Reconstruction of Ligand-Dependent Transactivation of Choristoneura fumiferana Ecdysone Receptor in Yeast

Hiep T. Tran, Hossein B. Askari, Salam Shaaban, Laura Price, Subba R. Palli, Tarlochan S. Dhadialla, Glenn R. Carlson and Tauseef R. Butt

LifeSensors, Inc. (H.T.T., H.B.A., S.S., T.R.B.) Malvern, Pennsylvania 19355
Rohm and Haas Company (L.P., S.R.P., T.S.D., G.R.C.) Spring House, Pennsylvania 19477


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Ecdysteroids play an important role in regulating development and reproduction in insects. Interaction of 20-hydroxyecdysone (20E) with ecdysone receptor (EcR) as a heterodimer with ultraspiracle (USP) protein triggers the activation of 20E-responsive genes. In this paper we describe a ligand-mediated transactivation system in yeast using the spruce budworm Choristoneura fumiferana ecdysone receptor (CfEcR). Coexpression of C. fumiferana USP (CfUSP) with CfEcR in yeast led to constitutive transcription of the reporter gene. However, deletion of the A/B domain of CfUSP abolished constitutive activity observed for the CfUSP:CfEcR complex. Replacement of USP with its mammalian homolog retinoid X receptors (RXRs) abolished the constitutive activity of the heterodimer but it did not restore EcR ligand-mediated transactivation. These data suggest that USP and its A/B domain play a role in the constitutive function of CfEcR:USP in yeast. A ligand-mediated transactivation was observed when GRIP1, a mouse coactivator gene, was added to EcR:RXR or EcR:{Delta}A/BUSP complexes. Deletion of the A/B domain of EcR in the context of {Delta}A/BEcR:RXR:GRIP1 or {Delta}A/BEcR:{Delta}A/BUSP:GRIP1 dramatically improved the ligand-dependent transactivation. This is the first example of highly efficient ligand-dependent transactivation of insect EcR in yeast. Analysis of transactivation activity of different ecdysteroidal compounds showed that the yeast system remarkably mimics the response observed in insect tissue culture cells and whole insect systems. The results open the way to develop assays that can be used to screen novel species-specific ecdysone agonist/antagonist insecticides.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Ecdysteroids play a crucial role in coordinating molting, metamorphosis, and reproduction in insects. These hormones, secreted mainly by prothoracic glands into the hemolymph, trigger the expression of a cascade of genes involved in the molding process(1). Ecdysteroids activate genes by interacting with the ecdysone receptor-ultraspiracle protein (EcR:USP) heterodimer bound to ecdysone response element (EcRE) that is located in the promoter region of ecdysone- inducible genes (2). Recently, several full-length cDNA encoding EcRs from Drosophila melanogaster (DmEcR) (3), Lucilia cuprina (LcEcR) (4), Bombyx mori (BmEcR) (5), Manduca sexta (MsEcR) (6, 7), Heliothis virescens (HvEcR) (8), Chironomus tentans (CtEcR) (9), Tenebrio molitor (TmEcR) (10), Choristoneura fumiferana (CfEcR) (11), Locusta migratoria (LmEcR) (12), and from mosquito, Aedes aegypti (AaEcR) (13) have been cloned. Due to alternative promoter and splicing, several isoforms of EcR were observed. For example, in D. melanogaster, there are three isoforms of EcR, A, B-1, and B-2, which result from alternative splicing and differences in the N-terminal AF-1 region (A/B domains). These isoforms are tissue specific and therefore the AF-1 domain is thought to play a crucial role in regulation of specific gene transcription (14). DNA-binding regions of EcRs share an exceptionally high level of homology, while other domains are less homologous. The most diverged region of EcR is AF-1 including the A and B domains (Refs. 11 and 15 and Fig. 1Go).



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Figure 1. Domain Comparison of EcR (A) and USP/RXR (B) Sequences

Percent amino acid identity (top number) and percent amino acid similarity (bottom number) were calculated using DNA star program (DNA Star Inc., Madison, WI) in comparison with CfEcR and CfUSP, respectively. The sequences used are DmEcR (Drosophila melanogaster EcR), LcEcR (Lucilia cuprina EcR). AaEcR (Aedes aegypti EcR), CtEcR (Chironomus tentansEcR), HvEcR (Heliothis virescens EcR), MsEcR (Manduca sexta EcR), BmEcR (Bombyx mori EcR), CfEcR (Choristoneura fumiferana EcR), LmEcR (Locusta migratoria EcR), Tm EcR (Tenebrio molitor EcR). EcRs were divided into Lepidopteran, Dipteran, and Coleopteran/Orthopretan groups. USP/RXR sequences used are HsRXR (Homo sapiens retinoid X receptor {alpha}), BmUSP (Bombyx mori USP), MsUSP (Manduca sexta USP), CfUSP (Choristoneura fumiferana USP), DmUSP (D. melanogaster USP), AsUSP (Aedes aegypti USP).

 
Of all the known nuclear steroid receptor superfamily members from insects, EcR is the only member with a known activating ligand. In insect cells, EcR forms a natural heterodimer with products of the ultraspiracle gene, USP, which is homologous to mammalian retinoid X receptors (RXRs) (2). It has been shown that mammalian RXRs are capable of substituting USP to form heterodimers with insect EcRs (16–18). While, neither CfEcR nor CfUSP alone binds to Hsp27-EcRE, their heterodimeric complex binds to EcREs even in the absence of ecdysteroids (15). Heterodimerization of EcR:USP is also required for ligand binding (17). The USP protein heterodimerizes not only with EcR but also with the DHR38 receptor (19), suggesting a broad role of USP in general transcription regulation. The USP in D. melanogaster has been shown to be required for normal eye morphogenesis, fertilization, egg-shell morphogenesis (20, 21), and adult thoracic development (22). Currently, insect USP has been cloned from D. melanogaster (23–25), B. mori (26), M. sexta (27), A. aegypti (28), C. fumiferana (15), and C. tentans (29). To date, a ligand for USP is unknown.

One of the goals of the insecticide industry is to develop safe chemicals that are not only effective but also pest selective. The nonsteroidal ecdysone agonist RH5992 (tebufenozide) induces precocious molting in lepidopteran larvae (30). Similar to 20-hydroxyecdysone (20E), RH5992 induces the expression of M. sexta hormone receptor 3, a homolog of D. melanogaster DHR3 (31, 32), suppresses the 14-kDa larval cuticular protein (33), and prevents the expression of dopa decarboxylase (34). Using RH5992 as a testing ligand, we developed a ligand-mediated transactivation system in yeast for EcR. The availability of such a system may have both applied and basic scientific interest. On one hand, the system could provide a tool for structure function analysis of EcR in relation to ecdysteroids and other known analogs. On the other hand, an available system would provide an effective means to screen new chemicals and validate and improve potential insecticidal candidates.

In this study, we report reconstitution of a ligand-mediated transactivation system in yeast using EcR and USP from the spruce budworm C. fumiferana. The yeast system allowed us to characterize the role of CfEcR and CfUSP A/B domains in ligand-independent and ligand-dependent transactivation in yeast. We have demonstrated that the presence of glucocorticoid receptor interacting protein (GRIP1) is required for the ligand-mediated transactivation process. The yeast ligand-dependent transactivation system can be also applied in phase 1 for screening pesticide leads that target EcR or USP receptors. To validate the yeast transactivation system for such screenings, we compared the data obtained from in vitro ligand receptor binding assays, transactivation assays in insect cells, and whole insect toxicity assays with transactivation potency using one yeast system for a set of compounds. The data demonstrated that the yeast system could mimic the response to ligands observed in other insect systems. Thus, the present ligand-dependent system in yeast for EcR would provide valuable tools for both basic and applied research.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Ligand-Independent Transactivation of CfEcR:USP
Expression of full-length CfEcR or CfUSP alone in yeast did not induce the reporter gene coupled with the natural EcREs Drosophila melanogaster Hsp-27 EcRE in the presence or absence of ligands (Fig. 2Go). However, strong reporter activity was observed when CfEcR and CfUSP were coexpressed in yeast (Fig. 2Go) indicating that CfUSP and CfEcR can heterodimerize and bind to EcREs in the absence of ligands. The role of the A/B domains of CfEcR and CfUSP in transactivation was investigated. As shown in Fig. 2Go coexpression of Cf{Delta}EcR (CfEcR with deletion of A/B domains) and CfUSP also results in induction of the reporter gene in the absence of ligands, but we did observe a small effect of the ligand RH5992. Deletion of the A/B domain of CfUSP abolished the observed constitutive transcription activity of the reporter induced by the CfEcR:CfUSP complex (Fig. 2Go). The addition of RH5992 was not able to induce transcriptional activity of either CfEcR:Cf{Delta}USP or Cf{Delta}EcR:Cf{Delta}USP. Thus, in combination with either full-length or A/B-deleted CfEcR, the A/B domain of CfUSP is required to induce transcription of the reporter gene.



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Figure 2. Transcriptional Activation of C. fumiferana USP and EcR

The yeast strains carrying the reporter plasmid that contains six EcREs coupled with E. coli ß-Gal gene in the absence or the presence of different combinations of plasmids for expression of CfUSP, CfEcR, and A/B domain-deleted CfEcR. The relative transactivation activity in the absence or the presence of 10 µM RH-5992 was measured as described in Materials and Methods. The data are presented as mean ± SD of at least eight independent experiments.

 
A Role of Mammalian GRIP in Ligand-Dependent Transactivation of EcR
As we have shown above, the presence of the full-length CfUSP in combination with CfEcR (both full-length and truncated versions) results in constitutive transcriptional activity of the EcR:USP complexes. Therefore, the EcR:USP complex is not suitable for a ligand-dependent transactivation assay in yeast. We replaced CfUSP with its mammalian counterpart, RXRs. It has been shown that RXR forms a heterodimer with insect EcR in vitro (2, 16, 17, 18). To investigate possible interactions between CfEcR and RXR subtypes in yeast, we coexpressed CfEcR and mammalian RXR ({alpha}, ß, and {gamma} subtypes). In contrast to USP, coexpression of RXRs and EcR resulted in no induction of reporter gene in either the presence or absence of stable ecdysteroid agonist, RH5992 (Fig. 3AGo). The absence of transactivation activity could be due to a lack of communication between the EcR:RXR heterodimer and the yeast general transcription complex. It has been shown that RXRs interact with GRIP1 and bridge ligand-activated nuclear receptors bound to cognate hormone response elements to the transcription initiation apparatus (35, 36). GRIP1 also interacts both in vitro and in vivo with nuclear receptors such as glucocorticoid receptor (GR), estrogen receptor (ER), androgen receptor (AR), thyroid hormone receptor (TR), retinoic acid receptor (RAR), and RXR (35, 36, 37). Coexpression of GRIP1 and CfEcR:RXR induced reporter gene activity only slightly in the presence of RH5992 for RXR{alpha}, but not for RXRß and RXR{gamma} (Fig. 3AGo). An effect of GRIP in the transactivation assay of CfEcR:Cf{Delta}USP was also observed (Fig. 3AGo).



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Figure 3. Effect of GRIP1 and the CfEcR A/B Domain on Transactivation of EcR:RXR and EcR:USP Complexes

The yeast strains carry the reporter plasmid that contains six EcREs coupled with E. coli ß-Gal gene in the absence or presence of different combinations of plasmids for expression of RXRs, GRIP1, and full-length CfEcR (A) or CfEcR with deletion of A/B domain (B). The relative transactivation activity in the absence of the presence of 10 µM RH-5992 was measured as described in Materials and Methods. The data are presented as mean ± SD of at least eight independent experiments.

 
After having demonstrated the role of the A/B domain of CfUSP, we asked whether the A/B domain of CfEcR may also play a role in ligand-dependent function in yeast. The Cf{Delta}EcR was coexpressed with either RXR{alpha}, RXRß, RXR{gamma}, or Cf{Delta}USP, along with coactivator GRIP1. Surprisingly, in comparison with other combinations of the full-length CfEcR (Fig. 3AGo), truncation of the CfEcR A/B domain resulted in the induction of reporter gene activity in the presence of ligand (Fig. 3BGo). Ligand-dependent transactivation activity of Cf{Delta}EcR:RXRs:GRIP1 or Cf{Delta}EcR:Cf{Delta}USP:GRIP1 is much stronger than that observed for CfEcR:RXRs:GRIP1 or CfEcR:Cf{Delta}USP:GRIP1, respectively (Fig. 3Go).

Application Of Yeast Ligand-Dependent Transactivation Assays For Compound Profiling
One of the prime objectives in developing a ligand-dependent transactivation system in yeast for insect EcR is to apply a system for screening compounds that act as an EcR ligand. Furthermore, the ligands could be used as pesticides that target insect EcR. We have demonstrated above that the ligand-dependent function of spruce budworm CfEcR could be achieved in combination with RXR{alpha}, ß, {gamma}, or Cf{Delta}USP in the presence of GRIP for RH5992. To examine the potency and selectivity of the yeast systems, 10 different Rohm and Haas compounds: RH101523, RH125048, RH123709, RH141650, RH71528, RH84658, RH0345, RH2485, RH5849 and RH5992 were tested. All of these compounds were modifications of the original compound RH5849 (38) with combinatorial chemistry processes used to create modifications. Structures and properties of some of these compounds have been described previously (30). In Fig. 4Go the responses to 10 compounds by four yeast systems {Delta}EcR:RXR{alpha}: GRIP1, {Delta}EcR:RXRß:GRIP1, {Delta}EcR:RXR{gamma}:GRIP1 and {Delta}EcR:{Delta}USP:GRIP1 are presented. First of all, we observed that Cf{Delta}EcR:RXRß:GRIP1 has low sensitivity not only to RH5992 but also to all other compounds (Fig. 4Go), suggesting that the system with RXRß may not be suitable for examining the structure-activity relationship. [Previously, we showed that these RXR receptors are expressed equally in yeast (51)].



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Figure 4. Effect of EcR Heterodimeric Partners (RXRs or USP) and Different Ligands on Transactivation Activity of Cf{Delta}EcR:GRIP1 Complex

The yeast strains contain the reporter plasmid, which consists of six EcREs coupled with E.coli ß-Gal gene in the absence or presence of different combinations of plasmids for expression of RXRs, Cf{Delta}USP, Cf{Delta}EcR, and GRIP1. The relative transactivation activity in the absence or the presence of 10 µM ecdysteroid analogs was measured as described in Materials and Methods. The data are presented as mean ± SD of at least eight independent experiments.

 
In general, both the systems with Cf{Delta}EcR:RXR{alpha} and Cf{Delta}EcR:RXR{gamma} respond to all compounds in the same manner (Fig. 4Go and Table 1Go). The compounds were either active or inactive for both systems. Two of these systems were not only strongly responsive to RH5992 but also to three additional compounds, RH125048, RH123709, and RH2485. The above systems showed a weak response to RH101523, RH141650, RH71528, RH84658, RH0345, and RH5849. Alternatively, the system with Cf{Delta}USP (Cf{Delta}EcR:Cf{Delta}USP:GRIP1) was responsive not only to all four compounds that are active for Cf{Delta}EcR:RXR{alpha} and Cf{Delta}EcR:RXR{gamma}, but also very responsive to five of the six compounds that were inactive for the RXR systems (Fig. 4Go and Table 1Go). Only one compound, RH141650, did not respond to Cf{Delta}EcR:Cf{Delta}USP:GRIP1. Further study showed that this compound was also not active in the insect cell transactivation system as well (Table 1Go). Two natural hormones for EcRs, ponasterone A and muristerone A, that have a strong potency in the insect EcR:USP transactivation system were able to activate EcR:USP complex in yeast (2-fold induction), but not the EcR:RXR complexes (Table 1Go). Thus, despite all 10 Rohm and Haas compounds being derivatives of RH5849, a ligand for EcR, and two natural ligands, ponasterone A and muristerone A, the ability of these ligands to induce transactivation is dependent on the EcR heterodimer partners, RXR or USP. These data clearly suggest that there are USP and RXR subtype-specific differences in response to ligands. These differences are important in future screenings for EcR ligands.


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Table 1. Comparison between Data of Transactivation Activity (Fold Induction) in Drosophila Cells, L57, with Data from Yeast Systems

 
Furthermore, 9-cis retinoic acid (9-cis RA), a ligand for RXR receptors, induced transactivation of Cf{Delta}EcR:RXR{alpha} and Cf{Delta}EcR:RXR{gamma} systems in yeast, but did not induce transactivation of EcR:USP complexes in yeast and insect cells (Table 1Go). These results indicate that the above EcR:RXR complexes can also be used to identify ligands for RXRs. However, such a system would not discriminate between ligands that transactivate via EcR or RXR, except for the obvious and known ligands for the two receptors. Based on the data using 9-cis RA for activation of EcR:RXR complexes in yeast, it should be possible to discover ligands for USP using an EcR:USP system in yeast. Using a combination of yeast strains containing EcR:RXR or EcR:USP, it should be possible to identify functional ligands for EcR, USP, or RXR.

Improvement of Ligand-Dependent Transactivation by Mutations in the ABC Transporter Pathway
The above results show that different compounds have different capabilities to induce EcR receptor-mediated transactivation (Fig. 4Go). There are at least two possible explanations for these differences. A simple explanation is that different compounds have different affinities and transactivation potentials relative for EcR:USP or EcR:RXR complexes. It is also possible that the intracellular concentrations of these compounds are different, although all cells were incubated with 10 µM of compounds for the same time periods.

One of the drawbacks of the yeast system is its thick cell wall that may act as a permeability barrier to small molecules (39). The potency of some chemicals in the yeast-based transactivation system might be compromised if these chemicals do not penetrate into the yeast cell or are actively exported. Yeast strains with mutations in certain genes such as SNQ2 and PDR5 that are involved in the ABC transporter pathway become hypersensitive to unrelated drugs (40) such as mycotoxins (49), or cycloheximide (50). In our study we examined the transactivation activity induced by 10 compounds using different yeast mutants (snq2, pdr5, and snq2 pdr5) and compared the activity of the same compounds in the isogenic wild-type yeast strain by using different transactivation systems: Cf{Delta}EcR:Cf{Delta}USP:GRIP1 (Fig. 5AGo), Cf{Delta}EcR:RXR{alpha}:GRIP1 (Fig. 5BGo), and Cf{Delta}EcR:RXR{gamma}:GRIP1 (Fig. 5CGo).



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Figure 5. Transcriptional Activation Induced by Different Ecdysteroid Agonists in ABC Cassette Transporter Mutant Yeast Strains using USP (A and D) or RXRs (B and C) as EcR Partners

A, Transactivation system Cf{Delta}USP:Cf{Delta}EcR:GRIP was used in the presence of 1 µM ecdysteroid analogs. B and C, The RXR{alpha}:Cf{Delta}EcR:GRIP and RXR{gamma}:Cf{Delta}EcR:GRIP complexes were used for transactivation assays in the presence of 1 µM and 10 µM ecdysteroid analogs, respectively. D, Dose response-dependent transactivation curves for RH5992 using the Cf{Delta}USP:Cf{Delta}EcR:GRIP system in the yeast wild-type and pdr5 snq2 double mutant strains. The relative transactivation activity in the absence or presence of ecdysteroid analogs was measured as described in Materials and Methods. The data are presented as mean ± SD of at least eight independent experiments. WT, Wild-type strain; snq2, strain with a deletion of SNQ2 gene; pdr5, strain with a deletion of the PDR5 gene; and snq pdr5, strain with a double mutation in SNQ2 and PDR5 genes.

 
As presented in Fig. 5AGo using Cf{Delta}EcR:Cf{Delta}USP:GRIP1, the snq2 mutation did not increase the transactivation activity by any of the compounds. In the wild-type and snq2 yeast strains, RH101523, RH71528, RH0345, and RH5849 did not induce transactivation activity at 1 µM concentration (Fig. 5AGo). However, the pdr5 and snq2 pdr5 mutants clearly showed markedly improved transactivation activity over the snq2 and wild-type strains for these compounds. The sensitivity of double-mutant snq2 pdr5 is similar to the pdr5 strain. At concentration of 1 µM, for compounds such as RH5992, the sensitivity of the pdr5 and pdr5 snq2 strains was not greater than that obtained using the wild-type strain. However, at lower concentrations, differences in sensitivity to RH5992 in these yeast strains is clearly observed. We examined dose-dependent response to RH5992 (10 pM to 10 µM) using Cf{Delta}EcR:Cf {Delta}USP:GRIP1 in the wild-type and the pdr5 snq2 double-mutant strains. At a concentration of 1 nM, the relative activity of ß-galactosidase was 5-fold higher than the basal background with the pdr5 snq2 double-mutant strain. For the wild-type yeast strain, the transactivation response to RH5992 was first detected at a concentration of 100 nM (Fig. 5DGo). Thus, the mutations in SNQ2 and PDR5 genes can improve the sensitivity to the RH5992 by 100-fold. As there are 29 genes involved in the ABC transporter pathway in yeast (41), further mutation in those genes could result in even higher sensitivity.

The data in Fig. 5AGo where Cf{Delta}USP was used as the partner of Cf{Delta}EcR are consistent with data in Fig. 5BGo. The strains pdr5 and snq2 pdr5 double mutant are more sensitive to the compounds in comparison to snq2 and wild-type strains when using RXR{alpha}. It should be noted that in the RXR{alpha} assay the improvement in sensitivity to all compounds except RH141650 and RH5849 is observed in the pdr5 and snq2 pdr5 strains. A similar picture appears when RXR{gamma} is used (Fig. 5CGo). Thus, in three yeast systems, both pdr5 and snq2 pdr5 mutants showed better sensitivity in comparison to the wild-type and snq2 strains.

Correlation of Yeast and Insect Transactivation Assays
One of the primary applications for the development of a yeast-based assay is its use in screening chemical leads that can be used as insecticides. Three yeast transactivation systems, Cf{Delta}EcR:RXR{alpha}:GRIP1, Cf{Delta}EcR:RXR{gamma}:GRIP1, and Cf{Delta}EcR:Cf{Delta}USP:GRIP1 were developed. We compared reporter gene activation response to 10 compounds using yeast (snq2 pdr5 mutant strain) and insect cell (Drosophila L57 cells) transactivation assays (Table 1Go). Insect cells were more sensitive than the yeast system. In general, at a given concentration, the compounds induced 3- to 10-fold higher transactivation in insect cells than in yeast cells. However, the pattern of transactivation by different compounds was similar. For example, RH141650 was inactive in transactivating reporter genes in both insect cell- and yeast-based cell assays. The other nine Rohm and Haas compounds and the two ecdysteroids, ponasterone A and muristerone A, that were potent in L57 insect cells were also active in the yeast system with {Delta}EcR:{Delta}USP (Table 1Go). Thus, the yeast assay with {Delta}EcR:{Delta}USP remarkably mimics the response to ligands observed in the insect cells.

Replacement of USP in the EcR:USP complex with its mammalian homolog RXR resulted in a different response to EcR ligands and responded to 9-cis RA. Two yeast systems with RXR{alpha} and RXR{gamma} have a similar pattern of response to the compounds (Table 1Go). Five compounds (RH101523, RH71528, RH84658, RH0345, and RH5849) that were active in the insect cell assay and yeast system with Cf{Delta}EcR:{Delta}USP were almost inactive in yeast cells transfected with EcR:RXR{alpha} or RXR{gamma} (Table 1Go). However, four compounds (RH125048, RH123709, RH2485, and RH5992) that were potent in the EcR:RXR assays were active in insect cells and in the {Delta}EcR:{Delta}USP yeast assay. As we have shown above, in terms of discovering a lead chemical, the yeast system using a combination EcR:RXR for chemical screening could generate both a false positive (responsive to RXR ligands) and potentially a false negative (loss of EcR ligands). To estimate the percentage of potential loss in future screenings, 86 compounds that were derivatives of the original ecdysteroid agonist RH5849 (38) were in the Cf{Delta}EcR:RXR{gamma}:GRIP1-containing yeast assay. The data were compared with insect cell transactivation assays, receptor ligand binding assays, and whole insect toxicity assays. It is necessary to compare the data from yeast assays with toxicity data in whole-insect experiments to validate the yeast system.

Figure 6AGo shows a response comparing transactivation of 86 RH5849 analogs using the pdr5 snq2 yeast strain transformed with Cf{Delta}EcR:RXR{gamma}:GRIP1 with that in the L57 insect cell line transformed with CfEcR and EcRE reporter. Although the fold induction of reporter gene expression was much higher for the insect cell system, there was a good correlation between transactivation activity induced by the compounds in both assays. Three of the 86 compounds were not active in both systems. Twenty-three compounds were weak in the yeast system (<2-fold induction) but active in insect cells (2- to 20-fold induction). Among the 86 compounds, only one was inactive in the insect system but active in the yeast assay (up to 7-fold). Thus, more than two-thirds of the compounds are positive in both systems.



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Figure 6. Correlation between Yeast Transactivation Assay Data with Data from Transactivation in Insect Cell Line, L57 (A), in Vitro Binding Assay (B), and Whole-Animal Toxicity Assay (C)

A, Correlation of insect cell transactivation assay of CfEcR:USP vs. reporter gene activation (fold induction) in yeast containing Cf{Delta}EcR:RXR{gamma}:GRIP1. The test compounds were tested at 10 µM concentration in the presence of 10 µM copper in the yeast assay. The insect assays were done as described in Materials and Methods at 0.1 µm test concentrations of compounds. B, Correlation of binding data [log(1/IC50); Plodia cell line nuclear extracts] vs. reporter gene activation (fold induction) in yeast containing Cf{Delta}EcR:RXR{gamma}:GRIP1. The compounds were tested at 10 µM concentration in the presence of 10 µM copper in the yeast assay. The binding assays were done as described in Materials and Methods. C, Correlation of reporter gene activation in the yeast assay vs. toxicity of third instar southern army worm larvae 48 h after treatment with 86 nonsteroidal ecdysone agonists. The compounds were tested at 10 µM concentration in the presence of 10 µM copper in the yeast assay. Conditions for toxicity assays for southern armyworm were as described in Materials and Methods.

 
Figure 6BGo shows the comparison of yeast transactivation assay data with ligand binding assay data using Plodia (a lepidopteran like spruce budworm) nuclear extracts as the source of EcR and USP. The relative displacement of compounds by tritiated ponasterone A was determined as described in Materials and Methods. There was good correlation of ligand binding activity to crude nuclear extracts with the ability of these compounds to induce transactivation of reporter gene in the yeast system. All but 11 compounds with a log(1/IC50) more than 6 resulted in at least a 4-fold transactivation activity in yeast. Eight compounds with a log(1/IC50) of less than 6 induced more than 4-fold transactivation in yeast. [Log (1/IC50) equal to 6 = IC50 of 1 µM].

The data presented in Fig. 6Go, A and B, were obtained in artificially reconstructed systems using insect cells and in vitro binding assays. We then analyzed toxicity data (EC50 values) for third instar southern armyworm larvae 48 h after treatment using the 86 compounds for correlation with transactivation data from the yeast assay (Fig. 6CGo). In general, we observed that compounds with a higher potency in the yeast system manifested higher toxicity in the whole-larval assay. The more potent the compound is in the yeast system, the lower the dose of the compound required to kill the insect. As shown above in Figs. 6Go, there was reasonably good correlation among the potencies of this select group of compounds in four different assay systems; yeast and insect cell line transactivation assays, ligand binding assay, and whole-insect assay. Therefore, considering the correlative data and the simplicity of the yeast-based assay, we feel confident that the yeast assay can be used to discover new and novel compounds with a ecdysone mode of action. The yeast system was used to screen 700 nonspecific, random compounds. Only one compound was detected with ecdysone agonist activity, suggesting that the system is very highly selective and can be used for high throughput screening of random libraries of compounds.


    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Role of A/B Domains of EcR and USP in Transcriptional Regulation
Overall alignment of insect EcR and USP receptors, respectively, revealed a high level of homology in the DNA and, to a lesser extent, ligand binding and the A/B domains (Refs. 11, 15 and Fig. 1Go). The divergence in the N-terminal A/B domain of nuclear receptors is proposed to play an essential role in tissue- and species-specific transcriptional regulation of specific genes (14). It is thought that the A/B domain plays a role in ligand-independent transcription induction, although its function is unknown. Expression of D. melanogaster EcR (42, 43) and Aedes aegypti EcR (44), but not C. fumiferana EcR, in yeast can induce ligand-independent transcription of a reporter gene. However, expression of the N-terminal truncated versions of these receptors in yeast failed to induce transcription of the reporter, suggesting that the A/B domains of D. melanogaster and A. aegypti EcR possess ligand-independent transactivation activity (Ref. 44 and H. T. Tran and T. Butt, unpublished data). Interestingly, both the fruit fly, D. melanogaster and the mosquito A. aegypti, are dipterans. While truncation of the A/B domain of DmEcR and AaEcR eliminated constitutive transcription (Ref. 44 and H. T. Tran and T. Butt, unpublished data), deletion of the A/B domain of CfEcR enhanced ligand-dependent transactivation activity (Fig. 3Go, A and B). As shown in Fig. 3Go, ligand-dependent transactivation of CfEcR:GRIP1 in combination with any EcR partner RXRs or Cf{Delta}USP was much weaker than transactivation of those corresponding combinations with truncated CfEcR. There are several plausible explanations for a role of CfEcR A/B domain in transcription activity of CfEcR:RXRs heterodimeric complexes in yeast. It is possible that the CfEcR A/B domain possesses a transcription repression activity, although additional data are required to support this hypothesis.

While expression of CfUSP alone did not result in transcription of the reporter gene, coexpression of CfUSP with either CfEcR or Cf{Delta}EcR induced constitutive transactivation (Fig. 2Go). Deletion of the A/B domain of CfUSP eliminated this constitutive transcriptional activity (Fig. 2Go). These observations allowed us to ascribe constitutive transcriptional activity to the A/B domain of C. fumiferana USP. The role of CfUSP in constitutive transcription is further demonstrated when combined with expression of A/B domain-deleted DmEcR or AaEcR even in the absence of a coactivator (44). In these instances coexpression of insect EcRs with CfUSP always leads to constitutive transcription of the reporter gene. Thus, the A/B domain of CfUSP might play an important role in ligand-independent transactivation of the EcR:USP complex in yeast. This is different from what we expect in insect cells, where the presence of ecdysteroids is required for transcriptional activity (45). We speculate that there may be a corepressor(s), which in the absence of ligands interacts with the EcR:USP complex to suppress transcriptional activity. A potential candidate for such a suppressor is the insect homolog of vertebrate signal mediator and receptor of transcription (SMRT), SMRTER, which interacts with EcR. SMRTER mediates repression by interacting with Sin3A, a repressor known to form a complex with histone deacetylase Rpd3/HDAC (46). It would be interesting to determine whether expression of SMRTER in yeast can suppress constitutive activity of EcR:USP.

Role of Coactivator GRIP-1 in Ligand-Dependent Transactivation
Ever since EcR cDNA was first cloned from D. melanogaster in 1991 (3), several groups have tried in vain to develop an EcR-based ligand-dependent transactivation assay in yeast. Dela Cruz and Mak (42) have reported that in yeast DmEcR caused induction of reporter gene activity in the absence of ligand. While CfEcR:CfUSP induced ligand-independent constitutive activity of the ecdysone reporter gene, CfEcR:RXR or CfEcR:Cf{Delta}USP were unable to induce transactivation. The lack of transactivation induction was probably due to the lack of heterodimer interaction with the yeast transcription complex. Previously, Walfish and co-workers showed that transcription of a corresponding reporter gene could not be induced via RXR{gamma} alone. However, in the presence of GRIP1, strong ligand-dependent induction was observed (35). A similar situation was observed for the EcR:RXR complex (Fig. 3Go). This is the first known example demonstrating that mammalian coactivator functions in concert with an insect receptor. Of all the three subtypes of RXRs, the strongest transactivation activity was obtained with RXR{alpha}, while the lowest transactivation was observed with RXRß. [Previously, we have shown that these mammalian RXR receptors are equally expressed in yeast (51).] There are two possible explanations for this. Coactivator GRIP1 can have different affinities toward different RXR subtypes with the strongest interaction being with RXR{alpha}. However, Walfish and co-workers showed that RXRß:GRIP1 gave the highest transactivation response to all-trans RA, while RXR{alpha}:GRIP1 gave the least response (35). Therefore, it is possible that the different responses in the presence of RXR subtypes are the result of differential interactions between EcR and RXRs as heterodimers. We also noted that the presence of the AF-1 domain of CfEcR seems to have an inhibitory effect on transcription activity of the reporter gene. As shown in Fig. 3BGo, deletion of the AF-1 domain strongly enhanced ligand-dependent transactivation. There are couple of possible explanations for this. First, the EcR AF-1 domain contains repression activity that inhibits transcription of the reporter gene. AF-1 could play a prominent role in transactivation, e.g. the differences between DmEcR and CfEcR in the AF-1 region. Second, the presence of the AF-1 domain might prevent optimal interaction between EcR and RXRs to form a heterodimer or interrupt a proper interaction between CfEcR:RXRs with coactivator GRIP1 or with yeast transcription machinery.

The p160 family of coactivators, SRC-1 [p160/NcoA-1/ERAP-160], GRIP-1 [TIF2/SRC-2], and p/CIP [ACTR/RAC-3/AIB/TRAM-1/SRC-3] mediate transcriptional activation by nuclear hormone receptors (reviewed in Ref. 47). These coactivators share domain structural homology predicting common modes of action by the individual members. p160 family members have C-terminal domains that contain histone acetyltransferase activity, suggesting that they modify chromatin. In the transactivation system in yeast, SRC-1 has been used as a substitute for GRIP1 (48). These authors demonstrated that SRC1 acts in a similar manner as GRIP1. We hypothesize that other members of the p160 coactivator family such as SRC-1 and p/CIP would function in the EcR transactivation assay. Our preliminary data showed that SRC-1 could substitute GRIP1 in EcR transactivation in yeast (data not shown). At the present moment, a homolog of mammalian GRIP1 coactivator in insects is yet to be found. BLAST search for GRIP1 homologs using fruit fly genome database yielded several potential candidates with unknown function. One of the candidates was a TGO gene product that has 23% identity (40% of similarities) with GRIP1 protein. However, such a level of homology is too low to predict any possible role for the TGO gene as a coactivator. Similar level of homology with GRIP1 was also found for Drosophila SS, CYC, and SIM gene products.

Compound Profiling Using EcR Transactivation Assay
One of the goals in developing a ligand-dependent transactivation system in yeast is its application for screening of chemical libraries. Figures 4Go and 5Go demonstrated that different ligands have different abilities to induce EcR transactivation in yeast. To validate the yeast system, we undertook comparative analyses of data obtained in yeast and insect cell systems. The first set of experiments involved 13 compounds (Table 1Go). The data in these figures indicated that the closest correlation between insect and yeast systems was observed when USP was used (Table 1Go). All 11 compounds active in the yeast system were able to induce transactivation in the insect cell line. RH141650 was the only compound examined that was not active in both systems. The risk of using yeast system EcR:RXR is obtaining both a false positive and a false negative—or loss of potential candidates for screening (Table 1Go). 9-cis RA, a ligand for RXR receptors, was able to activate the EcR:RXR complex in yeast (Table 1Go). The EcR:RXRs systems are weakly responsive to EcR natural ligands such as ponasterone A and muristerone A, as well as to several other compounds that are active in insect cells and for the EcR:USP system in yeast (Table 1Go). The response of yeast EcR:RXRs to 9-cis RA suggests the same system can be used to identify not only ligands for EcR but also ligands for RXR receptors. However, this make the system less powerful for insecticide screening as both EcR and RXR ligands would be identified in assays. To estimate the potential risk of using EcR:RXR systems, we tested 86 analogs of RH5849, the first nonsteroidal ecdysone agonist. The data presented in Fig. 6Go were obtained from the yeast system with RXR{gamma} and comparison of data from in vitro ligand binding, insect cell transactivation, and whole-animal assays, respectively. The data in Fig. 6AGo demonstrated that up to one-third of the compounds possess EcR receptor transactivation activity in the insect cell line, L57, but have weak activity in the yeast ligand-dependent transactivation system. When data from ligand displacement radiometric assays are compared with yeast transactivation data, all but 11 compounds that have an IC50 value less than 1 µM resulted in at least a 4-fold transactivation activity in yeast. Eight compounds that resulted in more than 4-fold transactivation in yeast have an IC50 > 1 µM. In both yeast and insect cell in vitro ligand displacement assays, the majority of the compounds (more than two-thirds) possess both transactivation activity in yeast and insect cells or have a low IC50 value in the ligand binding assay. This confirms that the yeast assay is valuable for screening chemicals targeting CfEcR. Because both the transactivation assay using insect cell lines and the in vitro ligand displacement assay are artificial, they might not adequately reflect the toxicity of these compounds to insects. It is necessary to compare the potency in the yeast transactivation assay with data from toxicity studies of test compounds on live insects. As spruce budworm, C. fumiferana (Lepidoptera), is not easily available for toxicity assay, we used another representative from the same order of insects, the southern armyworm, Spodoptera eridania, to determine the relative toxicity of the same 86 Rohm and Haas compounds. The data presented in Fig. 6CGo demonstrate that a strong correlation exists between the toxicity level of compounds and their transactivation induction ability in the yeast assay. The more potent the compound in the yeast assay the less the amount of compound needed to kill larvae. Thus, when different insect systems were compared with the yeast system using EcR/RXR{gamma}, the yeast system appeared adequate for screening for insecticidal compounds with a ecdysone mode of action in the future. Initial screening of more than 700 random compounds using the yeast EcR/RXR{gamma} system demonstrated only one compound with ecdysone agonist activity, suggesting that the system is very highly selective. However, in a future screening one would have to be wary of false positives caused by activation via RXR and false negatives, compounds that are EcR ligands but cannot transactivate via the EcR:RXR systems.

In summary, we developed a yeast transactivation system for insect EcR that can be used as a genetic screen to identify potential insecticides with ecdysone mode of action. The available yeast system also provides a tool to study the structure and function of EcR and USP receptors and to test potential ligands for these receptors. These data also encourage us to use yeast systems for human therapeutic and diagnostic nuclear receptor targets. Furthermore, the yeast system could be used to identify coactivators from both mammals and insects that can work in concert with EcR:USP as well as other accessory proteins.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Media, Strains, and Plasmids
Standard yeast and Escherichia coli media were prepared as described previously (52, 53). The yeast strain Y4727: Mat{alpha} his3-{Delta}200 leu2-{Delta}0 lys2-{Delta}0 met5-{Delta}0 trp1-{Delta}63 ura3-{Delta}0 was used as a host (gift from Dr. Jeff Boeke). The Y4727 snq2::kanMX, Y4727 pdr5::kanMX and Y4727 snq2::{Delta} pdr5::kanMX strains were obtained from the original Y4727 strain by deleting the whole open reading frame of the SNQ2 or the PDR5 or of both the SNQ2 and the PDR5 genes, respectively, using a PCR-transformation technique as described previously (54).

Yeast transformation was performed according to procedures outlined in Ref. 55 . Yeast transformants with plasmids were maintained in corresponding drop-out selective media. Multicopy yeast-E. coli shuttle plasmids containing the full-length and the A/B domain deleted receptor CfEcR, and full-length CfUSP plasmids were constructed as described below. The human RXR{alpha} and mouse RXRß or RXR{gamma} subtypes (56) were expressed under regulation of the CUP1 promoter in 2 µM multicopy plasmids with the LEU2 selective marker. The Nsi I-BamHI (GRIP1 gene with ADH1 promoter) fragment from the pGRIP812 (36) was blunt-ended and cloned into the PvuII site of the pRS423 (57). The ß-galactosidase reporter gene containing six natural D. melanogaster Hsp-27 EcREs was also constructed in this work (see below).

Plasmid Constructions
Plasmid pBRSS 6x EcRE-lac Z is a reporter plasmid carrying six copies of EcRE derived from the D. melanogaster heat shock protein gene, Hsp27 (58), located in the upstream of the iso-1-cytochrome C (CYC1) promoter that is coupled with the E. coli ß-galactosidase gene (lacZ). This is a yeast-E. coli multicopy shuttle plasmid containing URA3 as a yeast transformation marker. Full-length or A/B domain-deleted versions of CfEcR were fused at the N terminus in frame with human ubiquitin, which is under CUP1 promoter (plasmids YEpCfEcR and YEpCf{Delta}EcR, respectively). The multicopy yeast expression plasmids YEpCfEcR and YEpCf{Delta}EcR have been constructed based on the plasmid YEpE12 (59) that contains the TRP1 as a yeast-selective marker and ubiquitin-fused human estrogen receptor under CUP1 promoter. The following pair of primers, CfEcR-SalI and CfEcR-SacI, were used for amplification of full-length CfEcR A-form from the cDNA clone (11). The CfEcR-SalI primer: 5'-AGGAGTCGACCTTACATCTTGTCTTAAGACTAAGAGGTGGTatggacctgaaaca cgaagtggcttaccg-3', where uppercase letters indicate the nucleotide sequence belonging to human ubiquitin, the SalI site available in the ubiquitin is underlined, and the lowercase letters denote the sequence belonging to CfEcR A-form starting from ATG. The CfEcR-SacI primer: 5'-AAGGGAGCTCtaatctcccgcgcattc-3', where the lowercase letters indicate the nucleotide sequences belonging to the 3'-terminus of the CfEcR, and the SacI restriction site is underlined. For amplification of the A/B domain- deleted CfEcR, the following primers were used: Cf{Delta}EcR-SalI: 5'-GGAGTCGACCTTACATCTTGTCTTAAGACTAAGAGGTGGTatgcggcagcagga ggaactgtgtctg-3' (uppercase indicates the nucleotide sequence belonging to human ubiquitin, the SalI site available in the ubiquitin is underlined, and lowercase denotes the sequence belonging to the DNA binding domain of the CfEcR starting from amino acid RQQEELCLV) and the previously described CfEcR-SacI primer. The A/B domain-deleted EcR ({Delta}EcR) started from the beginning of the DNA binding domain with amino acid sequence RQQEELCLV. After protein translation and ubiquitin cleavage, the arginine residue "R" in the N terminus would be exposed, which is a proposed signal for short life protein (60). Therefore, an additional methionine was added before the RQQEELCVL to stabilize the protein. The DNA fragments were amplified in 30 cycles (96 C, 30 sec; 54 C, 1 min; and 72 C, 3 min) using high replication fidelity Deep Vent Polymerase (New England Biolabs, Inc., Beverly, MA). The PCR products for both full-length CfEcR and Cf{Delta}EcR were digested with SalI and SacI and subsequently recloned into the SalI and SacI sites of the plasmid YEpE12 (59). Similarly, the yeast expression vectors for spruce budworm C. fumiferana USP-CfUSP and CfUSP with deletion of A/B domain (pRS425-CfUSP and pRS425-Cf{Delta}USP, respectively) were constructed. Initially, the full-length CfUSP or Cf{Delta}USP was amplified from a cDNA clone (11) using the following pairs of primers:CfUSP-5' and CfUSP-3' and Cf{Delta}USP-5' and CfUSP-3', respectively. CfUSP-5': 5'-AGGAGTCGACCTTACATCTTGTCTTAAGACTAAGAGGTGGTatgtcaagtgt- ggcgaag-3'; Cf{Delta}USP-5': 5'-GGAGTCGACCTTACATCTT-GTCTTAAGACTAAGAGGTGGTatgtacccgcctaat caccccctgagt-3'. (Uppercase letters correspond to the nucleotide sequence of ubiquitin. The lowercase letters denote the nucleotide sequence corresponding to the 5'-terminus of the CfUSP starting from the ATG or from the DNA binding domain of the CfUSP, respectively. The ubiquitin SalI site is underlined) and CfUSP-3': 5'-CCTTCCATGGgaatgtcaataatgcccgtg-3'. (The NcoI site is underlined. The lowercase letters indicate the nucleotide sequence of the 3'-terminus of CfUSP cDNA.) The PCR products were purified and digested with SalI and NcoI and subsequently subcloned into the SalI-NcoI sites of a yeast expression vector containing a LEU2-selective marker, pRS425-ER{alpha}. [The plasmid pRS425-ER{alpha} has been constructed as follows: the BamHI-Pml I fragment containing CUP1 promoter, ubiquitin, fused with ER{alpha} and CYC1 terminator from the YEpE12 plasmid (59) was blunt ended and then ligated into the PvuII site of the pRS425 (57).]

Receptor-Mediated Transactivation Analysis in Yeast
The ecdysone-responsive transactivation system in yeast is composed of a reporter vector- E. coli ß-galactosidase gene under the control of CYC1-truncated promoter fused with six EcREs derived from the hsp27 ecdysone-responsive heat shock gene (58) and yeast vectors for expression of CfEcR, CfUSP, or RXRs and/or coactivator GRIP1. The reporter gene is located on the multicopy yeast 2-µm vector with URA3 marker. The insect receptor EcR is located on yeast in a multicopy vector and under the yeast CUP1 promoter with a TRP1 marker. Similarly, the RXRs or USP are also located on the 2-µm plasmid and under CUP1 promoters with a LEU2 marker. Both USP and EcR can form a heterodimer in vivo and bind to the EcREs and interact with the transcription initiation complex (TIC), which results in transcription of reporter gene (E.coli lacZ). Previously, it has been shown that the mammalian homolog of USP, RXR, forms a heterodimer with EcRs (16). Therefore, in our system we replaced USP with RXRs. Mouse GRIP1 has been shown to interact with RXR and enhance ligand-mediated transactivation of TR:RXR and RAR:RXR in yeast (35, 36). In this work a role of GRIP1 in ligand-dependent transactivation via RXR:EcR is investigated.

ß-Galactosidase Activity Assay
A method to measure ß-galactosidase activity was developed to estimate the potency of compounds and be simple enough to apply to high throughput screening. The transformed yeast cells with plasmids were grown overnight in selective liquid media at 30 C and diluted in prewarmed selective liquid media to 0.1 at OD600 (OD culture). CuSO4 is added to the media to a final concentration of 10 µM due to the fact that all insect EcR and USP are under the CUP1 promoter. One hundred microliters of the cell culture were spiked to each well of a 96-well microtiter plate. To each well, 2 µl of test ligand [diluted in dimethylsulfoxide (DMSO)] was added such that the final concentration of DMSO was 2%. For control wells, 2 µl of DMSO were also added. The final concentration of the tested compounds in the media was 1 µM, 10 µM, or 100 µM. Yeast cells were incubated in the presence of ligand in a shaker at 30 C. After 4 h of incubation, 100 µl of 2x "Z" Sarcosine-ONPG buffer (120 mM Na2HPO4, 80 mM NaH2PO4, 20 mM KCl, 2 mM MgSO4, 100 mM ß-mercaptoethanol, pH 7.0, 0.4% lauroyl sarcosine, 4 mg/ml ONPG) were added to each well, and the plate was incubated further at 37 C. The 2x "Z" Sarcosine-ONPG buffer is freshly prepared or stored at -20 C before use. After incubation at 37 C for 1 h, the reaction was stopped by adding 100 µl of quenching solution, 0.5 M Na2CO3, and ß-galactosidase activity at OD405 (OD reaction) was measured in a micro plate reader (BioTek, Winooski, VT). The relative activity of ß-galactosidase was calculated as 1000x OD reaction (as all the cell suspension started from the same OD and the time incubation has been standardized).

Crude Preparations of EcR and USP Proteins from Imaginal Wing Disc Cells of the Indian Meal Moth, Plodia interpunctella
Nuclear extracts were prepared from a cell line derived from imaginal wing discs of the Indian meal moth, Plodia interpunctella. Cells were maintained as described by Lynn and Oberlander (61). Cells grown to a density of 106 cells/ml in 300 ml of medium were harvested by centrifugation. The resulting cell pellet was suspended in 35 ml TMT buffer (10 mM Tris-HCl, pH 7.2, 5 mM MgCl2, 0.1% Triton X-100) and homogenized using 20 strokes on a Dounce homogenizer at 4 C. The homogenate, containing nuclei and cell debris, was kept on ice for 15 min, before centrifugation at 3,000 x g for 10 min at 4 C. The resulting supernatant was discarded, and the pellet was washed with 15 ml cold TM buffer and reextracted by centrifugation. The supernatant was discarded, and the pellet was extracted again with 4 ml TMK buffer (10 mM Tris-HCl, pH 7.2, 5 mM MgCl2, 800 mM KCl) by crushing the pellet thoroughly with a glass rod to a thick gelatinous slurry. The slurry was kept on ice for 15 min and then centrifuged at 100,000 x g for 60 min at 4 C. The resulting supernatant (~8 ml) was desalted over Econo-Pak 10DG desalting columns (Bio-Rad Laboratories, Inc., Hercules, CA) equilibrated with cold T buffer (10 mM Tris-HCl, pH 7.2). The desalted extract was used directly for binding assays.

Tritiated Ponasterone A Ligand Displacement Radiometric Assays
The assays were done essentially as described by Kapitskaya et al. (28) except that 100 µl Plodia nuclear extracts were used as the source for EcR and USP proteins. Total and nonspecific binding was obtained by carrying out the assays in the presence of 1% ethanol or 10-4 M 20E dissolved in ethanol (1% of reaction volume). Specific binding was determined by subtracting nonspecific binding from the total binding activity. The relative displacement of competitor ecdysone agonists was determined by running the ponasterone A binding reactions in the presence of various concentrations of each competitor. The fraction of tritiated ponasterone A bound for each compounds dose response was analyzed using IGOR software to obtain IC50 values.

Whole-Insect Toxicity Assays Using Larvae of the Southern Armyworm, Spodoptera eridania
Newly enclosed third instar larvae were fed on leaves sprayed with different concentrations of ecdysone agonists and evaluated for mortality 2 days after the start of feeding (0R2). Varying concentrations of ecdysone agonists dissolved in an acetone- methanol-water (containing 1% Triton X-100) system (5:5:90 vol/vol/vol) were used for spraying lima bean leaves. The leaves were placed bottom side up on moistened pieces of filter paper in Petri dishes. The leaves were sprayed with test or control (no test compound) solutions and allowed to dry. Sprayed leaves were kept on moist cotton in a Petri dish before placing third instar larvae on the leaves. For each dose tested 10 larvae were used. Petri dishes with armyworm were kept at 27 C. Armyworm mortality was rated by visual inspection after 48 h. The data were analyzed by probit analysis to obtain LC50 values in parts per million for each compound tested.

Transactivation Assay in L57 Cells
L57 cells created by eliminating functional EcR from D. melanogaster Kc cells were a gift from Drs. Peter and Lucy Cherbas of Indiana University. L57 cells were transfected with CfEcR (CfEcR placed under the control of baculovirus, Autographa californica multicapsid nucleopolyhedrovirus IE1 promoter) and reporter (ß-galactosidase reporter placed under 6X EcRE and minimal promoter), and the cells were cultured in the medium containing compounds. The reporter activity was measured at 48 h after adding ligands using TROPIX chemiluminescent assay kit. Fold activation was calculated by dividing reporter gene activity in the presence of ligands with that obtained in the absence of a ligand.


    ACKNOWLEDGMENTS
 
We would like to thank Dr. M. Stallcup for the clone with GRIP1 coactivator, Dr. J. Boeke for the yeast strain BY 4727, Drs. Peter and Lucy Cherbas of Indiana University for the gift of L57 cells, and Dr. P. Walfish for commenting on this work. We thank Jenny Li and Jayma Mikes for help in preparation of the figures.


    FOOTNOTES
 
Address requests for reprints to: Tauseef R. Butt, LifeSensors Inc., 271 Great Valley Parkway, Malvern, Pennsylvania 19355. E-mail: butt{at}lifesensors.com

Received for publication January 9, 2001. Revision received March 5, 2001. Accepted for publication March 12, 2001.


    REFERENCES
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 

  1. Riddiford L 1993 Hormones and Drosophila development. In Bate M, Martinez-Arias A (eds): The Development of Drosophila melanogaster. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, pp 899–939
  2. Yao TP, Segraves WA, Oro AE, McKeown M, Evans RM 1992 Drosophila ultraspiracle modulates ecdysone receptor function via heterodimer formation. Cell 71:63–72[Medline]
  3. Koelle MR, Talbot WS, Segraves WA, Bender MT, Cherbas P, Hogness DS 1991 The Drosophila EcR gene encodes an ecdysone receptor, a new member of the steroid receptor superfamily. Cell 67:59–77[Medline]
  4. Hannan GN, Hill RJ 1997 Cloning and characterization of LcEcR: a functional ecdysone receptor from the sheep blowfly Lucilia cuprina. Insect Biochem Mol Biol 27:479–488[CrossRef][Medline]
  5. Swevers L, Drevet JR, Lunke MD, Iatrou K 1995 The silkmoth homolog of the Drosophila ecdysone receptor (B1 isoform): cloning and analysis of expression during follicular cell differentiation. Insect Biochem Mol Biol 25:857–866[CrossRef][Medline]
  6. Fujiwara H, Jindra M, Newitt R, Palli SR, Hiruma K, Riddiford LM 1995 Cloning of an ecdysone receptor homolog from Manduca sexta and the developmental profile of its mRNA in wings. Insect Biochem Mol Biol 25:845–856[CrossRef][Medline]
  7. Jindra M, Malone F, Hiruma K, Riddiford LM 1996 Developmental profiles and ecdysteroid regulation of the mRNAs for two ecdysone receptor isoforms in the epidermis and wings of the tobacco hornworm, Manduca sexta. Dev Biol 180:258–272[CrossRef][Medline]
  8. Martinez A, Scanlon D, Gross B, Perara SC, Palli SR, Greenland AJ, Windass J, Pongs O, Broad P, Jepson I 1999 Transcriptional activation of the cloned Heliothis virescens (Lepidoptera) ecdysone receptor (HvEcR) by muristerone A. Insect Biochem Mol Biol 10:915–930
  9. Imhof MO, Rusconi S, Lezzi M 1993 Cloning of a Chironomus tentans cDNA encoding a protein (cEcRH) homologous to the Drosophila melanogaster ecdysteroid receptor (dEcR). Insect Biochem Mol Biol 23:115–124[CrossRef][Medline]
  10. Mouillet JF, Delbecque JP, Quennedey B, Delachambre J 1997 Cloning of two putative ecdysteroid receptor isoforms from Tenebrio molitor and their developmental expression in the epidermis during metamorphosis. Eur J Biochem 248:856–863[Abstract]
  11. Kothapalli R, Palli SR, Ladd TR, Sohi SS, Cress D, Dhadialla TS, Tzertzinis G, Retnakaran A 1995 Cloning and developmental expression of the ecdysone receptor gene from the spruce budworm, Choristoneura fumiferana. Dev Genet 17:319–330[Medline]
  12. Saleh DS, Zhang J, Wyatt GR, Walker VK 1998 Cloning and characterization of an ecdysone receptor cDNA from Locusta migratoria. Mol Cell Endocrinol 143:91–99[CrossRef][Medline]
  13. Cho WL, Kapitskaya MZ, Raikhel AS 1995 Mosquito ecdysteroid receptor: analysis of the cDNA and expression during vitellogenesis. Insect Biochem Mol Biol 25:19–27[CrossRef][Medline]
  14. Talbot WS, Swyryd EA, Hogness DS 1993 Drosophila tissues with different metamorphic responses to ecdysone express different ecdysone receptor isoforms. Cell 73:1323–1337[Medline]
  15. Perera SC, Palli SR, Ladd TR, Krell PJ, Retnakaran A 1998 The ultraspiracle gene of the spruce budworm, Choristoneura fumiferana: cloning of cDNA and developmental expression of mRNA. Dev Genet 22:169–179[CrossRef][Medline]
  16. Thomas HE, Stunnenberg HG, Stewart AF 1993 Heterodimerization of the Drosophila ecdysone receptor with retinoid X receptor and ultraspiracle. Nature 362:471–475[CrossRef][Medline]
  17. Yao TP, Forman BM, Jiang Z, Cherbas L, Chen JD, McKeown M, Cherbas P, Evans RM 1993 Functional ecdysone receptor is the product of EcR and Ultraspiracle genes. Nature 366:476–479[CrossRef][Medline]
  18. Suhr ST, Gil EB, Senut MC, Gage FH 1998 High level transactivation by a modified Bombyx ecdysone receptor in mammalian cells without exogenous retinoid X receptor. Proc Natl Acad Sci USA 95:7999–8004[Abstract/Free Full Text]
  19. Crispi S, Giordano E, D’Avino PP, Furia M 1998 Cross-talking among Drosophila nuclear receptors at the promiscuous response element of the ng-1 and ng-2 intermolt genes. J Mol Biol 275:561–574[CrossRef][Medline]
  20. Oro AE, McKeown M, Evans RM 1992 The Drosophila retinoid X receptor homolog ultraspiracle functions in both female reproduction and eye morphogenesis. Development 115:449–462[Abstract]
  21. Perrimon N, Engstrom L, Mahowald AP 1985 Developmental genetics of the 2C-D region of the Drosophila X chromosome. Genetics 111:23–41[Abstract/Free Full Text]
  22. Henrich VC, Szekely AA, Kim SJ, Brown NE, Antoniewski C, Hayden MA, Lepesant JA, Gilbert LI 1994 Expression and function of the ultraspiracle (usp) gene during development of Drosophila melanogaster. Dev Biol 165:38–52[CrossRef][Medline]
  23. Henrich VC, Sliter TJ, Lubahn DB, MacIntyre A, Gilbert LI 1990 A steroid/thyroid hormone receptor superfamily member in Drosophila melanogaster that shares extensive sequence similarity with a mammalian homologue. Nucleic Acids Res 18:4143–4148[Abstract]
  24. Oro AE, McKeown M, Evans RM 1990 Relationship between the product of the Drosophila ultraspiracle locus and the vertebrate retinoid X receptor. Nature 347:298–301[CrossRef][Medline]
  25. Shea MJ, King DL, Conboy MJ, Mariani BD, Kafatos FC 1990 Proteins that bind to Drosophila chorion cis-regulatory elements: a new C2H2 zinc finger protein and a C2C2 steroid receptor-like component. Genes Dev 4:1128–1140[Abstract]
  26. Tzertzinis G, Malecki A, Kafatos FC 1994 BmCF1, a Bombyx mori RXR-type receptor related to the Drosophila ultraspiracle. J Mol Biol 238:479–486[CrossRef][Medline]
  27. Jindra M, Huang JY, Malone F, Asahina M, Riddiford LM 1997 Identification and mRNA developmental profiles of two ultraspiracle isoforms in the epidermis and wings of Manduca sexta. Insect Mol Biol 6:41–53[Medline]
  28. Kapitskaya M, Wang S, Cress DE, Dhadialla TS, Raikhel AS 1996 The mosquito ultraspiracle homologue, a partner of ecdysteroid receptor heterodimer: cloning and characterization of isoforms expressed during vitellogenesis. Mol Cell Endocrinol 121:119–132[CrossRef][Medline]
  29. Vogtli M, Elke C, Imhof MO, Lezzi M 1998 High level transactivation by the ecdysone receptor complex at the core recognition motif. Nucleic Acids Res 26:2407–2414[Abstract/Free Full Text]
  30. Dhadialla TS, Carlson GR, Le DP 1998 New insecticides with ecdysteroidal and juvenile hormone activity. Annu Rev Entomol 43:545–569[CrossRef][Medline]
  31. Palli S, Riddiford L, Hiruma K 1991 Juvenile hormone and "retinoic acid" receptors in Manduca epidermis. Insect Biochem Mol Biol 21:7–15
  32. Palli SR, Hiruma K, Riddiford LM 1992 An ecdysteroid-inducible Manduca gene similar to the Drosophila DHR3 gene, a member of the steroid hormone receptor superfamily. Dev Biol 150:306–318[Medline]
  33. Rebers JE, Riddiford LM 1988 Structure and expression of a Manduca sexta larval cuticle gene homologous to Drosophila cuticle genes. J Mol Biol 203:411–423[Medline]
  34. Hiruma K, Riddiford L 1990 Regulation of dopa decarboxylase gene expression in the larval epidermis of the tobacco hornworm by 20-hydroxyecdysone and juvenile hormone. Dev Biol 138:214–224[Medline]
  35. Walfish PG, Yoganathan T, Yang YF, Hong H, Butt TR, Stallcup MR 1997 Yeast hormone response element assays detect and characterize GRIP1 coactivator-dependent activation of transcription by thyroid and retinoid nuclear receptors. Proc Natl Acad Sci USA 94:3697–3702[Abstract/Free Full Text]
  36. Hong H, Kohli K, Garabedian MJ, Stallcup MR 1997 GRIP1, a transcriptional coactivator for the AF-2 transactivation domain of steroid, thyroid, retinoid, and vitamin D receptors. Mol Cell Biol 17:2735–2744[Abstract]
  37. Hong H, Kohli K, Trivedi A, Johnson DL, Stallcup MR 1996 GRIP1, a novel mouse protein that serves as a transcriptional coactivator in yeast for the hormone binding domains of steroid receptors. Proc Natl Acad Sci USA 93:4948–4952[Abstract/Free Full Text]
  38. Wing KD, Slawecki RA, Carlson GR 1988 RH 5849 a non-steroidal ecdysone agonist: effect on larval Lepidoptera. Science 241:470–471
  39. Zysk JR, Johnson B, Ozenberger BA, Bingham B, Gorski J 1995 Selective uptake of estrogenic compounds by Saccharomyces cerevisiae: a mechanism for antiestrogen resistance in yeast expressing the mammalian estrogen receptor. Endocrinology 136:1323–1326[Abstract]
  40. Mahe Y, Lemoine Y, Kuchler K 1996 The ATP binding cassette transporters Pdr5 and Snq2 of Saccharomyces cerevisiae can mediate transport of steroids in vivo. J Biol Chem 271:25167–25172[Abstract/Free Full Text]
  41. Decottignies A, Goffeau A 1997 Complete inventory of the yeast ABC proteins. Nat Genet 15:137–145[Medline]
  42. Dela Cruz F, Mak P 1997 Drosophila ecdysone receptor functions as a constitutive activator in yeast. J Steroid Biochem Mol Biol 62:353–359.43[CrossRef][Medline]
  43. Dela Cruz FE, Kirsch DR, Heinrich JN 2000 Transcriptional activity of Drosophila melanogaster ecdysone receptor isoforms and ultraspiracle in Saccharomyces cerevisiae. J Mol Endocrinol 24:183–191[Abstract/Free Full Text]
  44. Tran HT, Shaaban S, Askari H, Raikhel AS, T R But 2001 Requirement of co-factors for the ligand-mediated activity of the insect ecdysteroid receptor in yeast. J Mol Endocrinol, in press
  45. Antoniewski C, Laval M, Dahan A, Lepesant JA 1994 The ecdysone response enhancer of the Fb:1 gene of Drosophila melanogaster is a direct target for the EcR/USP nuclear receptor. Mol Cell Biol 14:4465–4474[Abstract]
  46. Tsai CC, Kao HY, Yao TP, McKeown M, Evans RM 1999 SMRTER, a Drosophila nuclear receptor coregulator, reveals that EcR-mediated repression is critical for development. Mol Cell 4:175–186[Medline]
  47. McKenna NJ, Nawaz Z, Tsai SY, Tsai MJ, O’Malley BW 1998 Distinct steady-state nuclear receptor coregulator complexes exist in vivo. Proc Natl Acad Sci USA 95:11697–11702[Abstract/Free Full Text]
  48. Anafi M, Yang Y-F, Berlev NA, Berger SL, Butt TR, Walfish PG 2000 GCN5 and ADA adapter proteins regulate T3/GRIP coactivator dependent gene activation by human thyroid hormone receptor. Mol Endocrinol 14:718–732[Abstract/Free Full Text]
  49. Bissinger PH, Kuchler K 1994 Molecular cloning and expression of the Saccharomyces cerevisiae STS1 gene product. A yeast ABC transporter conferring mycotoxin resistance. J Biol Chem 269:4180–4186[Abstract/Free Full Text]
  50. Balzi E, Wang M, Leterme S, Van Dyck L, Goffeau A 1994 PDR5, a novel yeast multidrug resistance conferring transporter controlled by the transcription regulator PDR1. J Biol Chem 269:2206–2214[Abstract/Free Full Text]
  51. Kephard DD, Walfish PG, DeLuca H, Butt TR 1996 Retinoid R receptor isotype identity directs human vitamin D receptor heterodimer transactivation from 24-hydroxylase vitamin D response elements in yeast. Mol Endocrinol 10:408–419[Abstract]
  52. Sherman F, Fink G, Hicks J 1986 Methods in Yeast Genetics. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY
  53. Sambook J, Fritsch E, Maniatis T 1989 Molecular Cloning: A Laboratory Manual, ed 2. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY
  54. Storici F, Coglievina M, Bruschi CV 1999 A 2-microm DNA-based marker recycling system for multiple gene disruption in the yeast Saccharomyces cerevisiae. Yeast 15:271–283[CrossRef][Medline]
  55. Gietz D, St. Jean A, Woods RA, Schiestl RH 1992 Improved method for high efficiency transformation of intact yeast cells. Nucleic Acids Res 20:1425[Medline]
  56. Allegretto EA, McClurg MR, Lazarchik SB, Clemm DL, Kerner SA, Elgort MG, Boehm MF, White SK, Pike JW, Heyman RA 1993 Transactivation properties of retinoic acid and retinoid X receptors in mammalian cells and yeast. Correlation with hormone binding and effects of metabolism. J Biol Chem 268:26625–26633[Abstract/Free Full Text]
  57. Sikorski RS, Hieter P 1989 A system of shuttle vectors and yeast host strains designed for efficient manipulation of DNA in Saccharomyces cerevisiae. Genetics 122:19–27[Abstract/Free Full Text]
  58. Riddihough G, Pelham H 1987 An ecdysone response element in the Drosophila melanogaster. EMBO J 21:181–197[Abstract/Free Full Text]
  59. Graumann K, Wittliff JL, Raffelsberger W, Miles L, Jungbauer A, Butt TR 1996 Structural and functional analysis of N-terminal point mutants of the human estrogen receptor. J Steroid Biochem Mol Biol 57:293–300[CrossRef][Medline]
  60. Bachmair A, Finley D, Varshavsky A 1986 In vivo half-life of a protein is a function of its amino-terminal residue. Science 234:179–186[Medline]
  61. Lynn D, Oberlander H 1983 The establishment of cell lines from imaginal wing discs of Spodoptera furgiperda and Plodia interpunctell. J Insect Physiol 29:591–596[CrossRef]