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
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ABSTRACT
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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:
A/BUSP
complexes. Deletion of the A/B domain of EcR in the context of
A/BEcR:RXR:GRIP1 or
A/BEcR:
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.
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INTRODUCTION
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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. 1
).

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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 ), BmUSP (Bombyx
mori USP), MsUSP (Manduca sexta USP), CfUSP
(Choristoneura fumiferana USP), DmUSP (D.
melanogaster USP), AsUSP (Aedes aegypti USP).
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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 (1618).
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 (2325), 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.
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RESULTS
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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. 2
). However,
strong reporter activity was observed when CfEcR and CfUSP were
coexpressed in yeast (Fig. 2
) 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. 2
coexpression of Cf
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. 2
). The addition of RH5992 was not able to
induce transcriptional activity of either CfEcR:Cf
USP or
Cf
EcR:Cf
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.
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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 (
, ß, and
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. 3A
). 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
, but not for RXRß and RXR
(Fig. 3A
). An effect of GRIP in
the transactivation assay of CfEcR:Cf
USP was also observed (Fig. 3A
).

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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.
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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
EcR was coexpressed with
either RXR
, RXRß, RXR
, or Cf
USP, along with coactivator
GRIP1. Surprisingly, in comparison with other combinations of the
full-length CfEcR (Fig. 3A
), truncation of the CfEcR A/B domain
resulted in the induction of reporter gene activity in the presence of
ligand (Fig. 3B
). Ligand-dependent transactivation activity of
Cf
EcR:RXRs:GRIP1 or Cf
EcR:Cf
USP:GRIP1 is much stronger than
that observed for CfEcR:RXRs:GRIP1 or CfEcR:Cf
USP:GRIP1,
respectively (Fig. 3
).
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
, ß,
, or
Cf
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. 4
the responses to 10
compounds by four yeast systems
EcR:RXR
: GRIP1,
EcR:RXRß:GRIP1,
EcR:RXR
:GRIP1 and
EcR:
USP:GRIP1 are
presented. First of all, we observed that Cf
EcR:RXRß:GRIP1 has low
sensitivity not only to RH5992 but also to all other compounds (Fig. 4
), 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)].
In general, both the systems with Cf
EcR:RXR
and Cf
EcR:RXR
respond to all compounds in the same manner (Fig. 4
and Table 1
). 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
USP (Cf
EcR:Cf
USP:GRIP1) was
responsive not only to all four compounds that are active for
Cf
EcR:RXR
and Cf
EcR:RXR
, but also very responsive to five
of the six compounds that were inactive for the RXR systems (Fig. 4
and
Table 1
). Only one compound, RH141650, did not respond to
Cf
EcR:Cf
USP:GRIP1. Further study showed that this compound was
also not active in the insect cell transactivation system as well
(Table 1
). 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 1
). 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
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Furthermore, 9-cis retinoic acid (9-cis RA), a ligand for
RXR receptors, induced transactivation of Cf
EcR:RXR
and
Cf
EcR:RXR
systems in yeast, but did not induce transactivation of
EcR:USP complexes in yeast and insect cells (Table 1
). 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. 4
).
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
EcR:Cf
USP:GRIP1 (Fig. 5A
), Cf
EcR:RXR
:GRIP1 (Fig. 5B
), and
Cf
EcR:RXR
:GRIP1 (Fig. 5C
).
As presented in Fig. 5A
using Cf
EcR:Cf
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. 5A
). 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
EcR:Cf
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. 5D
). 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. 5A
where Cf
USP was used as the partner of Cf
EcR
are consistent with data in Fig. 5B
. 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
. It should be noted that in the RXR
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
is used (Fig. 5C
). 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
EcR:RXR
:GRIP1, Cf
EcR:RXR
:GRIP1, and
Cf
EcR:Cf
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 1
). 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
EcR:
USP
(Table 1
). Thus, the yeast assay with
EcR:
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
and RXR
have a similar
pattern of response to the compounds (Table 1
). Five compounds
(RH101523, RH71528, RH84658, RH0345, and RH5849) that were active in
the insect cell assay and yeast system with Cf
EcR:
USP were almost
inactive in yeast cells transfected with EcR:RXR
or RXR
(Table 1
). However, four compounds (RH125048, RH123709, RH2485, and RH5992)
that were potent in the EcR:RXR assays were active in insect cells and
in the
EcR:
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
EcR:RXR
: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 6A
shows a response comparing
transactivation of 86 RH5849 analogs using the pdr5 snq2
yeast strain transformed with Cf
EcR:RXR
: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.
Figure 6B
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. 6
, 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. 6C
). 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. 6
, 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.
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DISCUSSION
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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. 1
). 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. 3
, A and B). As shown in Fig. 3
, ligand-dependent transactivation
of CfEcR:GRIP1 in combination with any EcR partner RXRs or Cf
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
EcR induced constitutive transactivation (Fig. 2
).
Deletion of the A/B domain of CfUSP eliminated this constitutive
transcriptional activity (Fig. 2
). 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
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
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. 3
). 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
, 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
. However, Walfish and
co-workers showed that RXRß:GRIP1 gave the highest transactivation
response to all-trans RA, while RXR
: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. 3B
, 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 4
and 5
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 1
). The data in these figures indicated that the closest
correlation between insect and yeast systems was observed when USP was
used (Table 1
). 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 negativeor loss of potential candidates for screening (Table 1
). 9-cis RA, a ligand for RXR receptors, was able to activate the
EcR:RXR complex in yeast (Table 1
). 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 1
). 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. 6
were obtained from the yeast system with RXR
and comparison
of data from in vitro ligand binding, insect cell
transactivation, and whole-animal assays, respectively. The data in
Fig. 6A
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. 6C
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
, 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
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
|
---|
Media, Strains, and Plasmids
Standard yeast and Escherichia coli media were
prepared as described previously (52, 53). The yeast strain Y4727:
Mat
his3-
200 leu2-
0
lys2-
0 met5-
0 trp1-
63
ura3-
0 was used as a host (gift from Dr. Jeff
Boeke). The Y4727 snq2::kanMX, Y4727
pdr5::kanMX and Y4727
snq2::
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
and mouse RXRß or
RXR
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
EcR, respectively). The multicopy yeast expression
plasmids YEpCfEcR and YEpCf
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
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 (
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
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
USP, respectively) were
constructed. Initially, the full-length CfUSP or Cf
USP was amplified
from a cDNA clone (11) using the following pairs of
primers:CfUSP-5' and CfUSP-3' and Cf
USP-5' and CfUSP-3',
respectively. CfUSP-5':
5'-AGGAGTCGACCTTACATCTTGTCTTAAGACTAAGAGGTGGTatgtcaagtgt-
ggcgaag-3'; Cf
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
. [The plasmid pRS425-ER
has
been constructed as follows: the BamHI-Pml I
fragment containing CUP1 promoter, ubiquitin, fused with ER
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.
 |
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