, but not

GRV575M, displayed Bud-dependent GRIP1 binding to the NR interaction domain that

includes all three NR box motifs (LXXLL) (59). Taken together, the results from

molecular dynamic simulations using a TIF2 peptide (figure 9), and the in vivo and in

vitro functional assays using GRIP1 expression plasmids (figure 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).

DISCUSSION

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 NFkB 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, NFkB transrepression function was found to be normal (35% compared to

31% for the wild-type parent Ch-P10). While we do not know what accounts for the

difference in NFkB 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 (figure 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 (figure 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 to all five Budr mutants (Table 1), and GR cDNA produced an altered DHPLC

elution profile in the B region (amino acids 373-584). 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 (SK and RM, 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 (figure 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 (figure 9) and protein interaction assays using the p160 coactivator

GRIP1/TIF2 (figure 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 NFkB signaling though the interleukin-8 gene regulatory

region. Since we found that GRV575M was defective in mediating maximal repression of

NFkB signaling in transfected Ch-Bd2 cells (figure 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 ~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 GRa receptor based on cDNA

sequence analysis (figure 6). If this crude measure of GRa and GRV575M transcript ratios

were correct, then one way to explain the BudR phenotype would be if the GRV575M

mutation had a inhibitory effect on GRa activity. This type of dominant negative

activity would be similar to what Vottero et al. (50) found when they co-transfected the

GRI747M mutant with GRa at a 1:1 ratio in CV-1 cells. To test this idea, we recently used

transient co-transfection assays of GRV575M and GRa into CV-1 or Ch-Bd2 cells at

various molar ratios and measured Bud-dependent transactivation using the MMTV-

Luc reporter (SK and RM, unpublished data). Results from these co-transfection assays

were inconclusive, however, since GRV575M inhibitory effects on GRa activity appeared

to be additive, rather than synergistic, using molar ratios of up to 5:1 of pCMX-GRV575M

relative to pCMX-GRa. 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 GRa 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 GRa protein. For example, if coactivator

binding destabilizes the GRa receptor complex as a mechanism of negative feedback

signaling, then the level of GRV575M protein in the cell would accumulate relative to GRa

because of differences in sensitivity to such feedback mechanisms. Interestingly,

transfection of equal amounts of pCMX-GRa and pCMX-GRV575M plasmid DNA into

COS-7 cells resulted in higher steady-state levels of GRV575M protein than GRa protein in

cell extracts as determined by Western blotting (figure 7). Therefore, it is possible that

GRV575M protein is inherently more stable than GRa 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 co-transfection assays (SK and RM,

unpublished data). Reconstitution experiments using stable transfections of GRa 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. While 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, for

example, mutations in GRIP1/TIF2, SRC1 and RAC3/AIB1. 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.

MATERIALS AND METHODS

Cell Culture

The Chago K1 cell line was obtained from the American Type Culture Collection

(ATCC, Rockville, MD) and cultured in RPMI 1640 media with L-glutamine (Irvine

Scientific, Santa Ana, CA), plus 10% defined calf bovine serum (CBS, Hyclone, Logan,

UT), 100U/ml penicillin and 0.1mg/ml streptomycin (Sigma, St. Louis, Mo). Cultures

were maintained in a 37oC 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 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,

Madison, WI) using XhoI and HindIII (46). pNFkB-luc was obtained from Stratagene.

The pCMX-hGRa expression vector (63) was used to construct the GR V575M and GRg

variants using QuikChange TM XL Site- directed mutagenesis Kit (Stratagene, La Jolla,

CA) and appropriate mutagenic primers. The GRIP1 bacterial expression vector

pGEX2TK/GRIP 563-1121 and eukaryotic expression vector pSG5.HA-GRIP were obtained

from M. R. Stallcup and have been described (59). The GR templates for in vitro

coupled transcription/translation for the GST pull-down assays were created by cloning

the Kpn1/Xho1 fragments of pCMX-hGRa or pCMX-hGR V575M into the MCS 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/plate and grown in RPMI media supplemented with 10% CBS

and antibiotics for 24h. The following day, each plate was rinsed with PBS and 4 ml

RPMI media (no serum or antibiotics) was added before transfection with 10mg

linearized pMM-GFP plasmid DNA in DOTAP:DOPE transfection reagent (Avanti

Lipids, Alabaster, AL) Lipofectamine (Invitrogen, Carlsbad, CA) using a lipid to DNA

ratio of 4:1 (wt/wt). After a 6 h incubation at 37o C in 5% CO2, the lipid mixture was


aspirated and replaced with RPMI growth media. Following an overnight recovery,

media was aspirated and replaced with selection media containing 200mg/ml G418

(Geneticin, CalBiochem). Selection media was changed every fourth day. After 15

days, 48 Neo-resistant colonies were picked and plated in fresh media without G418.

Fifteen expanded colonies were split to 10cm plates and treated with 10-7M Budesonide

(AstraZeneca) for 48h. 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 48h before addition of RPMI selection media containing 50mg/ml Zeocin

(Invitrogen) and 10% CBS. Media was changed every 3-4 days and Zeo-resistant

colonies were picked and expanded in 12-well plates. Ten Zeo-resistant cell lines were

screened for sensitivity to Budesonide by treating with 1mM Ganciclovir sodium

(Cytovene-IV; Hoffmann-La Roche Nutley, NJ) with or without steroid (10-7M

Budesonide). Two Budesonide-sensitive subclones (Ch-P8 and Ch-P10) were selected

for mutational analysis.

Analysis of GFP by Fluorescent Activated Cell Sorting (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 (10-7M Budesonide), cells were harvested 24 or 48 hours later with Trypsin-

EDTA , washed once with PD buffer (137mM NaCl, 2.7mM KCl, 1.5 mM KH2PO4, 8.1

mM Na2HPO4, pH7.2) and fixed for 30 min in 4% paraformaldehyde. After a final wash,

cells were either stored at 4o C overnight or examined immediately by FACS (Becton-

Dickinson FACScan with Lysis II software).


Quantitation of GR levels by Immunoblotting

Whole cell protein extracts were prepared from ~2x106 cells that had been harvested by

trypsinization, washed with ice cold PBS, and resuspended in 200ml cold PBSTDS (1%

Triton X-100, 0.5% sodium deoxycholate, 0.1% SDS), with protease inhibitors (1mg/ml

leupeptin, 1mg/ml aprotinin, 1mM EDTA, and 0.5mM PMSF) in phosphate buffered

saline. After 10 min on ice, the cell lysate was cleared by centrifugation at 14,000xg, for

10 min at 4oC. Cell extracts (30 mg protein) were separated by SDS-PAGE on a 7.5%

polyacrylamide gel, and transferred to nitrocellulose by electroblotting in transfer

buffer at 4oC for 1h at 90Volts. Non specific sites on the membrane were blocked for 1h

in 3% nonfat dry milk solution in TBST (100mM Tris pH8.0, 0.9% NaCl, 0.05% Tween

20). GR was detected using anti-hGR polyclonal antibody PA1-512 (Affinity Bioreagents,

Golden CO) at a 1:1200 dilution for 1h. After washes, peroxidase-labeled goat anti-

rabbit IgG secondary antibody (Life Technologies, Grand Island, NY) was applied at a

1:20,000 dilution for 30 min. SuperSignal chemiluminescent substrate (Pierce,

Rockford,IL) and Bio Max autoradiographic film (Kodak) were used to identify GR

protein on the membrane.

Hormone Binding Assay

Cells were grown in RPMI media containing 10% charcoal-stripped calf bovine serum 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 ( 20mM Tris-

HCl, pH 7.5;10mM EDTA in phosphate buffered saline). After 10 min at room

temperature, cells were removed by repeated pipeting and centrifuged at 1500 rpm for

5 min at 4oC. Approximately 2 X 107 cells were resuspended in PBS, pelleted, quick-

frozen in liquid N2 and stored at –80o C. Pellets were thawed on ice in 250 ml TEGN50


(50mM NaCl, 1mM EDTA, 12% vol/vol glycerol, 1mM 2-mercaptoethanol, 10mM Na-

molybdate, 1mM PMSF and 10mM TRIS-HCl, pH 7.5 at 4oC). 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 10,000xg for 10 min at 4o C. Soluble protein

concentration was determined by colorimetric assay (BCA, Pierce). Binding assays were

set up in triplicate, with each reaction containing 65ml cell extract and saturating

amounts of 3H-Dex (10nM), specific activity 81Ci/mM (Amersham, Piscataway, NJ).

Non-radioactive ligand was added at 1000-fold molar excess to one tube of each set.

Samples were incubated on ice for 2 hours. Unbound hormone was removed by

addition of 100ml of a charcoal-dextran suspension in TEGN50 (10mg/ml activated

charcoal and 1mg/ml dextran) followed by passage through a 0.45mm 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/well in 12-well tissue culture plates in

RPMI media supplemented with 10% charcoal-stripped calf bovine serum, and 100U/ml

each penicillin and streptomycin. After overnight recovery, media was aspirated and

cells rinsed once with PBS. One ml of serum-free RPMI media was added to each well.

Plasmid DNA (2 mg reporter gene; pMMTV-Fluc and 0.5 mg control gene; pTk-Rluc/

well) was added to the cationic lipid DOTAP:DOPE (Avanti Polar Lipids, Alabaster, AL)

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 6h at 37o C. The lipid mixture

was then replaced with growth media and cells 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 1ng/ml tumor necrosis factor-a (TNF-a; R&D Systems, Minn, MN)

for the NFkB transrepression studies. Forty-eight hours after transfection, cells were

harvested for Dual Luciferase Assay (Promega, Madison, WI) using passive lysis buffer.

Lysates were assayed for firefly and Renilla luciferase activity by addition of

appropriate substrate and measurement of fluorescence using a Turner Designs Model

TD-20/20 Luminometer (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 10cm 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 of

pSG5.HA-GRIP, 50 ng of pCMX, pCMX-hGRa or pCMX-hGRV575M , and the same

amounts of luciferase reporter plasmids as described above. The relative luciferase

units for pCMX-hGRa or pCMX-hGRV575M transfections shown in figure 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 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

progesterone receptor (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 (CAS 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 ERa/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 (Sidechain placement With a

Rotamer Library; University of California San Francisco, San Francisco CA 94143-0450)

(66) version 2.9 was used to assign the side chain conformations of the amino acid

residues in the GR/budesonide/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 budesonide (CAS registry number

[51333-22-3]) using the structure builder of Maestro version 4.1.012 (Schrödinger, Inc.;

New York, NY 10036-4041) and minimized in the presence of the rigid receptor using

the AMBER* force field. Molecular dynamics (MD) simulations with the CHARMM

force field with a 600 ps equilibration phase and 1000 ps collection phase were

performed on the GR/budesonide/Ac-LAALL-NHMe model complex. An explicit

water sphere of radius 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 alpha carbons of the protein, while 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 LIE 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 IPTG for 3 hours. Bacterial cells were harvested, resuspended

in NETN buffer (100mM NaCl, 1mM EDTA, 20mM Tris-HCl, pH 8.0,) containing 0.5%

NP-40 detergent plus a protease inhibitor cocktail (Sigma). Cells were then lysed using

a probe sonicator (Branson) @ 60% duty cycle for 15 sec x 2 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 4oC. Purification of GST proteins from these extracts was

performed by incubation of the supernatant for 30 min at 4o C with gentle agitation

using prewashed Glutathione Sepharose 4B (Amersham Biosciences). GST-bound

beads were washed by centrifugation (500 X g for 5 min at 4o 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 [3 5S]

methionine-labeled GRa, GRV575M and firefly luciferase proteins were synthesized in the

presence or absence of 10-7M Bud using the TNT-T7 coupled Reticulocyte Lysate System

(Promega) according to the manufacturer’s instructions. The binding assay was

conducted essentially as described (59). Briefly, 40ml of bead slurry containing GST or

GST-GRIP563-1121 fusion protein was incubated with 10 ml of the in vitro synthesis

reaction and 50ml NETN Binding Buffer. Tubes were rotated slowly at 4oC for 2 hours

and then the beads were washed four times by centrifugation and resuspended in

NETN Binding Buffer at 4oC. The Bud concentration was maintained at 10-7M for all


+Bud samples during the binding and washing steps. Finally, GST proteins were eluted

from the beads using 25 ml of 10mM reduced glutathione and protein samples were

analyzed by SDS-PAGE and autoradiography using Amplify fluorographic reagent

(Amersham).

ACKNOWLEDGMENTS

The authors 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 budesonide-resistance, Dr. Ron Evans for the gift of human GRa 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 Labs Division of Biotechnology for help with the

Denaturing HPLC analysis. This work was supported by grants to RLM from

AstraZeneca, Inc., the NIH (HL-60201) and the Jack Findlay Doyle II Charitable Fund.

PC was supported by the Foundation for Knowledge and Competence Development

(KK-stiftelsen) and Karo Bio AB, Huddinge, Sweden.


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Table 1. Characterization of Chago variants containing GR signaling defects. Three

of the cell lines were identified as spontaneous mutants (Ch-Bd1, Ch-Bd2, ChBd-3) and

two were obtained from mutagenized cell populations (Ch-BdE4, Ch-BdE5). Ch-P8 is

the parental wild-type line for Ch-Bd2, ChBd3, Ch-BdE4 and Ch-BdE5, whereas, Ch-P10

is the parental wild-type line for Ch-Bd1 (see fig. 1). The abbreviation "n.d." refers to not

determined.

Glucocorticoid Signaling Relative GR levels

Cell Line BudesonideSensitivitya

Fold-activationb

Percentrepressionc

ProteinLeveld

Bindingactivitye

Ch-P10 wild-type 38.0 33% 100 2.1

Ch-Bd1 resistant 4.3 37% 26 1.4

Ch-P8 wild-type 37.6 36% 100 2.1

Ch-Bd2 resistant 3.3 3% 36 1.7

Ch-Bd3 resistant 5.2 n.d. 37 1.5

Ch-BdE4 resistant 8.5 n.d. 76 0.8

Ch-BdE5 resistant 9.2 13% 87 2.4

aBased on growth in ganciclovir + budsonide media.bMMTV-luciferase transactivation data, similar to figure 3a.cTransrepression of NFkB data, similar to figure 4b.dQuantitation of band and intensity from western blot in figure 3b.eSpecific 3H-dexamethasone binding expressed as cpm/mg protein.


FIGURE LEGENDS

Figure 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 following 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.

Figure 2. Characterization of the Bud-sensitive phenotype in the Ch-P8 cell line. A)

Budesonide 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 budesonide.

Data for cell viability is presented as mean ± SEM.

Figure 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 is presented as mean ± SEM. B) Western blot analysis of GR protein

expression in Chago cells using an anti-GRa antibody.


Figure 4. Complementation of the BudR phenotype in Ch-Bd1 and Ch-Bd2 cells by

transiently transfected GR cDNA. A) Defects in transcriptional activation functions in

Ch-Bd1 and Ch-Bd2 could be complemented by transfection of GRa cDNA. B) The

defect in transrepression of NFkB function following TNFa stimulation could be

corrected in Ch-Bd2 cells by expression of GRa cDNA. Ch-Bd1 cells had no loss of

transrepression function relative to the parental line Ch-P10. Data is presented as mean

± SEM.

Figure 5. Detection of sequence variations in the GR coding sequence using RT-PCR

and Denaturing High Performance Liquid Chromatography (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 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 TM DNA Fragment

Analysis System (Transgenomic Inc., Omaha, NE).

Figure 6. Sequence analysis of GR coding sequences spanning the B region. A) DNA

and inferred protein sequence of the two sequence alterations (GRg and GRV575M)

identified in cloned GR sequences obtained from the B region RT-PCR reactions (see

figure 5). B) Functional map of the B region showing the location of the GRg and

GRV575M mutations relative to the DBD and exon boundaries. C) Summary of DNA

sequence data from independent plasmid isolates of the GR cDNA B region of the Ch-

P8, Ch-Bd2 and Ch-BdE5 cell lines. The total number of random cDNA isolates with the

indicated sequence organization are shown in parentheses.


Figure 7. Functional expression of GRa, GRg and GRV575M proteins in transiently

transfected COS-7 cells. A) Western blot of GR protein levels in COS-7 cells that were

transfected with pCMX expression plasmids containing the full-length GRa, GRg and

GRV575M coding sequences. The relative levels of a-tubulin expression in the COS-7

extracts was determined using an a-tubulin specific antibody. B) Specific 3H-Dex

binding activity in the same COS-7 extracts shown in "A." Binding assays were

performed as described in Materials and Methods except that total Dex concentration

was varied between 10-10 and 10-8 M as indicated. Data is presented as mean ± SEM.

Figure 8. Transcriptional activation and transrepression functions of GRa, GRg and

GRV575M coding sequences in transiently transfected Ch-Bd2 cells. A) Fold-induction

of the MM-Luc reporter gene using increasing amounts of Bud. B) Percent

transrepression of an NFkB reporter gene following treatment with TNF-a and Bud.

Data is presented as mean ± SEM.

Figure 9. Molecular modeling predicts that GRV575M is defective in p160 coactivator

binding. A) ClustalW alignment of amino acid residues in the region of GRV575 from a

variety of intracellular receptors. This 28 residue stretch corresponds to most of helix 3

and all of helix 4 of the human GR (51), ERa (53), PPARg (55), and TRb (56) ligand

binding domains as defined by x-ray crystallography. The "*" identifies the

corresponding residues in hERa (I358), hPPARg (T297) and hTRb (V284) that have been

shown by these same studies to make van der Waals contacts with Box II peptides of

p160 coactivators. The "•" denotes residues that contribute to a shallow groove within

the p160 coactivator binding site. B) Molecular modeling of the GRV575M mutation. Top


view of the hydrophobic coactivator binding pocket for the wild-type complex (left)

and mutated complex (right). The GR surface is shown as a green mesh. The

coactivator peptide is indicated by a blue tube, having the C-terminus to the right. The

LxxLL leucines are shown as ball-and-stick models, and the mutated residue at position

575 is shown as space-filling atoms under the mesh. Residue 575 directly interacts with

the Leu!+1 and Leu!+4 side chain residues in the coactivator peptide. Both pictures are

representative snapshots of the equilibrated complexes after 1600 ps of simulation.

Figure 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-GRa or pCMX-GRV575M, pMMTV-Fluc, pTk-Rluc, with

or without pSG5.GRIP1 in the presence or absence of 10-7M 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 [3 5S] methionine-labeled GRa, 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 10-7M Bud as indicated and eluted proteins

were separated by SDS PAGE and visualized by autoradiography. The input

radiolabeled proteins present in 2 ml 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, 6-8) binding reactions containing 10 ml of reticulocyte lysate.

Kunz et al., Figure 1

pMMTV-HSV-tk-Zeo

Ch-GFP.9

Ch-P8

Ch-Bd1

Ch-BdE4 Ch-BdE5

Ch-Bd2

Ch-P10

ChagoK1

Select colonies in 4 µM ganciclovir, 0.1 µM budesonide, 50 µg/ml Zeo

Screen for GFP fluorescence in 0.1 µM budesonide by FACS

pMMTV-GFP-neo

EMS mutagenesis

BudS foundercell lines

Isolation of 36 BudR cell lines of which 3 were GFP-

Isolation of 60 BudR cell lines of which 2 were GFP-

Ch-Bd3

Kunz et al. figure 2

A

0

20

40

60

80

10024 hr48 hr

10-10 10-9 10-8 10-7 10-6

Budesonide (M)

%G

FP

Pos

itive

B

-Bud

+Bud

0 1 2 3

8

6

7

5

4

2

1

3

4 5 6 7 8 9 10

Days in Culture

Via

ble

Cel

ls/m

l (x

104 )

Kunz et al. Figure 3

Fo

ld-I

nd

uct

ion

Cell Line

Ch-

P8

Ch-

Bd2

Ch-

Bd3

Ch-

BdE4

Ch-

BdE5

Ch-

P10

Ch-

Bd1

40

30

20

10

0

B

A

Ch-

P8

Ch-

Bd2

Ch-

Bd3

Ch-

BdE4

Ch-

BdE5

Ch-

P10

Ch-

Bd1

- GRα


B

A

20

40

60

80

100

120

Ch-P10 Ch-Bd1 Ch-Bd1+ GRα


Cell Line

Fold

-in

du

ctio

n (M

MT

V)



10

20

30

40

50

60

70

80

90

Perc

ent

Rep

ress

ion

(NFk

B)

Cell Line


0 100 200 300 400 500 600 700 777

BGH E

AF-1 DBD Ligand Binding

P8/P8

Bd2/P8

BdE5/P8

BdE4/P8

4.03.0 4.5 5.03.52.54.53.5 5.0 5.54.03.04.53.5 5.0 5.54.03.04.53.5 5.0 5.54.03.0

Kunz et al Figure 6

DNA Binding Domain Hinge + Ligand Binding Region373 584

R (GRγ) V575M

Exon 4 Exon 5Exon 3B

Cwild type GR

(18)

GRγ(2)

Ch-P8

wild type GR(17)

GRγ(2)

Ch-Bd2

Ch-BdE5wild type GR

(8)

GRγ V575M

(1)

V575M

(11)

A GRγ (3 bp insertion at exon 3 splice site)449 450 451 452 453 454GTG GAA GGT AGA CAG CAC AAT V E G R Q H N

V575M (G to A transition)573 574 575 576 577GCA GCA ATG AAA TGG A A M K W

GR

Tubulin

No

DN

AG

Rα

V575

M

GRγ

121

79

53

kDa

A

B


GRα

GRγ

V575M

Bin

din

g A

ctiv

ity

(cp

m/µ

g)

0

100

200

300

400

500

600

10-8 M 10-9 M 10-10 M

[3H-Dexamethasone]


0

10

20

30

40

50

60

70GRα

GRγ

V575M

10-8 M 10-7M 10-9 M 10-10 M

[Budesonide]

Perc

ent

Rep

ress

ion

B

GRα

GRγ

V575M

Fold

-in

du

ctio

n

0

20

40

60

80

100

120

10-8 M 10-7M 10-9 M 10-10 M

[Budesonide]

A


GR LNMLGGRQVIAAVKWAKAIPGFRNLHLD 590MR LNRLAGKQMIQVVKWAKVLPGFKNLPLE 796PR LNQLGERQLLSVVKWSKSLPGFRNLHID 745TRa FTKIITPAITRVVDFAKKLPMFSELPCE 245TRb FTKIITPAITRVVDFAKKLPMFCELPCE 299RARa FSELSTKCIIKTVEFAKQLPGFTTLTIA 255RARb FSELATKCIIKIVEFAKRLPGFTGLTIA 255RARg FSELATKCIIKIVEFAKRLPGFTGLSIA 257RXRa ICQAADKQLFTLVEWAKRIPHFSELPLD 295RXRb ICQAADKQLFTLVEWAKRIPHFSSLPLD 366RXRg ICHAADKQLFTLVEWAKRIPHFSDLTLE 296 PPARa CQCTSVETVTELTEFAKAIPGFANLDLN 303PPARb CQCTTVETVRELTEFAKSIPSFSSLFLN 276PPARg CQFRSVEAVQEITEYAKSIPGFVNLDLN 340 VDR LADLVSYSIQKVIGFAKMIPGFRDLTSE 257ERb LTKLADKELVHMISWAKKIPGFVELSLF 325 ERa LTNLADRELVHMINWAKRVPGFVDLTLH 373

GRV575M

•• • • • • •*

Helix 3 Helix 4

GRα V575M

A

B

A

B

Rela

tive

Lu

cife

rase

Un

its

0

2

4

6

8

10

12

14

BudGRIP-1

GRα V575M

+ + +++ + + +

- -- - - -

- -

GRα

GRα

GRα

GRα

V575

M

V575

M

V575

MLu

cif.

Luci

f.

+ +++ + + +

++ + +- - -- - -

- - - - --

---

-

35S Protein

GST-GRIPGST

Bud


- 120

- 84

- 66

- 39- 50

MW (kDa)

1 2 3 4 5 6 7 8 9

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