An intramolecular folding sensor for imaging
estrogen receptor–ligand interactions
Ramasamy Paulmurugan* and Sanjiv S. Gambhir*
Departments of Radiology and Bioengineering, Bio-X Program, Molecular Imaging Program at Stanford (MIPS), Stanford University School of Medicine,
James H. Clark Center, 318 Campus Drive, East Wing, First Floor, Stanford, CA 94305-5427
Communicated by Michael E. Phelps, University of California School of Medicine, Los Angeles, CA, August 24, 2006 (received for review May 17, 2006)
Strategies for high-throughput analysis of interactions between
various hormones and drugs with the estrogen receptor (ER) are
crucial for accelerating the understanding of ER biology and
the human ER (hER) ligand-binding domain (hER–LBD) in complex
lecular folding pattern could be used to distinguish ER agonists
from selective ER modulators and pure antiestrogens. We there-
fore constructed and validated intramolecular folding sensors
encoding various hER–LBD fusion proteins that could lead to split
of the appropriate ligands. A mutant hER–LBD with low affinity for
circulating estradiol was also identified for imaging in living
subjects. Cells stably expressing the intramolecular folding sensors
expressing wild-type and mutant hER–LBD were used for imaging
hER–LBD intramolecular folding sensor suited for high-throughput
quantitative analysis of interactions between hER with hormones
and drugs using cell lysates, intact cells, and molecular imaging of
small living subjects. The strategies developed can also be ex-
tended to study and image other important protein intramolecular
complementation ? optical imaging ? split reporters
of these hormones are mediated by the estrogen receptor (ER),
which is a ligand-inducible nuclear transcription factor (1). In the
classical pathway of steroid hormone action, 17?-estradiol (E2),
hormones, and a variety of other estrogens bind to the ligand-
binding domain (LBD) of ER and lead to its dimerization and
subsequent binding to a specific regulatory sequence in the pro-
(2, 3), which then trigger activation or repression of many down-
stream target genes (4). The deficiency or excess of estrogens can
lead to various pathological conditions including osteoporosis and
breast carcinomas (5), making ER a major cellular therapeutic
Elegant crystallographic studies with ER–LBD have shown that
various ER ligands (4, 6, 7). The conformation of H12 behaves as
a ‘‘molecular switch’’ that either prevents or enhances ER from
binding to an array of coactivator proteins, which then activates
transcription of many downstream estrogen-regulated genes re-
sponsible for cell growth. Given the critical role of H12 in ER
signaling, we reasoned that it may be feasible to develop an
intramolecular ER folding sensor with specific split reporter
complementation patterns to study ligand pharmacology based
directly on the conformational changes of H12 in response to
different ligands (Fig. 1).
We used a split synthetic Renilla luciferase (RLUC) and firefly
luciferase (FLUC) complementation system, which we previously
developed and validated (8, 9), to test this hypothesis by assaying
ligand-induced RLUC?FLUC complementation in cell lysates,
strogens are responsible for the growth, development, and
maintenance of the reproductive, skeletal, neuronal, and im-
intact cells, and cell implants in living mice by noninvasive biolu-
minescence optical imaging. The validated ER intramolecular
different ER ligands, agonists, selective ER modulators (SERMs),
low affinity to E2 was identified and used as an ER sensor for
characterization of ER ligand interaction in living mice without
significant competition from endogenous circulating estrogens.
Author contributions: R.P. and S.S.G. designed research; R.P. performed research; R.P. and
S.S.G. analyzed data; and R.P. and S.S.G. wrote the paper.
The authors declare no conflict of interest.
Abbreviations: RLUC, Renilla luciferase; FLUC, firefly luciferase; N-RLUC, N-fragment of
ligand-binding domain; SERM, selective ER modulator; DES, diethylstilbestrol; 4-OHT,
4-hydroxytamoxifen; E2, 17?-estradiol; H12, helix 12; ICI, ICI182,780.
*To whom correspondence may be addressed. E-mail: firstname.lastname@example.org or
© 2006 by The National Academy of Sciences of the USA
induced intramolecular folding of ER that leads to split RLUC complementa-
tion. The N- and C-terminal fragments of split RLUC were fused to the N and
C terminus, respectively, of the hER? of various lengths (amino acids 281–549
and 281–595). Binding of ER ligands to the intramolecular folding sensor
(N-RLUC-hER-C-RLUC) induces different potential folding patterns in the LBD
for ER antagonist (b) (H12 and ligands are colored green), low complemen-
tation for ER agonist (a) (H12 and ligands are colored blue), and no comple-
mentation for partial ER agonist?antagonist (c) (H12 and ligands are colored
N- and C-RLUC fragments after binding with partial agonist (c) is smaller than
that of agonists (b), this model depicts the importance of the orientations of
the split RLUC fragments in complementation. The yellow spheres are hydro-
phobic amino acids located between helix 3 and helix 5 of LBD.
Schematic representation of the hypothetical model of ligand-
October 24, 2006 ?
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RLUC Complementation as Sensors of ER Ligand-Induced Intramolec-
ular Folding. Several factors need to be considered to achieve
efficient ligand-induced split RLUC complementation, and also
ligand-induced complementation that distinguishes agonists from
antagonists. Key factors include (i) the distance between the
complementing N- and C-RLUC fragments in the intramolecular
folding sensor before and after the binding of ligands, (ii) the
the substrate binding pockets through complementation after li-
gand binding. By carefully considering these factors along with the
crystal structures of different ER ligand complexes (bound with
agonists, SERMs, and pure antiestrogens) and the importance of
H12 within the LBD, we constructed a series of vectors that
expresses fusion proteins with split RLUC fragments and different
portions of the hER (Fig. 2a) (4, 10). We also confirmed the
orientation of split RLUC fragments needed for efficient comple-
mentation (Fig. 6, which is published as supporting information on
the PNAS web site). All of these vectors were studied in 293T cells
(ER-negative cells, transiently transfected with ER) treated with
several ER ligands, including E2, tamoxifen, raloxifene, genistein,
diethylstilbestrol (DES), and 4-hydroxytamoxifen (4-OHT) (struc-
tures of ligands are in Fig. 7, which is published as supporting
information on the PNAS web site). Among the different portions
of hER examined, the fusion protein containing hER of amino
E), and domain F] showed a significant level of ligand-induced
was used for constructing all vectors in this study. The level of
and agonists was 80 ? 15 times greater than in cells exposed to
carrier controls (P ? 0.001). The cells exposed with genistein, a
very-low-affinity ER? agonist, showed no complementation, sim-
ilar to untreated cells (Fig. 2b). At the same time, the cells
amino acids 281–549 of hER (hER281–549: domains D and E) show
complementation that clearly distinguishes agonists from SERMs.
In transfected cells treated with SERMs and agonists, 80-fold ?
served, respectively, relative to untreated cells (P ? 0.05) (Fig. 2c).
Up-regulation of the multidrug-resistant P-glycoprotein by ER
ligands and associated change in the signal by coelenterazine (11)
were ruled out by different experiments in cell lysates and intact
cells (Fig. 8, which is published as supporting information on the
PNAS web site).
Complementation of ER–LBD Intramolecular Folding Sensor Is Inde-
pendent of the Levels of Endogenous ER? Expression. To determine
whether ligand-induced hER intramolecular-folding-assisted
RLUC complementation in transfected 293T cells is due to in-
creased protein expression of the folding sensor N-RLUC-hER281–
549-C-RLUC, Western blot analysis was performed by using the
RLUC antibody before and after treatment with different ER
ligands. Treatment of 293T cells with E2, DES, genistein, and
raloxifene did not lead to significant changes in the protein level of
the folding sensor (Fig. 2d Inset Lower). Interestingly, even though
the levels of sensor protein were significantly reduced in the
transfected cells treated with the SERM tamoxifen and two anti-
cancer drugs, epigallocatechin gallate and cisplatin (both non-ER
ligands), the RLUC signal in tamoxifen-treated cells was still
significantly greater than that in the cells treated with the agonists
to any RLUC signal, compared with carrier control-treated cells
(P ? 0.05). These results show that the variations in the RLUC
through changes in the level of sensor protein but were more likely
to be caused by different complementation patterns within the
receptor induced by the ligands. The ER? protein level in trans-
fected MCF-7 cells was also estimated with Western blot analysis
before and after the cells were treated with different ER ligands.
Treatment of MCF-7 cells with ER ligands did not lead to signif-
icant changes in the intracellular ER? protein levels (Fig. 2d Inset
Upper). Also, ligand-induced intramolecular folding of ER studied
and 293T) cell lines showed no significant relation with the intra-
cellular ER level (Fig. 3 d–f).
various ER ligands. (a) Schematic diagram of different intra-
molecular folding sensor constructs with the split RLUC frag-
side of the ER–LBD were used to identify a vector that leads to
tation and can distinguish ER agonists (A), antagonists (B), and
mentation. (b) ER ligand-specific intramolecular folding sensor.
293T cells were transiently transfected to express the intramo-
lecular folding sensor N-RLUC-hER281–595-C-RLUC and treated
with the indicated ER ligands or carrier control for 18 h. Treat-
ment with ER antagonists and agonists led to similar levels of
intramolecular-folding-assisted complementation that was sig-
nificantly higher than that of carrier control-treated cells (P ?
0.001). (c) Antagonist-specific intramolecular folding sensor.
293T cells were transiently transfected to express the intramo-
ligands or carrier control as in b. Treatment with ER antagonists
ern blot analysis of MCF7 cells using anti-ER? antibody after
treatment with different ligands (Inset Upper). Shown are ER
ligand antagonist-specific and agonist-specific intramolecular-
folding-assisted RLUC complementation in 293T cells and the
Western blot analysis of corresponding sample using RLUC an-
averages of triplicate samples ? SEM.
Intramolecular folding sensors and their response to
www.pnas.org?cgi?doi?10.1073?pnas.0607385103Paulmurugan and Gambhir
Time- and Dose-Dependent Induction of hER–LBD Intramolecular RLUC
Complementation. To determine the kinetics of ligand-induced
RLUC complementation, 293T cells transiently transfected to
express the fusion protein N-RLUC-hER281–549-C-RLUC were
exposed to 1 ?M of three representative ligands (the agonists E2
and DES and the SERM 4-OHT) and assayed for complemented
RLUC activity at 6, 12, 18, and 24 h. The results showed significant
levels of RLUC complementation in transfected cells exposed to
the 4-OHT at all time points studied (P ? 0.002 relative to carrier
control-treated?untreated cells). The agonists E2 and DES led to
RLUC complementation that was significantly lower than 4-OHT
but higher than that of carrier control?untreated cells at all time
points studied (P ? 0.001). Furthermore, the maximum level of
exposure to ligands (Fig. 4a). The potency of each ligand to induce
RLUC complementation in transiently transfected 293T cells ex-
pressing N-RLUC-hER281–549-C-RLUC was studied at different
ligand concentrations with RLUC activities determined by lumi-
nometer assays. A dose-dependent increase in complemented
RLUC activity was observed in all cases, with maximum induction
at 1 ?M except for the agonist DES, which showed maximum
activity at 1.5 ?M relative to carrier control-treated?untreated cells
Competitive Binding of ER Agonists and SERMs in Ligand-Induced
Intramolecular-Folding-Assisted RLUC Complementation. To study
ER intramolecular folding in living mice, the 293T cells were stably
transfected to express the intramolecular folding sensor N-RLUC-
hER281–549-C-RLUC. No significant difference in RLUC activity
was observed between the stable and transiently transfected cells
with all ligands studied (data not shown). These stable cells were
used for studying the competitive binding of an agonist (E2) and a
were assayed for complemented RLUC activity 18 h after being
simultaneously exposed to a fixed concentration of E2 (1 ?M) with
cells expressing the intramolecular folding sensor were exposed to
a fixed concentration of tamoxifen (1 ?M) and various concentra-
tions of E2 (0.008–1 ?M). E2-induced complemented RLUC
activity was significantly enhanced in the presence of tamoxifen
(P ? 0.05) (Fig. 4c), and the tamoxifen-induced complemented
RLUC activity was reduced in the presence of E2 (P ? 0.05)
The Pure Antiestrogen Fulvestrant [ICI182,780 (ICI)] Leads to RLUC
Complementation Signal That Is Neither Like an Agonist nor a SERM.
The drug ICI is a pure antiestrogen (Fig. 3a) that is currently in
clinical use for the treatment of both ER-positive and ER-negative
breast cancers (12, 13). Crystallographic studies with ER??pure
antiestrogen complex suggest unique structural features of this
receptor complex leading to its interesting pharmacology (14). In
our system with 293T cells transfected to express the sensors
RLUC-ER (281–549) or RLUC-ER (281–595), ICI led to dose-
dependent RLUC complementation with maximum induction at
by ICI was distinct from that of ER SERMs or agonists (Fig. 3f).
It is of note that no significant degradation of the fusion sensor
(Fig. 3c) (15).
A Single Amino Acid Change at Glycine-521 with Threonine Selectively
Abolishes the E2-Mediated Complementation of Folding Sensor With-
out Significantly Affecting the Complementation Induced by Other ER
Ligands. Binding of endogenous E2 to the intramolecular folding
sensor in living animals will limit its use in tumor models or a
transgenic model for studying hER interactions with ligands and
drugs. Hence, to overcome this issue, we generated single amino
acid mutations at position 521 of hER? that are analogous to the
mutation generated in mouse ER? at amino acid position 525
(G525R), which was reported to reduce affinity to E2 by 1,000-fold
Structure of the ER ligand ICI used for the study. (b)
Dose-dependent RLUC complementation of ICI. 293T
cells were transiently transfected to express the in-
tramolecular folding sensor N-RLUC-hER281–549-C-
RLUC and treated with various concentrations of ICI
for 18 h. RLUC activity was determined as in b. (c)
Expression of the intramolecular folding sensors in
expression of N-RLUC-hER281–549-C-RLUC in transiently
or ICI was determined by Western blotting as de-
scribed in Fig. 4c. (d–f) Ligand-induced intramolecular
folding by different ER agonists and antagonists in
comparison with ICI. The intramolecular folding sen-
sor was transiently transfected into ER-positive MCF-7
cells and ER-negative MDA-MB-231 and 293T cells and
18 h. RLUC activity was determined as in b. The error
bars represent SEM of triplicate determinations.
Study of ER-ligand-induced intramolecular
Paulmurugan and Gambhir PNAS ?
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(16). We constructed 19 different mutants at position 521 within
N-RLUC-hER281–595-C-RLUC, which leads to RLUC comple-
of constructs screened with six different ER ligands are presented
in Table 1, which is published as supporting information on the
the glycine-to-threonine (G521T) transition led to a 94% reduction
in the intramolecular folding-mediated RLUC complementation
induced by E2 (P ? 0.001); an only 12–22% reduction for DES,
4-OHT, and raloxifene (P ? 0.05); and no significant changes in
response to genistein and ICI (P ? 0.05) (Fig. 9, which is published
as supporting information on the PNAS web site). The sensor with
mutant hER (N-RLUC-hER281–549/G521T-C-RLUC), which distin-
guishes ER antagonists from SERMs (Fig. 2d), and the sensor with
mutant mouse ER (N-RLUC-mER281–549/G525R-C-RLUC) were
ligands, and RLUC complementation was determined by lumi-
nometer assay. Our folding sensor with mutant hER (G521T)
appeared less sensitive to the endogenous ligand E2 and retained
more of the DES and raloxifene effects compared with the folding
sensor with the mutant mER (G525R) (Fig. 10, which is published
as supporting information on the PNAS web site).
The ER Intramolecular Folding System with LBDs ER281–549?G521G,
ER281–549?G521T, and ER281–595?G521G Using Split FLUC Enzyme
Fragments. To evaluate the ER intramolecular folding system’s
utility and generalizability, we also constructed vectors with im-
ER constructs and various ligands (Fig. 11, which is published as
supporting information on the PNAS web site). The results show
similar patterns as achieved when using the corresponding system
with split RLUC fragments (Fig. 12, which is published as support-
ing information on the PNAS web site).
Imaging of ER Intramolecular Folding in Living Mice. We studied the
interactions between hER with ligands in living mice using the ER
intramolecular folding-mediated RLUC complementation system,
by implanting 293T cells stably expressing wild-type (N-RLUC-
hER281–549-C-RLUC) or mutant (N-RLUC-hER281–549/G521T-C-
mice (n ? 3). Bioluminescence imaging of RLUC activity was
performed immediately after cell implantations and 18 h after i.p.
in RLUC complementation induced by the SERMs 4-OHT and
raloxifene in different experiments; we used raloxifene for many of
our animal experiments. Significant RLUC activity was observed
hER sensor (wild-type hER, 2.16 ? 0.52 ? 103photons per second
per square centimeter per steradian; mutant hER, 9.7 ? 1.2 ? 103
photons per second per square centimeter per steradian) (P ? 0.01
relative to the site with wild-type hER) (Fig. 5a). Similar FLUC
activity pattern was observed with wild-type and mutant ER
constructs (Figs. 11 and 12). The lower level of signal produced
from implanted cells expressing the wild-type hER sensor is most
availability of raloxifene. The split FLUC system showed signifi-
cantly more signal in living animals than the same system with the
RLUC fragments, so this sensor was used for studying the agonist-
and antagonist-specific induction of complementation in living
Imaging of ER Intramolecular Folding Sensor to Distinguish ER Ligands
in Living Mice. We show the utility of our system in differentiating
pharmacological classes of ER ligands in living animals based on
receptor conformations by implanting 293T cells stably expressing
the wild-type ER sensor (281–549) with FLUC fragments and the
mutant form of ER–LBD (N-RLUC-hER281–549/G521T-C-RLUC)
on either side of the lower back of the male nude mice (n ? 2; two
implants in each animal) and imaged 24 h after implantation
(before ligand administration) and every 24 h after administration
of 20 ?g of DES or 4-OHT with adjuvant (sesame oil) or adjuvant
in a 50-?l volume (17). These results show a significantly greater
SERM than the animals that received the agonist or adjuvant only
We have developed and validated two hER intramolecular folding
sensors that can be used to distinguish ER ligand pharmacology.
These receptor sensors can be directly translated from cell culture
studies to molecular imaging in small living subjects. In this study
we used an ER-based split reporter complementation strategy to
follow the position of H12 within the ER–LBD to detect changes
in the receptor structural folding in response to ligand binding. The
longer construct with the F domain (281–595) appears ligand-
pharmacology-independent (Fig. 2b), whereas the shorter con-
mentation of the intramolecular folding sensor. (a) 293T cells transiently
transfected with the intramolecular folding sensor (N-RLUC-hER281–549-C-
RLUC) were assayed for RLUC activity at different time points after exposure
to E2, 4-OHT, and DES. The maximum fold ligand induction of split RLUC
(*). (b) Concentration-dependent activation of ligand-induced RLUC comple-
mentation. 293T cells were transiently transfected with the intramolecular
folding sensor and exposed to increasing concentrations of the indicated
ligands for 18 h. RLUC activity was determined by luminometer assays as in a
significantly in transfected cells treated with the ER antagonists 4-OHT (?),
tamoxifen (Œ), and raloxifene (■). The ER agonist DES (‚) led to maximum
induction of complemented RLUC activity at 1.5 ?M. All other ligands led to
a dose-dependent increase in complemented RLUC activity with maximum
induction at 1 ?M, relative to carrier control-treated cells. (c) Competitive
binding of tamoxifen to the intramolecular folding sensor. 293T cells tran-
siently transfected with the intramolecular sensor N-RLUC-hER281–549-C-RLUC
were treated with various concentrations of the ER antagonist tamoxifen in
activity was determined as in b, and the samples were normalized for trans-
fection efficiency by cotransfecting with FLUC. (d) Competitive binding of E2
to the intramolecular folding sensor. 293T cells stably transfected with the
intramolecular sensor N-RLUC-hER281–549-C-RLUC were treated with various
concentrations of E2 in the presence of a fixed concentration of tamoxifen (1
?M). RLUC activity was determined as in b.
Antagonists- and agonists-specific induction of split RLUC comple-
www.pnas.org?cgi?doi?10.1073?pnas.0607385103 Paulmurugan and Gambhir
luciferase complementation in response to SERMs, moderate
levels for agonists, and minimal levels for pure antiestrogens (Fig.
2c). We validated these intramolecular folding sensors with various
ER ligands in both transiently and stably transfected 293T cells and
transiently transfected MDA-MB-231 (ER-negative) and MCF-7
(ER-positive) cells. To extend the folding sensor for applications in
hER (G521T) into the folding sensor that was insensitive to
circulating endogenous estrogen but retained its ability to distin-
guish SERMs from synthetic agonists. Alternatively, ovariecto-
competition from endogenous estrogens while retaining the ability
compare strategies with the wild-type and mutant systems.
ER ligands by using either purified ER? protein or ER isolated
from cell lysates (18–21). Limited fluorescence-based assays (22)
have been developed to measure receptor conformational changes
(23) and recruitment of coactivator peptides (22, 24, 25) in the
full-length hER? within cell culture (26). Other assays have been
designed to study the effects of synthetic ligands on ER transcrip-
tion through the activation of downstream target genes (27).
However, most of these reported assays are not suitable for
and especially in living subjects through noninvasive molecular
A nontranscriptional assay containing fusion chimeras of either
Flp recombinase (28) or Cre recombinase (29) with a truncated
mouse ER? (amino acids 281–599) has been reported and used for
regulating the recombination of reporter genes in cells and living
animals. This system demonstrates high background activity even
before the addition of ER ligands, mainly through enzymatic
amplification, thus limiting its dynamic range in response to differ-
ent ER ligands. We developed an analogous fusion chimera by
fusing a truncated version of hER (amino acids 281–595) with
background (mock-transfected cells) even before the addition of
ligands (unpublished data). The addition of ligands generates
FLUC activity that is only 5- to 6-fold higher than that of carrier
control-treated cells (unpublished data). These results clearly sup-
port the notion that these nontranscriptional chimeras containing
ER–LBD are not optimal for studying concentration-dependent
interactions between ER and their ligands.
To our knowledge, only one study has reported the construction
of mutant versions of hER (G521R and G521V) for selective ER
ligand binding using a fusion chimera containing hER251–595with
Flp recombinase enzyme (28). Incorporation of the same mutation
into our intramolecular folding sensor (N-RLUC-hER281–595-C-
RLUC) led to nearly complete abolishment of signal for all ER
ligands (hERG521R) and a significant reduction in signal (77–89%)
(Table 1). We constructed intramolecular folding sensors using the
hERG521mutants with 19 different possible amino acids. We found
that the replacement of hERG521with threonine leads to nearly
complete abolishment of the E2-induced RLUC complementation
and to only a 10–20% reduction for all other ER ligands studied.
Subsequently, 293T cells stably expressing this intramolecular fold-
ing sensor (N-RLUC-hER281–549/G521T-C-RLUC) were generated
for imaging hER??ligand complexes in living animals.
The advantages of the intramolecular folding sensor strategy
developed and validated include the following: (i) it is real-time
(because RLUC exhibits flash kinetics) and quantitative; (ii) it can
be used to distinguish binding of agonists, SERMs, and pure
antiestrogens; (iii) it can be adapted for studying ligand binding to
hER in living animal models by molecular imaging, and thus
pharmacokinetic properties of each drug?ligand can be examined;
(iv) it allows for a high-throughput strategy for screening?
comparing different ER ligands and drugs in multiple cell lines; (v)
it allows direct transition from cell culture studies to small living
subjects because it is based on a bioluminescence split reporter
strategy; and (vi) it will allow for applications using transgenic
models that incorporate the intramolecular folding sensor. In
addition, the availability of other split reporters with different
properties and substrate specificity should allow multiplexing with
other reporter assays.
The limitations with using split RLUC as the reporter gene
regarding efflux of its substrate coelenterazine were resolved by
showing experiments that resulted in no significant relation be-
tween the RLUC complementation and the multidrug resistance
systems (Fig. 8) (11). In addition, the intramolecular folding system
was also studied with the improved split FLUC fragments by
replacing RLUC fragments. Both systems showed equal sensitivity
in different cell culture experiments. The FLUC fragments showed
nist-induced intramolecular folding in a mouse
model. (a) Shown is optical CCD camera imaging
of 293T cells stably expressing intramolecular
folding sensors N-RLUC-hER281–549-C-RLUC and
N-RLUC-mutant-hER281–549/G521T-C-RLUC in living
female nude mice before and after treatment
with antagonist raloxifene (0.5 mg per mouse)
and the corresponding quantitative graph. (b)
Similar imaging conducted by using the same
sensors with the split FLUC fragment system (N-
FLUC-hER281–549-C-FLUC and N-FLUC-mutant-
hER281–549/G521T-C-FLUC). The site implanted with
the cells expressing the intramolecular folding
sensor with the mutant hER (G521T) shows a
ifene treatment compared with that of the wild-
Bioluminescence imaging of ER antago-
Paulmurugan and GambhirPNAS ?
October 24, 2006 ?
vol. 103 ?
no. 43 ?
more detectable signal in mouse experiments than RLUC because
of more light penetration through tissues due to the more red-
shifted wavelengths of FLUC. Also, the FLUC-based folding
system showed greater efficiency in differentiating ER ligands in
living mice. It is also possible that the exact locations (cytosolic vs.
nuclear) of our fusion reporter proteins may affect the results
obtained, and this will need to be explored in future studies. In
have to be investigated with testing of additional drugs.
The strategies developed in this study can also be extended to
FRET and bioluminescence resonance energy transfer (30) by
replacing the split RLUC?FLUC reporter fragments with the
appropriate choice of donors and acceptors. This system will help
to validate a new class of molecular ‘‘switch’’ for imaging drug–
receptor interactions in living subjects that was previously not
feasible. This study will eventually translate to improved methods
for understanding the underlying estrogen biology, preclinical drug
development, and target validation, as well as investigation of other
important intramolecular folding systems.
The methods used for constructing different plasmid vectors and
the procedures used for cell culture, transfection, and luciferase
assays for RLUC and FLUC are in Supporting Materials and
Methods, which is published as supporting information on the
PNAS web site (Figs. 2a and 6a).
The Ligand-Concentration-Dependent Intramolecular-Folding-Assisted
Complementation Study. To determine the concentration of dif-
ferent agonists and antagonists of ER? required for inducing
efficient intramolecular folding of the ER–LBD sensors, 293T
cells transiently transfected with pcDNA-N-RLUC-hER281–549-
C-RLUC were exposed to different ligands including E2, ralox-
ifene, tamoxifen, DES, 4-OHT, and genistein at six different
for RLUC activity after 18 h of incubation and normalized as
mentioned in Supporting Materials and Methods.
Kinetics of Ligand-Induced Intramolecular Folding of hER281–549 and
Split RLUC Complementation. To determine the time point for
maximum ER ligand-induced split RLUC complementation, 293T
cells transiently transfected with pcDNA-N-RLUC-hER281–549-C-
RLUC were exposed to E2, 4-OHT, and DES (1 ?M). The cells
were assayed for RLUC activities at 6, 12, 18, and 24 h after ex-
posure to ligands as described in Supporting Materials and Methods.
Competitive Binding of ER Agonists and Antagonists in Induction of
Intramolecular-Folding-Assisted RLUC Complementation. To deter-
mine the effect of competitive binding on ER ligand-mediated split
RLUC complementation, 293T cells transiently transfected to
express the fusion protein N-RLUC-hER281–549-C-RLUC were
exposed to agonist E2 (1 ?M) with different concentrations of
antagonist tamoxifen (0.008–2 ?M) or to tamoxifen (1 ?M) with
different concentrations of E2 (0–1 ?M) for 18 h. RLUC activities
were determined as described in Supporting Materials and Methods.
The Ligand Agonist- and Antagonist-Specific Intramolecular Folding in
ER-Positive and ER-Negative Cell Lines. To determine the specificity
of ligand agonists and antagonists in induction of intramolecular
folding, ER positive (MCF-7) and negative (MDA-MB-231) cell
lines were transfected with pcDNA-N-RLUC-hER281–549-C-
in DMSO (1 ?M) or carrier control (DMSO). Complemented
RLUC activities and expression of the folding sensor were deter-
mined 18 h after transfection as described in Supporting Materials
Selection of 293T Cells Stably Expressing N-RLUC-hER281–549-C-RLUC
and N-RLUC-mutant hER281–549-C-RLUC for in Vivo Imaging Studies.
293T cells stably expressing the intramolecular folding sensor with
mutant (G521T) and wild-type hER? were selected by transfection
puromycin hydrochloride (1.5 ?g?ml). Stable clones were propa-
gated in MEM containing puromycin hydrochloride and used for
imaging studies in living mice.
Optical CCD Imaging of ER Ligand-Induced Intramolecular Folding in
Living Mice. All animal handling was performed in accordance with
Stanford University Animal Research Committee guidelines. For
imaging in living nude mice (nu?nu), 293T cells stably expressing
one of the fusion proteins N-RLUC-hER281–549-C-RLUC,
FLUC, or N-FLUC-hER281–549/G521T-C-FLUC were used. In vivo
31 (see Supporting Materials and Methods for details).
We acknowledge Anobel Tamrazi and Carmel Chan for help in improv-
ing the manuscript and Tim Doyle and Shay Keren for the help with
instrumentation. This work was supported by National Cancer Institute
In Vivo Cancer Molecular Imaging Centers Grant P50 CA114747,
National Cancer Institute Small Animal Imaging Resource Program
Grant R24 CA92865, and National Institutes of Health Grant R01
CA82214 (to S.S.G.).
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www.pnas.org?cgi?doi?10.1073?pnas.0607385103 Paulmurugan and Gambhir