The influence of heterodimer partner ultraspiracle/retinoid X
receptor on the function of ecdysone receptor
Subba R. Palli
1
, Mariana Z. Kapitskaya
2
and David W. Potter
2
1 Department of Entomology, University of Kentucky, Lexington, KY, USA
2 RheoGene Inc. Norristown, PA, USA
Steroid hormones, ecdysteroids, regulate insect develop-
ment, reproduction and several other physiological
processes. The most active form of ecdysteroids is 20-hy-
droxyecdysone (20E). The 20E transduces its signal
through a heterodimeric complex of two nuclear recep-
tors, the ecdysone receptor (EcR) [1] and the ultraspira-
cle (USP), an ortholog of the vertebrate retinoid X
receptor (RXR) [2–4]. Both EcR and USP are members
of the nuclear receptor superfamily [5] and exhibit a typ-
ical modular structure comprising the N-terminal
A ⁄ B domain, the DNA-binding or C domain, the hinge
or D domain, the ligand-binding or E domain, and the
C-terminal F domain. The ligand-binding domain sup-
ports ligand-dependent dimerization and transactivation
functions. A ⁄ B and F domains support ligand-inde-
pendent transactivation. The DNA-binding domain and
the N-terminal region of the hinge region are known to
support dimerization of two receptors.
The EcR:USP heterodimers bind to the ecdysteroid
response elements (EcRE) present in the promoter
regions of ecdysteroid response genes and regulate

on either end of helix 9 are responsible for improved activity of LmRXR.
The EcR:Lm-HsRXR chimera heterodimer induced reporter genes with
nanomolar concentration of ligand compared with the micromolar concen-
tration of ligand required for activating the EcR:HsRXR heterodimer. The
EcR:Lm-HsRXR chimera heterodimer, but not the EcR:HsRXR hetero-
dimer, supported ligand-dependent induction of reporter gene in a
C57BL ⁄ 6 mouse model.
Abbreviations
CfEcR, Choristoneura fumiferana EcR; DMSO, dimethylsulfoxide; 20E, 20-hydroxyecdysone; ECD, ecdysteroid; EcR, ecdysone receptor;
EcRE, ecdysone response element; G:CfEcR(DEF), GAL4:CfEcR(DEF); RLU, relative light units; RXR, retinoid X receptor; SEAP, secreted
alkaline phosphatase; VP:Hs–LmRXR(EF), VP16:HsbRXR (helices 1–8) LmRXR (helices 9–12 + F); V:MmRXR(EF), VP16:MmRXR(EF);
V:CfUSP(EF), VP16:CfUSP(EF); V:LmRXR(EF), VP16:LmRXR(EF); USP, ultraspiracle; WT, wild-type.
FEBS Journal 272 (2005) 5979–5990 ª 2005 The Authors Journal Compilation ª 2005 FEBS 5979
(ligand-dependent regulation of transgenes) in various
biotechnology applications. Several nuclear receptors,
including the glucocorticoid receptor (GR), the pro-
gesterone receptor (PR), the estrogen receptor (ER),
and the EcR are being used to develop gene switches
for applications in medicine and agriculture. Because
the EcR and its ligands are not found in vertebrates,
they are attractive targets for the development of gene
switches for applications in humans. The EcR gene
switch is being developed for use in various applica-
tions including gene therapy, expression of toxic pro-
teins in cell lines and cell-based drug discovery assays
[6–14].
EcRs function as an ecdysteroid-dependent tran-
scription factor in cultured mammalian cells [15,16].
No et al. [17] used DmEcR and human RXRa to
develop an EcR gene switch and demonstrated its

However, the ligand sensitivity of this two-hybrid for-
mat EcR gene switch is not very high and requires a
micromolar concentration of ligand for induction of
genes. To improve the ligand sensitivity of EcR gene
switch, we tested insect RXR and chimeras between
human and insect RXRs as partners for EcR and
discovered that the partner of EcR affects functioning
of EcR in gene switch applications. The ligand sensi-
tivity of EcR gene switch was improved by  100-fold
by replacing HsRXR with a chimera between HsRXR
and an insect RXR from Locusta migrotoria
(LmRXR).
Results
Use of invertebrate RXR improves the function
of EcR in mammalian cells
Alignment of USP and RXR sequences showed that
the RXR homologs, USPs from lepidopteran and dip-
teran insects fall into one group and the RXR homo-
logs identified from insects belonging to other orders
(e.g. Heteroptera, Locusta migratoria and Coleoptera,
Tenebrio molitor, as well as from crab and tick group
with vertebrate RXRs) (Fig. 1). In other words, the
RXR homologs identified in insects belonging to
orders other than Lepidoptera and Diptera as well as
from crab and tick are closer to vertebrate RXRs than
to their counterparts in lepidopteran and dipteran
insects.
As shown in Fig. 2A, use of USP from the lepidop-
teran insect Choristoneura fumiferana [V:CfUSP(EF)]
as a partner for EcR from Choristoneura fumiferana

with the G:CfEcR(DEF) + V:LmRXR(EF) switch,
showing that the ligand sensitivity of the G:CfEcR-
(DEF) + V:LmRXR(EF) switch is higher than that
of the G:CfEcR(DEF) + V:HsRXR(EF) switch. Pro-
teins isolated from 3T3 cells transfected with
V:LmRXR(EF), V:CfUSP(EF) or V:HsRXR(EF) were
analyzed using western blots and VP16 antibodies. As
shown in Fig. 2B, all three fusion proteins are
expressed in similar quantities suggesting that the dif-
ference observed in ligand sensitivity of LmRXR,
HsRXR and CfUSP switches is due to structure of
these proteins rather than due to differences in their
expression levels.
When the green fluorescence protein (GFP; placed
under the control of GALRE) and G:CfEcR(DEF) +
V:CfUSP(EF), G:CfEcR(DEF) + V:HsRXR(EF) or
G:CfEcR(DEF) + V:LmRXR(EF) constructs were
transfected into 3T3 cells, the cells transfected with
G:CfEcR(DEF) + V:CfUSP(EF) switch constructs
showed GFP fluorescence in the cells treated with
dimethylsulfoxide (DMSO), 1.0 or 10 lm RG-102240
(Fig. 3). Low levels of GFP fluorescence were detected
in 3T3 cells transfected with G:CfEcR(DEF) +
V:LmRXR(EF) constructs and exposed to DMSO.
However, upon exposure to 1.0 or 10 lm RG-102240,
these cells showed higher GFP fluorescence (Fig. 3). In
contrast, the GFP activity was not observed in 3T3
cells transfected with G:CfEcR(DEF) + V:HsRX-
R(EF) constructs and exposed to DMSO. Upon expo-
sure to 1.0 or 10 lm RG-102240, these cells showed

which regions of LmRXR are responsible for this
improved activity, we prepared five chimeras of
LmRXR and HsRXR by sequentially replacing helix 6
with helix 12 of HsRXR with the corresponding
regions of LmRXR. The chimeric RXRs, HsRXR and
LmRXR were assayed as partners for CfEcR in
ligand-dependent induction of reporter activity in 3T3
cells. The luciferase reporter gene regulated by GAL-
RE (pFRLUC), G:CfEcR(DEF) and V:HsRXR(EF)
or V:LmRXR(EF) or VP6 fusion of each Hs–
LmRXR(EF) chimera shown in Fig. 4A were trans-
fected into 3T3 cells and the transfected cells were
exposed to RG-102240. Luciferase activity was
measured at 48 h after addition of ligand. The
G:CfEcR(DEF) + V:HsRXR(EF) switch induced lu-
ciferase activity at 1 lm or higher concentration of
RG-102240 and the G:CfEcR(DEF) + V:LmRXR-
(EF) switch induced luciferase activity at 0.2 lm or
higher concentration of RG-102240 (Fig. 4A). Repla-
cing RXR with Hs–LmRXR(EF) chimera containing
helices 1–7 of HsRXR and 8–12 of LmRXR or helices
1–8 of HsRXR and 9–12 of LmRXR resulted in an
increase in ligand sensitivity of the EcR switch. Luci-
ferase activity was induced with a 0.04 lm or higher
concentration of RG-102240 (Fig. 4A) in the presence
of these chimeras. The other three chimeras performed
similar to HsRXR. Proteins isolated from 3T3 cells
that were transfected with chimera constructs were
analyzed using Western blots and VP16 antibodies. As
shown in Fig. 4B, fusion proteins for all five chimeras

in which HsRXR amino acid residues were replaced
with LmRXR residues in helix 9 as well as in loops on
either side of helix 9 performed better than wild-type
HsRXR as partners of EcR in supporting ligand-
dependent induction of reporter activity. One partic-
ular mutant, in which three amino acids present in the
loop between helix 8 and 9 of HsRXR were replaced
with three amino acids present in the same region of
LmRXR (D450E ⁄ A451V ⁄ K452R), performed even
better than LmRXR as a partner for EcR in ligand-
CH6 CH8 CH9 CH10 CH11
A
B
Fig. 4. (A) 3T3 cells were transfected with
pRLUC, pFRLUC, G:CfEcR(DEF) and
V:HsRXR(EF) or V:LmRXR(EF) or VP6 fusion
of one the Hs–LmRXR(EF) chimeras. Trans-
fected cells were exposed to DMSO, 0.04,
0.2, 1 or 5 l
M RG-102240 for 48 h. The cells
were harvested and assayed for luciferase
activity. The fly luciferase activity was nor-
malized using Renilla luciferase activity. The
values presented are mean ± SD (n ¼ 3).
(B) Twenty micrograms of proteins isolated
from 3T3 cells transfected with V:Hs–
LmRXR(EF) chimera constructs were separ-
ated on SDS ⁄ PAGE, transferred to nitrocel-
lose and analyzed using VP16 antibodies.
Arrow points to fusion protein bands.

HsRXR(EF) chimera switch induced the luciferase
activity with 1 lm or higher concentration of RG-
102240. This is similar to the ligand sensitivity of the
G:CfEcR(DEF) + V:HsRXR(EF) switch, but lower
than that of the G:CfEcR(DEF) + V:LmRXR(EF)
switch in which the luciferase activity was induced with
0.2 lm or higher concentration of RG-102240 (Fig. 7).
These data confirmed the results that the region of
LmRXR containing helix 9 and the two loops on
either side of helix 9 is responsible for improved per-
formance of LmRXR as a partner for EcR in ligand-
dependent induction of reporter activity.
123456789
A
B
Fig. 6. 3T3 cells were transfected with pRLUC, pFRLUC, G:CfEcR(DEF) and V:HsRXR(EF) or V:LmRXR(EF) or mutants of HsRXR. (A) Trans-
fected cells were exposed to DMSO, 0.04, 0.2, 1 or 5 l
M RG-102240 for 48 h. The cells were harvested and assayed for luciferase activity.
The fly luciferase activity was normalized using Renilla luciferase activity. The values presented are mean ± SD (n ¼ 3). (B) Twenty micro-
grams of proteins isolated from 3T3 cells transfected with V:HsRXR(EF) mutant constructs were separated on SDS ⁄ PAGE, transferred to
nitrocellulose and analyzed using VP16 antibodies. The arrow points to fusion protein bands. Mutant 1, D450E ⁄ A451V ⁄ K452R; mutant 2,
S455K ⁄ N456S ⁄ P457A ⁄ S458Q; mutant 3, V462L; mutant 4, S470A; mutant 5, T473E; mutant 6, C475T ⁄ K476R ⁄ Q477T ⁄ K478T ⁄ Y475H;
mutant 7, E481D ⁄ Q482E ⁄ 483P; mutant 8, A495S; mutant 9, A528S.
Influence of RXR on EcR function S. R. Palli et al.
5984 FEBS Journal 272 (2005) 5979–5990 ª 2005 The Authors Journal Compilation ª 2005 FEBS
To determine whether the region of LmRXR
(helix 9 and two loops either side of it) that improved
EcR performance would also affect RXR perform-
ance mediated through 9-cis-retinoic acid, we com-
pared the performance of HsRXR, LmRXR and the

CfUSP(EF) pulled down by EcR in the presence of
DMSO or 1 lm RG-102240, suggesting that EcR
and USP can heterodimerize in the absence of ligand
(Fig. 9). In contrast, the amount of HsRXR,
Fig. 7. Comparison of two parent RXRs and Lm-HsRXR(EF) chi-
mera in transactivation assays. 3T3 cells were transfected with
pRLUC, pFRLUC, G:CfEcR(DEF) and V:HsRXR(EF) or V:LmRXR(EF)
or VP6 fusion of Lm–HsRXR(EF) chimera (LmRXR helix 9 and loops
on either side of it were replaced with the corresponding region of
HsRXR). The transfected cells were exposed to DMSO, 0.04, 0.2,
1or5l
M RG-10240 for 48 h. The cells were harvested and
assayed for the luciferase activity. The fly luciferase activity was
normalized using Renilla luciferase activity. The values presented
are mean ± SD (n ¼ 3).
Fig. 8. Comparison of two parent RXRs, Lm-HsRXR(EF) and
Hs–LmRXR(EF) chimeras in 9-cis-retinoic acid induced transactiva-
tion assays. 3T3 cells were transfected with pRLUC, pFRLUC,
G:CfEcR(DEF) and V:HsRXR(EF) or V:LmRXR(EF) or VP6 fusion of
Lm-HsRXR(EF) chimera (LmRXR helix 9 and loops on either side of
it were replaced with the corresponding region of HsRXR) or
Hs–LmRXR(EF) chimera 9. The transfected cells were exposed to
DMSO, 0.04, 0.2, 1, 5 or 25 l
M 9-cis-retinoic acid for 48 h. The
cells were harvested and assayed for the luciferase activity. The fly
luciferase activity was normalized using Renilla luciferase activity.
The values presented are mean ± SD (n ¼ 3). Asterisks on top of
the bars indicate significant difference from DMSO-treated cells at
P < 0.5 determined by t-test.
Fig. 9. GST:CfEcRDEF and [

when compared with the EcR:HsRXR switch that
requires 1 lm RG-102240 to initiate induction of the
luciferase reporter gene and the reporter activity rea-
Fig. 10. Dose-dependent induction of reporter gene by gene
switch receptors. (A) 3T3 cells were transfected with G:Cf(DEF),
V:Hs–LmRXR(EF), pFRLUC and pRLUC. The transfected cells were
grown in the medium containing 0, 0.04, 0.2, 1 or 5 l
M concentra-
tion of RG-102240. The cells were collected at 48 h after adding
ligand and reporter activity was quantified. The fly luciferase activity
was normalized using Renilla luciferase activity. The values presen-
ted are mean ± SD (n ¼ 3). (B) 3T3 cells were transfected with
G:CfEcR(DEF) + V:HsRXR(EF) gene switch. 3T3 cells were trans-
fected with G:Cf(DEF), V:HsRXR(EF), pFRLUC and pRLUC. The
transfected cells were grown in the medium containing 0, 0.2, 1, 5
and 25 l
M concentration of RG-102240. The cells were collected at
48 h after adding ligand and reporter activity was quantified. The fly
luciferase activity was normalized using Renilla luciferase activity.
The values presented are mean ± SD (n ¼ 3).
Fig. 11. (A) Time course of induction of reporter gene by gene
switch plasmids. (A) 3T3 cells were transfected with G:Cf(DEF),
V:Hs–LmRXR(EF), pFRLUC and pRLUC. The transfected cells were
grown in the medium containing 1 l
M concentration of RG-102240.
The cells were collected at 0, 1, 3, 6, 12, 24, 48 and 72 h after add-
ing ligand and reporter activity was quantified. The fly luciferase
activity was normalized using Renilla luciferase activity. The values
presented are mean ± SD (n ¼ 3). (B) 3T3 cells were transfected
with G:Cf(DEF), V:Hs–LmRXR(EF), pFRLUC and pRLUC. The trans-

phate (SEAP) in mouse sera was evaluated at various
time points after ligand administration. The G:CfEcR-
(DEF) + V:Hs–LmRXR(EF) chimera switch induced
SEAP activity that reached peak levels at five days
after the administration of ligand (Fig. 12). By con-
trast, the G:CfEcR(DEF) + V:HsRXR(EF) switch did
not cause induction of SEAP activity up to 25 days
after the administration of ligand (Fig. 12). Thus, the
G:CfEcR(DEF) + V:Hs–LmRXR(EF) chimera switch
but not G:CfEcR(DEF) + V:HsRXR(EF) switch is
sensitive enough to support ligand-dependent induction
of reporter gene expression in vivo.
Discussion
The EcR heterodimerizes with the nuclear receptor
USP, binds to ecdysteroids and ecdysone response ele-
ments and regulates the expression of ecdysteroid
responsive genes. Because ecdysteroids and their lig-
ands are absent in vertebrates, including humans, they
are being developed to regulate transgenes in various
applications including gene therapy, functional genom-
ics, drug discovery, and biopharmaceutical production
[24]. In mammalian cells, the CfEcR and CfUSP het-
erodimer induces the expression of reporter genes regu-
lated by EcRE even in the absence of ligand; therefore,
they are not useful for gene switch applications. Ver-
tebrate RXR such as human or mouse RXR have been
used in the place of USP in all EcR gene switches
developed to date. One of the major limitations of cur-
rent versions of EcR gene switches is the requirement
of micromolar concentrations of ligand for induction

helix 9 are responsible for improved performance of
Fig. 12. In vivo comparison of human RXRb and Hs-RXRb –Lm-
RXRb fusion protein in a C57BL ⁄ 6 mouse model. The gene swit-
ches, composed of plasmids containing pCMV ⁄ GAL4-pCfEcR(DEF),
pCMV ⁄ VP16-Hs-RXR(EF) (s) or pCMV ⁄ VP16-HsRXRb(H1-8)-
LmRXRb(H9-12) fusion (m), and 6xGAL4RE-TTR-SEAP, were elec-
troporated into the quadriceps of C57BL ⁄ 6 mice. Animals were
treated with 5 mg RG-102240 ⁄ 50 lL DMSO ⁄ mouse by IP injection
three days after electroporation of plasmid. SEAP in mouse sera
was evaluated for up to 17 days after ligand administration. Values
are the average from seven animals ± SD.
S. R. Palli et al. Influence of RXR on EcR function
FEBS Journal 272 (2005) 5979–5990 ª 2005 The Authors Journal Compilation ª 2005 FEBS 5987
LmRXR when compared with the performance of
HsRXR as a partner for CfEcR. Structural studies on
EcR:USP and RAR:RXR heterodimers showed that
helices 7 and 10, present in two nuclear receptors,
form heterodimerization interfaces and play critical
roles in heterodimerization [25]. The data presented
here showed that besides helices 7 and 10, amino acid
residues present in helix 9 and in the two loops present
on either side of helix 9 play critical roles in hetero-
dimerization of EcR:USP ⁄ RXR. In fact, there is only
one amino acid each in helix 7 and helix 10 that is dif-
ferent between HsRXR and LmRXR. Replacing these
two amino acids in HsRXR with the corresponding
amino acids present in LmRXR did not increase the
performance of HsRXR as a partner for CfEcR. In
contrast, replacing HsRXR amino acids present in
helix 9 and in the loops on either side of helix 9 with

HsRXR and helix 9 and the loops on either side of
helix 9 from HsRXR or mutants of HsRXR with a
change in just three amino acids (D450E ⁄
A451V ⁄ K452R) will definitely help in the develop-
ment of gene switches, especially those that require
in vivo applications.
Experimental procedures
Constructs
The construction of GAL4:CfEcR(DEF) [G:CfEcR(DEF)],
VP16:MmRXR(EF) [V:MmRXR(EF)] and VP16:CfUS-
P(EF) [V:CfUSP(EF) has been described previously [23]
VP16:LmRXR(EF) [V:LmRXR(EF)] was constructed by
amplifying EF domains of LmRXR using primers contain-
ing EcoRI and BamHI sites in the forward and reverse pri-
mer, respectively, followed by cloning of the PCR product
into EcoRI and Bam HI digested pVP16 vector (Clontech
Inc. Palo Alto, CA). pFRLUC reporter plasmid was pur-
chased from Stratagene (La Jolla, CA). pRLUC is reporter
plasmid expressing Renilla luciferase under the control of
thymidine kinase promoter (Promega, Madison, WI). The
GST fusion construct of MmR(EF) was made by cloning
MmR(EF) domain into pGEX-5X-1 vector (Amersham
Pharmacia Biotech, Piscataway, NJ) forward and reverse
primers, respectively.
Ligands
RG-102240 [N-(1,1-dimethylethyl)-N¢-(2-ethyl-3-methoxy-
benzoyl)-3,5-dimethylbenzohydrazide] also known as
GS
TM
-E and RheoSwitchÒ ligand 1 (RSL1) is a synthetic

gene).
Western blots
3T3 cells were transfected with V:LmRXR(EF) or V:CfUS-
P(EF) or V:HsRXR(EF). Twenty micrograms of proteins
isolated form these cells at 48 h after transfection were sep-
arated by SDS ⁄ PAGE and transferred to nitrocellulose
membranes. After blocking with 1% dry milk powder,
the membranes were exposed to VP16 primary antibody
Clontech and peroxidase-cojugated secondary antibody.
Peroxidase signals were detected using enhanced chemilumi-
nescence kit (Amersham BioSciences, Piscataway, NJ,
USA).
Pull-down assays
CfEcR DEF domains were cloned into pGEXT vector
(Amersham Pharmacia Biotech) and expressed in
pLysS ⁄ BL-21 Escherichia coli cells. The GST fusion pro-
teins were extracted as outline in protocol supplied by
Amersham Pharmacia Biotech. Hs–LmRXR chimera,
HsRXREF, LmRXREF and CfUSP proteins were labeled
with [
35
S]-methionine using TNT kit (Promega). Labeled
Hs–LmRXR chimera, HsRXREF, LmRXREF and CfUSP
were mixed with GST:CfEcR(DEF) and incubated in bind-
ing buffer containing DMSO or 1 lm RG-102240 and the
complexes were precipitated with glutathione agarose beads
(Amersham Pharmacia Biotech). The pellet was washed
and resolved on SDS ⁄ PAGE and the gel was dried and
exposed to X-ray film.
Acknowledgements

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