Expression of ecdysteroid receptor and ultraspiracle from Chironomus tentans (Insecta, Diptera) in E. coli and purification in a functional state

Insect Biochemistry and Molecular Biology 32 (2002) 167–174 www.elsevier.com/locate/ibmb

Expression of ecdysteroid receptor and ultraspiracle from Chironomus tentans (Insecta, Diptera) in E. coli and purification in a functional state Marco Grebe, Margarethe Spindler-Barth

*

Abteilung fu¨r Allgemeine Zoologie und Endokrinologie, Universita¨t Ulm, Albert-Einstein-Allee 11, 89081 Ulm, Germany

Abstract Full length clones of ecdysteroid receptor (EcR) and Ultraspiracle (USP) from Chironomus tentans were expressed as GST fusion proteins in E. coli and purified by affinity chromatography. The absence of detergents during the purification procedure is essential for retaining receptor function, especially ligand binding. Presence of USP is mandatory for ligand binding to EcR, but no other cofactors or posttranslational modifications seem to be important, since Scatchard plots revealed the same characteristics (two high affinity binding sites for Ponasterone A with KD1=0.24±0.1 nM and KD2=3.9±1.3. nM) as found in 0.4 M NaCl extracts of Chironomus cells. Gel mobility shift assays showed binding of the heterodimer to PAL and DR5 even after removal of the GST-tag, whereas EcR binding to PAL1 is GST-dependent. USP binds preferentially to DR5. Addition of unprogrammed reticulocyte lysate improves ligand binding only slightly. Removal of GST has no effect on 3H-ponasterone A binding, but alters DNA binding characteristics. Calculation of specific binding (5.3+3.0 nmol/mg GST EcR) revealed that 47±26% of purified receptor protein was able to bind ligand. The addition of purified EcR to cell extracts of hormone resistant subclones of the epithelial cell line from C. tentans, which have lost their ability to bind ligand, restores specific binding of 3H-ponasterone A.  2002 Elsevier Science Ltd. All rights reserved. Keywords: Nuclear receptor; Ligand binding; Hormone; DNA-binding; Insect

1. Introduction Moulting hormones and their intracellular receptor (EcR) play a crucial role in insect development (Lezzi et al., 1999; Spindler-Barth and Spindler, 2000; Spindler et al., 2001). Characterisation of its biochemical and physicochemical characteristics is impeded by difficulties in obtaining pure receptor protein in a functional state (Srinivasan, 1992) due to the size, the considerable hydrophobicity and sensitivity to detergents and high salt conditions. Meanwhile the DNA binding (NiedzielaMajka et al., 1998) and the ligand binding (Halling et al., 1999) domains of the Drosophila EcR and the dimerization partner Ultraspiracle (USP) were expressed successfully as fusion proteins in E. coli. However, the syn-

* Corresponding author. Tel.: +49-731-502-2593; fax: +49-731502-2581. E-mail address: [email protected] (M. Spindler-Barth).

thesised products were found to be insoluble proteins in inclusion bodies. A considerable improvement in yield and function of the receptor ligand binding domain was achieved by coexpression of nuclear receptors involved in heterodimerization (Li et al., 1997), and omission of hydrophobic C-terminal end of DmEcR. The solubility and yield of EcR are greatly enhanced and allowed the purification of milligram amounts of functional ligand binding domains of EcR/USP (Halling et al., 1999). Expression of Drosophila full length EcR in E. coli failed due to the instability of the expressed proteins and the size of the receptor molecule, but DmEcR was synthesised recently in SF9 cells (Arbeitman and Hogness, 2000). In contrast, the smaller EcR from Chironomus tentans could be expressed as full length protein in E. coli, however the functionality concerning ligand binding was poor (Elke et al., 1997). Chironomus EcR is smaller in size and lacks the extremely hydrophobic region at the N-terminal end. Although receptor domains exert their functions

0965-1748/02/$ - see front matter  2002 Elsevier Science Ltd. All rights reserved. PII: S 0 9 6 5 - 1 7 4 8 ( 0 1 ) 0 0 0 9 8 - 4

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autonomously, these activities are modulated by intramolecular signaling between different domains (Doesburg et al., 1997; Scheller et al., 1998; Kumar et al., 1999), dimerization with other nuclear receptors (Glass, 1996), binding of comodulators (McKenna et al., 1999) and interactions with other proteins. Our aim was to obtain pure full length receptor in a functional state to study the biochemical and biophysical properties of EcR and USP individually and in the presence of the heterodimerization partner.

2. Experimental procedures 2.1. Plasmid constructs and expression in E. coli The CtEcRH (Imhof et al., 1993) encoding DNA fragment cloned into the BamH1 site of the E. coli expression plasmid pGEX-KT (Hakes and Dixon, 1992) and the CtUSP-2 (Vo¨ gtli et al., 1999) encoding DNA fragment cloned into the HindIII site of the E. coli expression plasmid pGEX-KG (Guan and Dixon, 1991) were kindly provided by Dr Lezzi (ETH Zu¨ rich, Switzerland). Overnight cultures of E. coli strain BL21 (DE3) (Studier et al., 1990) freshly transformed with the expression plasmids were diluted 1:50 with 2×YT medium (16 g/l Trypton, 10 g/l yeast extract, 5 g/l NaCl, 50 mg/l ampicillin, 0.2% glucose, pH 7.0) and grown at 37°C to OD600=0.6. Expression was induced by addition of isopropyl ß-d-thiogalactoside (IPTG, final concentration 0.2 mM) and the culture was incubated further for 3 h at 28°C. Due to the instability of the GST-EcR vector only freshly transformed E. coli were used. 2.2. Affinity purification and thrombin cleavage of fusion proteins Four hundred millilitres of bacteria were collected by centrifugation, washed once with PBS (137 mM NaCl, 2.7 mM KCl, 8 mM Na2HPO4, 1.5 mM KH2PO4, pH 7.3) and resuspended in 10 ml TE-buffer (10 mM Tris– HCl pH 7.3, 1 mM EDTA, aprotinin, pepstatin and leupeptin each 1 µg/ml final concentration) supplemented with dithiothreitol (DTT, 5 mM final concentration) and phenylmethylsulfonyl fluoride (PMSF, 0.5 mM final concentration). Cells were disrupted under cooling by sonification (Branson Sonifier B-12, Branson, Danbury, CT) using a microtip for 3×3 s with 90 W and stored at ⫺80°C until use. The homogenate was centrifuged (30 000g, 20 min, 4°C) to remove cell debris. The supernatant was supplemented with NaCl to a final concentration of 150 mM and incubated with glutathione sepharose 4B (Pharmacia Biotech, Uppsala, Sweden). Two millilitres of 50% sepharose slurry per 10 ml supernatant were incubated for 45 min at 4°C followed by 45 min at room temperature with gentle agitation. After

centrifugation (500g, 5 min, 4°C) the gel was washed twice with 10 ml cold low salt buffer (20 mM HEPES, 20 mM NaCl, 20% glycerol, 1 mM EDTA, pH 7.3; aprotinin, pepstatin, leupeptin each 1 µg/ml final concentration), four times with 10 ml high salt buffer (20 mM HEPES, 400 mM NaCl, 20% glycerol, 1 mM EDTA, pH 7.3; aprotinin, pepstatin, leupeptin each 0.5 µg/ml final concentration) and finally four times with 10 ml Tris-buffer (100 mM Tris, 120 mM NaCl, 20% glycerol 1 mM EDTA, pH 8). The fusion proteins were eluted by incubation of the gel with glutathione buffer (10 mM reduced glutathione, 5 mM dithiothreitol, 100 mM Tris, 120 mM NaCl, 20% glycerol 1 mM EDTA, pH 8) and mixed gently for 45 min at 4°C followed by 20 min at room temperature. After centrifugation (500g, 5 min, 4°C) the supernatant containing the purified GST receptor protein was removed and aliquots were stored at ⫺80°C. The elution step was repeated twice with 20 min incubation at room temperature. To remove the GST label of fusion proteins the glutathione sepharose gel was resuspended after the washing procedure in cleavage buffer (20 mM HEPES, 20 mM NaCl, 20% glycerol, 1 mM EDTA, 1 mM 2-mercaptoethanol, pH 7.3) supplemented with 40 U/ml thrombin (from bovine plasma, Sigma, Steinheim, Germany) and 200 pg/ml aprotinin (Sigma) and incubated for 1 h at 4°C. Incubation by gentle agitation was continued for 2 h at room temperature and the gel centrifuged (500g, 4°C, 5 min). 2.3. Electrophoresis, silver staining and Western blots Protein was precipitated with the same volume of 15% trichloroacetacid (TCA) and incubated on ice. The TCA pellets were dissolved in loading buffer (100 mM Tris, 3% SDS, 1 mM 2-mercaptoethanol, pH 8.8, 0.1% bromphenol blue). Cell lysates were diluted with the same volume of 2× concentrated loading buffer. The samples were boiled for 5 min (La¨ mmli, 1970) and loaded on a SDS-gel (0.6×MDE gel solution, Boehringer, Ingelheim, Germany; AT Biochem., Minigel Twin, 8.6×7.2×0.1 cm3, Biometra, Go¨ ttingen, Germany). Gels were stained with silver according to Blum et al. (1987) or submitted to Western blots as described already in detail (Rauch et al., 1998). The membranes were probed either with antibody pABcE/D (Wegmann et al., 1995) directed against the D-domain of CtEcR (kind gift of Dr M. Lezzi, ETH Zu¨ rich, Switzerland) or with a monoclonal anti-GST-antibody (G-1160, Sigma, Deisenhofen, Germany). 2.4. Ligand binding assays Ligand binding was determined with [3H]-ponasterone A (specific activity=7.9 TBq/mmol; a kind gift of Prof. H. Kayser, Novartis, Switzerland) using a filter assay as

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described previously (Turberg and Spindler, 1992) with slight modifications (Rauch et al., 1998). Since NaCl impedes hormone binding, purified receptor preparations were desalted with Sephadex G-25 column (Pharmacia, Uppsala, Sweden) using a low salt buffer (20 mM HEPES, 20 mM NaCl, 20% glycerol, 1 mM EDTA, 1 mM 2-mercaptoethanol, pH 7.9). The buffer was supplemented with 1 mg/ml bovine serum albumin (BSA) and a mixture of protease inhibitors (aprotinin, leupeptin, pepstatin, final concentration 1 µg/ml each). Samples were incubated with radiolabelled ligand for 1 h at room temperature. Nonspecific binding was determined by addition of 0.1 mM nonlabelled 20-OH-ecdysone. KD values were calculated according to Scatchard (1949). For reconstitution experiments cell extracts of hormone resistant C. tentans subclones (Grebe et al., 2000) were mixed either with purified GST-CtEcR (50 ng/500 µl) or GST-CtUSP (100 ng/500 µl GST-USP), and incubated with 2 nM [3H]-ponasterone A under the same conditions. For competition experiments, aliquots of purified receptor were incubated with 5 nM [3H]-ponasterone A in the presence of various concentrations of 20-OH-ecdysone or the hormone agonist RH 5992. The purity of 3H-ponasterone A was checked routinely by HPLC analysis before use.

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3. Results and discussion CtEcR and CtUSP are expressed separately in E. coli as full length proteins fused to glutathion-S-transferase. After induction with IPTG induction, prominent bands appear (Fig. 1). Full length cEcR fused to GST has a molecular weight of 87 kDa, which may partially explain the low expression rates of 3±1.8 mg/l (n=14) for EcR and 3.2±1.4 mg/l for USP compared to about 60 mg/l for VDR with 48 kDa (Hsieh et al., 1995) but 22 mg/l for GST-TR with 80 kDa (Ball et al., 1995). Very low expression rates (less than 1 µg/l E. coli culture) are reported for GST-AR with a molecular weight of 137 kDa (Roehrborn et al., 1992). However, comparable low rates are also described for DmEcR and DmUSP ligand binding domain (Halling et al., 1999), which indicates that the ecdysteroid receptor poses specific problems independent of the molecular size. The predominant part of the expressed protein (90% or more) is stored in inclusion bodies, although for induction with IPTG the temperature is lowered to 28°C. Under these conditions most of the TR is shifted into the soluble fraction (Ball et al., 1995). No attempts are

2.5. Electrophoretic mobility shift assays (EMSA) DNA binding studies were performed essentially as described by Elke et al. (1999). The following oligonucleotides were labelled with [α-32P]dCTP to a specific activity of about 107 cpm/µg by fill-in reaction with Klenow polymerase: DR5,

5⬘-GATCTAGAGAGGTCAA CGAAAGGTCATGTCCAAG-3⬘; PAL1, 5⬘-GATCTAGAGAGGTC AATGACCTTGTCCAAG-3⬘; SINGLE, 5⬘-GATCTAGAGAGGTCAACGT TGACCTCCAAG-3⬘.

Approximately 10 fmol of labelled oligonucleotide (1.5 ng DNA) dissolved in binding buffer (20 mM HEPES, 100 mM KCl, 5% glycerol, 2 mM dithiothreitol, 0.1% NP-40, pH 7.4 and 10 mg/ml BSA) were used as probes; 1–20 ng EcR, USP or both and 1 µg non-specific poly[dI-dC] (Boehringer, Mannheim, Germany) were added. For supershift experiments the samples were supplemented with 1 µl of antibody mAB11 (Khoury Christianson et al., 1992; kind gift of Dr Kafatos). Radioactivity was quantitated with a phosphorimager using ImageQuant software (Molecular Dynamics, Amersham Pharmacia Biotech AB, Uppsala, Sweden).

Fig. 1. Siver stained SDS gels showing fractions of the purification procedure of GST-CtEcR (A) and GST-CtUSP-2 (B). 1=lysate of noninduced and 2=IPTG induced E. coli, 3=homogenate, 4=pellet and 5=supernatant before and 6=after absorption to the affinity gel, 7=first and 8=second elution, 9=purified receptor after removal of the GSTtag. The receptor concentration in 4 and 5 is below detection limit. GST-EcR (87 kDa) runs with an apparent MW of 97 kDa. GST-USP (77 kDa) has an apparent MW of 77 kDa.

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made to solubilize the nuclear receptors of the pellet, since previous experiments revealed that after treatment with detergents only residual ligand binding activity remained (Elke et al., 1997). Considerable amounts of EcR-LBD are obtained by treatment with detergents and urea, but ponasterone A binding experiments revealed that different classes of hormone binding sites are observed for Drosophila EcR/USP after renaturation compared with a single class using cell extracts (Halling et al., 1999). The yield of receptor, capable of hormone binding, is enhanced by several freeze/thaw cycles (Rauch, unpublished results), but the affinity of hormone binding is impaired. Purification by affinity chromatography poses a further problem. EcR and USP are expressed as fusion proteins with GST, since this tag allows purification under rather mild conditions. This is especially important, since ligand binding to the Chironomus receptor is even more sensitive to elevated salt concentrations than the Drosophila receptor (Turberg et al., 1988). Elevated temperatures and salt concentrations cause ligand independent activation of cEcR (Turberg and Spindler, 1992; Elke et al., 1999) and prevent one studying the influence of ligand on other functions of the purified receptors like DNA binding. Absorption to the affinity matrix is poor. This is expected since absorption of GST-fusion proteins to the gel matrix rapidly declines with increasing molecular weight (Frangioni and Neel, 1993). To compensate partially for the low absorption efficiency the amount of gel is enhanced about 10-fold compared with the recommendation of the manufacturer (Pharmacia Biotech, Uppsala, Sweden). According to silver gels only neglectable amounts of receptor are removed from the gel during the extensive washing procedure (Fig. 1). Elution is repeated three times, which enhanced the yield considerably. The purity of the second elution is even better. Ball et al. (1995) report that 50% of TR remains on the matrix and can be eluted only in the presence of detergents. Loss of receptor due to insufficient elution from the gel may explain the low yield in the absence of detergents. Only some minor contaminations are seen in silver stained SDS gels of the purified receptor preparation (Fig. 1). The main contamination is a band with an apparent molecular weight of about 70 kDa, which may be DnaK, a hsp 70 like protein (Swamy et al., 1999) involved in the regulation of protein degradation in E. coli (Yu et al., 1992) and might also influence receptor functionality (Arbeitman and Hogness, 2000). Additional proteins with an apparent molecular weight of 45 kDa are present in EcR preparations, which are also induced by IPTG, but do not contain GST (Fig. 1) After purification, 47±26% (n=6) of GST-EcR is able to bind 3H-ponasterone A after addition of USP. The presence of USP is an absolute requirement for ligand

binding to purified Chironomus EcR as is shown in Fig. 2. Higher concentrations of USP do not improve ligand binding further. Reconstitution of hormone binding by combination of separately expressed proteins is reported also using isolated ligand binding domains of DmEcR and DmUSP (Halling et al., 1999). In contrast, only coexpression of RAR and RXR results in correctly folded proteins, capable of ligand binding, which could not be restored by the subsequent mixing of both partners (Li et al., 1997). No other factors seem to be important for the quality of hormone binding, since purified material give essentially the same results as obtained with cell extracts (Fig. 3). Purified receptor preparations possess the same two high affinity hormone recognition sites (KD1=0.24±0.1 nM, n=3 and KD2=3.9±1.3 nM, n=8) obtained with hormone sensitive Chironomus cells (Rauch et al., 1998; Grebe et al., 2000) and are reported also from other species (Dinan, 1985; Handler and Maroy, 1989; Spindler et al., 1984). The same affinity constants where obtained by evaluation of the binding data with Scatchard plot analysis and first-order kinetics (Rauch et al., 1998; Rauch, 1999). Arbeitman and Hogness (2000) also report that ligand binding of EcR/USP expressed in Sf9 cells does not require additional factors. In contrast, VDR needs some uncharacterised factors from liver nuclear extracts for high affinity binding of the ligand (Hsieh et al., 1995) and the DBD-LBD fragment of mineralocorticoid receptor requires hsp 90 for ligand binding (Caamano et al., 1993). Hsp 90 prevents dissociation of the ligand, caused by Triton X-100 (Inano et al., 1994), and improves ligand binding to the estrogen receptor. Reticulocyte lysate, added as source of comodulators and heat shock proteins, enhances ligand binding of receptors purified with detergents to a considerably higher extent than BSA (Elke et al., 1997), but improved ligand binding to EcR purified in the absence of detergents only slightly (Fig. 2). This indi-

Fig. 2. Specific 3H-ponasterone A binding (means±SD, n=3) of (A) purified GST receptors and (B) purified receptor after thrombin cleavage. (A) E=GST-CtEcR, U=GST-ctUSP, 2U=twofold concentration of CtUSP, RL=reticulocyte lysate (50 ng GST-EcR and 100 and 200 ng GST-USP/500 µl test volume were used).

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Fig. 4. Western blot of purified receptors before and after cleavage with thrombin. (a) Detection of GST-CtEcR (+) and GST-CtUSP (∗) with anti-GST serum. (b) Detection of GST-cEcR (apparent MW: 97 000 Da) and GST-free CtEcR (apparent MW 66 000 Da) with antiserum pABCE/D.

Fig. 3. Scatchard plot of [3H]-ponasterone A binding with EcR/USP using (A) 0.4 M NaCl extracts from C. tentans cells (Rauch et al., 1998), (B) bacterially expressed and affinity purified GST receptors and (C) receptors after removal of the GST moiety (SD⬍10%).

cates that the three-dimensional structure of the receptor proteins was maintained under the mild conditions of the purification procedure. The GST moiety is removed by cleavage with thrombin. An excess of thrombin is chosen to ensure quantitative removal of GST. Western blots with specific antibodies show the identity of the expressed protein (Fig. 4). No change in ligand binding is observed after thrombin cleavage with Scatchard analysis (Fig. 3C). GST-tag is reported to alter receptor properties due to aggregation of receptor proteins (Niedziela-Majka et al., 1998), but no receptor oligomers is detected in gel mobility shift experiments (Figs. 5 and 6). Oligomerization of receptor molecules is favoured using high protein concentrations and leads to loss of hormone binding (Halling et al., 1999). The concentrations of CtEcR and CtUSP never

Fig. 5. DNA binding of purified receptors (A) with GST-tag and (B) after cleavage of the GST-tag. After cleavage of the GST-tag the binding specificity is changed. The heterodimer recognizes PAL1 and DR5 elements in both cases, but no shift is detected with EcR on PAL 1 in the absence of the GST-tag.

exceed 0.2 µg/ml, which may support the high functionality of the receptor preparation. The final yield of purified receptors is 17.5±10 µg/l GST-EcR (n=6) and 37.5±10 µg GST-USP/l culture (n=3). This seems low compared with 1 mg/l TR reported by Ball et al. (1995) and 1–5 mg/l of VDR (Hsieh et al., 1995) both purified in the presence of detergents. However, only 60–125 µg DmEcR-DBD and about 600 µg DmUSP-DBD are obtained (Rusin et al., 1996) despite the enhanced solubility of his-tagged receptor domains and high expression rates of 11% (EcR-DBD) and 16% (USP-DBD).

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Fig. 7. Ligand specificity of purified GST-CtEcR/GST-ctUSP: 5 nM [3H]-ponasterone A was compared with increasing concentrations of (䊐) 20-OH-ecdysone and (왕) RH 5992 for 1 h at room temperature (SD⬍13%). KD for RH 5992=30 nM and for 20-OH-ecdysone=500 nM calculated according to Cheng and Prusoff (1973) (SD⬍10%).

Fig. 6. The identity of the USP-DNA complex was verified by supershift with USP specific antibody AB11.

The specific activity of the purified GST-EcR is 5.3±3.0 nmol binding sites/mg EcR in the presence of an excess of USP. With the exception of VDR (Hsieh et al., 1995) this exceeds all previous reports on the functionality of purified full length receptors. Separate expression of the ligand binding domain of DmEcR and DmUSP purified in the presence of detergents produced 1.9 pmol binding sites/mg protein, which is improved however about 1000-fold (6 nmol/ligand binding sites/mg protein) by coexpression with USP. Calculation of the specific activity of purified full length DmEcR in SF9 cells using the data reported by Arbeitman and Hogness (2000) reveal that a maximum of 1% of the receptor protein is able to bind ligand. Ligand specificity of the main naturally occurring moulting hormone 20-OH-ecdysone and the hormone mimick RH 5992 is identical to Chironomus cell extracts (Fig. 7). KD values of purified receptors calculated according to Cheng and Prusoff (1973) correspond to the values determined for extracts from the Chironomus cell line (Quack et al., 1995).

Both, PAL 1 and DR elements bind EcR/USP heterodimer, independent of whether GST is present or not (Elke et al. 1997, 1999; Vo¨ gtli et al., 1999). GST-EcR binds to Pal 1 as reported previously (Elke et al., 1997). In contrast, no shift is observed with EcR on PAL1 after removal of the GST-tag, whereas USP shows pronounced binding to DR5, both in the presence and absence of GST-tag. This corresponds to the results obtained with in vitro translated EcR and indicates that PAL 1 recognition is due to GST-tag induced homodimerization (Elke and Spindler, unpublished). There are several indications that bacterially expressed, in vitro translated receptors and EcR complexes obtained from cellular extracts differ in their DNA binding characteristics (Elke et al., 1999; Elke and Spindler, unpublished) due to the presence of hsp’s and immunophilins (Song et al., 1997). High molecular weight complexes for CtEcR/CtUSP present in nuclear extracts of the epithelial cell line have been described previously (Spindler-Barth and Spindler, 1998). Nevertheless, purified CtEcR/CtUSP and CtUSP are able to bind to hormone responsive elements (Figs. 5 and 6). Purified GST-CtEcR interacts with PAL 1 due to GSTinduced dimerization; USP and EcR/USP bind to both hormone responsive elements after cleavage of the GST moiety. However, at least 20-fold concentrations of EcR (18 ng/20 µl) are necessary. For Drosophila EcR/USP expressed in Sf9 cells it has recently been shown that DNA binding occurs only in the presence of heat shock proteins hsp 90, hsp 70 and additional chaperones (Arbeitman and Hogness, 2000). The functionality of purified receptors is further tested with hormone resistant cells (Fig. 8). Addition of the purified GST-EcR, but not GST-USP, restores ligand binding in cell extracts from hormone resistant subclones of the epithelial cell line from C. tentans, which have lost their ability to bind ligand due to mutations in the

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Fig. 8. Rescue of specific 3H-ponasterone A binding (means±SD, n=3) in hormone resistant subclones of the epithelial cell line from C. tentans (Grebe et al., 2000). Cell extracts of 0.4 M NaCl were incubated with 2 nMol [3H]-ponasterone A. White=no receptors added, hatched=100 ng/ml purified GST-EcR added, black=200 ng/ml purfied GST-USP added. After addition of exogeneous GST-EcR, ligand binding is in a comparable range to ponasterone A binding of wild type cell extracts (Grebe et al., 2000).

ligand binding domain of EcR (Zo¨ llner, Glo¨ ggler and Spindler-Barth, unpublished) to different degrees.

Acknowledgements The work was supported by the “Deutsche Forschungsgemeinschaft” (Teilprojekt A5, SFB 351). The skilful technical assistance of Mrs K. Himmelberg is gratefully acknowledged. The plasmids for expression of GSTCtEcR and GST-CtUSP-2 were provided by Drs Lezzi and Elke. [3H]-ponasterone A was a kind gift of Dr Kayser (Novartis, Basel). Antiserum pABcE/D was obtained from Dr Lezzi (ETH Zu¨ rich) and antibody AB11 from Dr Kafatos (EMBL, Heidelberg). M.G. kindly acknowledges the help of Dr C. Elke with EMSAs. M.G. was supported by a DFG graduate fellow¨ kotoxikologie ship (Graduiertenkolleg: “O und Umwelthygiene”).

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