Metabolic Research Unit and Department of Medicine (F.S., W.L.,
J.D.B.) University of California San Francisco,
California 94143
Department of Pharmacology and Cancer
Biology (C.-y.C., D.P.M.) Duke University Medical Center
Durham, North Carolina 27710
Department of
Pathology and Program in Molecular Biology (S.K.N., Y.W.)
University of Colorado Health Sciences Center Denver,
Colorado 80262 Departments of Medicine and Cell Biology
(R.N.D.) National Science Foundation Center for Biological
Timing University of Virginia Health Sciences Center
Charlottesville, Virginia, 22908
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ABSTRACT |
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![]() |
INTRODUCTION |
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The hydrophobic cleft does not form properly upon ER binding to tamoxifen or raloxifene (7, 10), which may account for the antiestrogenic action of these ligands in some tissues. Conversely, tamoxifen and raloxifene have estrogenic effects on other tissues. Like estrogens, tamoxifen and raloxifene promote interaction of some cofactors or peptides with ER structures outside of the hydrophobic cleft (11, 12, 13, 14). These interactions probably contribute to the AF-2-independent estrogenic actions of tamoxifen and raloxifene. Novel ER ligands that possess estrogenic activities in most tissues and antiestrogenic activities in the breast and uterus will be clinically useful for reducing the estrogen-mediated increase in breast and endometrial tumors that accompanies otherwise beneficial postmenopausal hormone replacement therapies (15, 16, 17, 18, 19). Identification of such improved selective estrogen receptor modulators (SERMs) will be aided by the development of techniques that discern the effects of each putative SERM on the types and timing of ER interactions with ligand-selective ER-interacting targets.
Previously we used phage display to isolate a large number of
peptides that bound to different sites on nuclear receptors including
ER (12, 13, 14). Each peptide differed in their interactions with
specific nuclear receptors or in response to different ligands. Some of
the nuclear receptor-interacting peptides contained the LXXLL motif and
could be grouped into three classes based upon sequence conservation of
the two amino acids immediately amino terminal to LXXLL (12). All three
classes of LXXLL are naturally found in cofactors that interact with
AF-2. Some cofactors contain multiple LXXLL motifs predominantly of a
single class. Others contain LXXLL motifs of varying classes and even
LXXLL motifs that are distinct from these three classes. It is thought
that such divergence in LXXLL sequence (5, 6, 20), combined with
nuclear receptor- or ligand-specific divergences in the structure of
the hydrophobic activation function-2 cleft (21), and variations in the
interactions of cofactors to other nuclear receptor surfaces,
contributes to the divergent actions of different ligands and nuclear
receptors.
Although the molecular alterations that accompany ligand binding to
nuclear receptors have been intensely characterized (1, 2, 3), very little
is known of the specificity and order of those events within living
cells. Recent studies of fluorophore-labeled nuclear receptors and
their interacting cofactors (22, 23, 24, 25, 26, 27) demonstrated that the temporal and
spatial characteristics of nuclear receptors could be directly examined
within cells by fluorescence microscopy. Here, we used fluorescence
microscopy to measure in intact cells the ligand-specific interactions
of ER with the nuclear receptor cofactor GRIP1 and five peptides that
we recently selected from combinatorial libraries for their binding to
ligand-bound ER (12, 13, 14). Human ER expressed as a fusion with blue
fluorescent protein (28) (BFP) localized to discrete subdomains of the
nucleus. GRIP1 (glucocorticoid receptor-interacting protein 1) and the
peptides expressed in cells as fusions with the spectrally distinct
green fluorescent protein (28) (GFP) were more evenly distributed
throughout the nucleus; the GFP-peptide fusions were also present in
the cytoplasm. When coexpressed with ER
-BFP in cells not treated
with ER ligand, the GFP-peptides and GFP-GRIP1 exhibited the same
distributions as when expressed alone. When incubated with
E2, three peptides containing variants of LXXLL
relocalized to assume the intranuclear position of ER. A fourth,
unrelated peptide was selectively recruited in response to tamoxifen
whereas recruitment of a fifth peptide was promoted by any of
E2, tamoxifen, raloxifene, or the antiestrogen
ICI 182,780. GRIP1 was selectively recruited by
E2 or tamoxifen incubation. Simultaneous
incubation with an excess of ICI 182,780 blocked recruitment of GRIP1,
each LXXLL peptide, and the tamoxifen-specific peptide.
Recruitment of the peptides and GRIP1 to the intranuclear
location of ER in living cells mimicked their previously reported
ligand dependence and efficacy of ER
interaction. In addition to
confirming in living cells the ligand specificities of these
interactions, the intranuclear recruitment assay uniquely enabled us to
determine that each peptide and GRIP1 varied in the timing of
recruitment after ligand addition. Surprisingly, temporal studies of
dissociation showed that preformed complexes involving LXXLL
interactions with ER uniquely were not disrupted even after 4 h of
incubation with a 1,000-fold molar excess of ICI 182,780. Thus, we
report a novel procedure for investigating the ligand-specific
recruitment of labeled factors or peptides to nuclear receptors in
living cells. This allowed us to determine the unique timing of
different ligand-specific complexes formed with ER and to discover that
LXXLL-dependent interactions alter the availability of the receptor for
subsequent ligand binding in living cells.
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RESULTS |
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Oligonucleotides encoding peptide sequences representative of each of
the class I, II, and III peptides were fused in frame to the carboxy
terminus of GFP (see Fig. 1) and
expressed in mouse GHFT15 cells. The intracellular locations of GFP
and each GFP- labeled LXXLL peptide were identified by fluorescence
microscopy after their expression. GFP (not shown) and the three
GFP-LXXLL fusions were distributed throughout the cytoplasm and nucleus
(Fig. 2
, AC, left panels).
The proportion of GFP-LXXLL fluorescence in the nucleus and cytoplasm
varied from evenly distributed between nucleus and cytoplasm to some
nuclear preference. The variation in nuclear/cytoplasmic partitioning
was independent of expression level and was globally similar for GFP
and each GFP-LXXLL fusion.
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In cells grown in E2-free media, the dispersed
cellular distribution of each GFP-LXXLL fusion was unchanged upon
coexpression of ER-BFP (Fig. 2
, AC, no ligand). In contrast,
incubation of cells coexpressing ER
-BFP and any of the GFP-LXXLL
fusions with 10-8 M
E2 caused the GFP-LXXLL to assume the reticular
pattern characteristic of ER
-BFP in the nucleus (Fig. 2
, AC,
estradiol). Complete overlap of GFP-LXXLL with ER
-BFP in the
identical subnuclear compartment after E2
addition is indicated by the exclusively cyan-colored image obtained
when the separate blue and green images are merged (Fig. 2
, AC,
merge). This was observed in cells that express GFP-LXXLL in low
stoichiometry relative to ER
-BFP. In cells expressing more GFP-LXXLL
than ER
-BFP, colocalization of GFP-LXXLL and ER
-BFP was observed
as a concentration of green fluorescence at the site of blue
fluorescence (not shown). When ER
-BFP was not coexpressed, there was
no intranuclear redistribution of GFP-LXXLL in the presence of
E2 (Fig. 2
, AC, left panels) or any
other ER ligand (not shown). Similarly, GFP itself did not redistribute
to ER
-BFP upon incubation with E2 or any other
ER ligand (not shown). Thus, relocalization of GFP-LXXLL was
specifically dependent upon the LXXLL peptide, coexpression of
ER
-BFP, and addition of E2.
Intracellular Relocalization of Different LXXLLs to ER Parallels
Their Interaction Profiles
To further characterize the ligand dependence of GFP-LXXLL
colocalization with ER-BFP, we determined the
E2-induced relocalization kinetics of each of the
class I, class II, and class III GFP-LXXLLs to ER
. Each GFP-LXXLL
was coexpressed with ER
-BFP in cells grown in
E2-free media. One day after transfection,
parallel coverslips were incubated with no hormone, or with
10-10, 10-9,
10-8 or 10-7
M E2 for 24 h. We then
determined the fraction of cells in which GFP-LXXLL colocalized with
ER
-BFP for each E2 concentration.
By fluorescence microscopy, we scanned the coverglass using blue
fluorescence excitation and emission filters to first identify cells
expressing ER-BFP. We then rapidly switched to the green filter set
to determine whether the cell contained visible GFP-linked target. If
the GFP-linked target was also present, it was then scored as
colocalized if there was any concentration of green fluorescence at the
site of the ER. By scoring GFP-peptide or cofactor-expressing cells
only after determining which cells obviously contained ER
-BFP, we
avoided the bias in which a bright, reticular GFP fluorescence pattern
would inflate our detection of colocalized cells containing otherwise
undetectable levels of the generally less fluorescent ER
-BFP. By
setting the colocalization criterion as "any" colocalization, we
also removed any biases that would have resulted if we had attempted to
subjectively score cells for the variable extent of colocalization.
Since the proportion of non-colocalized cells decreases with increasing
colocalization, the recruitment of specific factors or peptides is
measured as the change in the proportion of cells that responded after
the addition of different concentrations of E2.
The validity of this approach was confirmed by the high reproducibility
of the data obtained from multiple independent experiments, which are
plotted in Fig. 3A
as the mean ±
SD in the percent of cells showing colocalization at each
ligand concentration. Half-maximal binding to ER
-BFP with each class
of GFP-LXXLL was reached at 37 x 10-10
M E2, approximately the concentration
of E2 needed for activation of ER-regulated
promoters in cell transfection studies (37).
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Delayed Temporal Kinetics of Class II LXXLL Recruitment to
ER
Sequence-specific differences in colocalization of the three
LXXLL peptides with ER were also evident in time course studies. We
conducted single cell recordings of the
E2-induced intracellular recruitment of LXXLL to
ER
. First, we identified cells, grown in the absence of
E2, that expressed both the class I GFP-LXXLL and
ER
fused to red fluorescent protein (RFP). The ER
-RFP fusion
protein was functionally active in the ligand-induced activation of
estrogen-responsive promoters (data not shown). ER
-RFP and GFP-LXXLL
digital images of the same cell were captured using red and green
fluorescent filter sets before the addition of ligand and at 1-min
intervals after the addition of 10-6
M E2. An example of one cell before
E2 addition and 20 min after
E2 addition is provided in Fig. 4A
. Appropriate controls, using cells
expressing only ER
-RFP or GFP-LXXLL of intensities equivalent to
those in the coexpressing cells, showed that there was no fluorescence
bleedthrough between the red and green images (not shown).
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The cell-to-cell variability in recruitment over short time periods
required that we score large numbers of cells at each time point to
obtain data in which we have confidence. This could not be accomplished
by recording individual cells for prolonged time periods. However, our
prior experience demonstrated that we could readily and reproducibly
score by visual inspection 50150 cells within a 10-min window (Fig. 3). We coexpressed each GFP-LXXLL together with ER
-BFP in cells
grown in the absence of ligand and then scored those cells, exactly as
for Fig. 3
, for colocalization in 10-min windows between 1525 min,
4050 min, and 8595 min at 24 h after the addition of
10-8 M E2
(Fig. 4C
).
Colocalization of the class I and class III GFP-LXXLLs with ER-BFP
was detected within 20 min after the addition of
10-8 M E2 and
increased thereafter. This represented the time required for
E2 to enter the cell, bind to ER
-BFP, and have
detectable amounts of freely diffusing GFP-LXXLL concentrate at the
intranuclear location of the liganded ER
-BFP. Whereas the
colocalization of GFP-class I LXXLL and GFP-class III LXXLL fusions
with ER
-BFP showed identical time courses and reached similar levels
in response to saturating levels of E2, the
redistribution of the class II peptide to ER
-BFP was much less rapid
(Fig. 4C
). The delayed time course of class II LXXLL intracellular
colocalization with ER
-BFP, which would not have been detected in
other assays, demonstrated that ER association with different LXXLL
sequences follows different temporal kinetics. An intriguing
possibility is that the different LXXLL temporal kinetics underlies a
previously proposed (38) sequential recruitment of cofactors to ER
after E2 addition.
Ligand-Specific Differences in Class I, II, and III LXXLL
Colocalization with ER
Incubation of cells coexpressing GFP-LXXLL and ER-BFP overnight
with 10-6 M tamoxifen resulted in a
slight concentration of green fluorescence emitted from the class I
GFP-LXXLL over the reticular pattern of ER
-BFP fluorescence (Fig. 2A
). This weak colocalization was reproducible and was quantified in
Table 1
as the percentage of cells in
which the indicated GFP-linked peptide showed any visible
colocalization with ER
-BFP in response to the indicated ER ligand.
Tamoxifen promoted class I GFP-LXXLL colocalization with ER
-BFP but
not class III GFP-LXXLL colocalization with ER
-BFP (Table 1
). Thus,
the class I and class III peptides, which behaved identically in
response to E2 (Figs. 3A
and 4C
), differed in
their response to tamoxifen. The class II GFP-LXXLL also did not
appreciably respond to tamoxifen (Table 1
).
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Ligand-Dependent Recruitment of the Full-Length ER-Interacting
Cofactor GRIP1 to the Intranuclear Compartment Containing ER
The ligand-specific and temporally distinct associations of
different ER-interacting peptides with ER suggested that a similar
approach could be employed to demonstrate the ligand specificity and
pattern of recruitment of full-length ER-interacting cofactors to ER
in vivo. Indeed, one ER coactivator SRC-1a fused to GFP
recently was shown to be recruited to the intranuclear location of
ER
fused to the cyan fluorescent protein upon
E2, but not tamoxifen or ICI 182,780, incubation
(27). We determined that the related ER-interacting cofactor GRIP1,
fused to GFP, was also recruited upon ligand addition to the
intracellular subcompartment containing ER
-BFP (Fig. 6
) and then detailed the ligand
specificity and kinetics of that recruitment (Fig. 7
).
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Temporal Variation in LXXLL Requirements for
E2 and Tamoxifen Recruitment of GRIP1 to
ER
E2 and tamoxifen-specific recruitment of
GFP-GRIP1 to ER-BFP was distinguished by their different time
courses and dependencies upon the LXXLL motifs in GRIP1 (Fig. 7
).
E2 recruitment was characterized by a rapid
LXXLL-dependent phase followed by a slow LXXLL-independent phase. Rapid
recruitment was detected as an initial plateau of 3040% of the cells
displaying colocalization of GFP-GRIP1 with ER
-BFP within 20 min
after E2 addition (Fig. 7A
, GRIP1-wt). This early
phase plateau was blocked (Fig. 7A
, GRIP1-
LXXLL) by mutation to
LXXAA of the two LXXLL motifs of GRIP1 required for interaction with
the ER ligand binding domain (5). A more gradual increase in
GRIP1/ER
colocalization that followed 1.5 h after
E2 addition was not abrogated by the LXXAA
mutations. These complex temporal kinetics and LXXLL dependencies
suggest time-dependent variations in the available types of
E2/ER/GRIP1 associations with some lagging
associations possibly dependent upon interim interactions and/or
enzymatic processes.
The weaker tamoxifen-dependent recruitment of GFP-GRIP1 to ER-BFP
also displayed a complex time course (Fig. 7B
) that was mechanistically
distinct from that induced by E2.
Tamoxifen-induced colocalization of GFP-GRIP1 and ER
-BFP was
statistically significant at 8 and 24 h after tamoxifen addition.
Before that, a slow gradual recruitment of GRIP1 was not statistically
significant. A precipitous drop in colocalization at 4 h after
tamoxifen addition may indicate some tendency toward a temporally
biphasic response, but this interpretation is questionable given that
the change in colocalization at the early time points was not
statistically significant. The statistically significant
tamoxifen-dependent colocalization at 8 and 24 h was disrupted by
the mutation of the GRIP1 LXXLL motifs to LXXAA (Fig. 7B
). Because it
is unlikely that the LXXLL motifs of GRIP1 interact directly with the
tamoxifen-bound ER, the LXXLL dependence of the slow,
tamoxifen-dependent GRIP1 recruitment may reflect a more indirect
recruitment of GRIP1 to the tamoxifen-bound ER or a dependence on
additional motifs present in GRIP1.
E2/ER/LXXLL Complexes Become Resistant
to Subsequent Challenge with Antiestrogen
Previous reports showed that ligand-induced binding of LXXLL to ER
in vitro caused an alteration in the rate by which the
ligand dissociates from the ER (39). To determine whether LXXLL
interaction with ER in vivo might similarly slow ligand
access to ER, we examined whether ER/cofactor or ER
/peptide
complex formation altered access to the antiestrogen ICI 182,780. ICI
182,780 did not promote ER
-BFP colocalization with GFP-GRIP1,
GFP-
/ßV, or any of the three GFP-LXXLL fusions (Table 1
).
Consistent with its role as an antiestrogen, simultaneous addition of
10-6 M ICI 182,780 with
10-9 M
E2 abrogated colocalization of ER
-BFP with
each of the three GFP-LXXLL fusions and GFP-GRIP1 (Table 1
, ICI
inhibition) whereas colocalization of the ICI 182,780-responsive,
II
peptide was unaffected. ICI 182,780 (10-6
M) also blocked colocalization of ER
-BFP and
GFP-
/ßV in response to 10-7
M tamoxifen (Table 1
).
Having showed that 10-6 M ICI
182,780 effectively blocked recruitment of GRIP1 and the /ßV,
LXXLL-I, LXXLL-II and LXXLL-III peptides to ER
, we next determined
the temporal kinetics of complex dissociation. To do so, we challenged
preformed complexes with an excess of ICI 182,780 to block the
reformation of transiently dissociated complexes. Initially, the
proportion of cells containing ER
colocalized with GFP-
/ßV
after 24 h incubation with 10-7
M tamoxifen was determined, 10-6
M ICI 182,780 was added to the media, and the cells
subsequently were scored for any colocalization at the indicated time
points after ICI 182,780 addition (Fig. 8A
). Complete disruption of
colocalization of GFP-
/ßV and ER
-BFP was observed within 2
h of ICI 182,780 addition. This established that 2 h was
sufficient time for ICI 182,780 to enter the cells, disrupt all
preformed complexes, and completely release all GFP-
/ßV
concentrated at the ER
subcompartment.
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DISCUSSION |
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In addition to confirming in living cells the ligand specificities of
these previously known interactions, the analysis of intranuclear
recruitment allowed us to follow complex formation with time after
ligand addition. Temporal variations in both recruitment and
dissociation were observed (Figs. 4, 5
, 7
, and 8
). Temporally delayed
recruitments may represent a secondary association of some complexes
via intermediary factors that are initially recruited in response to
ligand binding. Such an indirect interaction may be responsible for the
delayed (Fig. 7
) interaction of ER
with GRIP deleted of the two
LXXLL motifs previously described (5) to be necessary for direct GRIP1
interaction with ER
. Alternatively, GRIP1 is known to interact with
other regions of ER
(11), and the temporal delay of intranuclear
recruitment of GRIP1
LXXLL may result from time-dependent ER
interactions or modifications that may be required for the proper
folding of these alternative GRIP1 interaction sites.
The molecular basis for the delayed recruitment to ER of the
isolated class II LXXLL (Fig. 4C
) similarly remains to be defined.
However, the direct interaction of the class II peptide with AF-2 in
ER
is weak compared with the class I and class III LXXLL
interactions (12), and the delayed intracellular recruitment may arise
from class II LXXLL E2-induced associations with
other regions of ER
, other ER-interacting factors, or even with
ER
covalently modified by cofactors recruited to the
E2-bound ER. Although the mechanisms for these
temporal variations in intranuclear recruitment remain unknown, the
previously unrecognized variations in the timing and sequence of
complexes recruited to ER after ligand addition are likely to be a key
determinant of ligand response and adaptation and are uniquely detected
by the intranuclear recruitment assay.
Challenge of the preformed complexes with competitive antagonists
demonstrated that each peptide or cofactor was displaced from the
complexes with unique dissociation kinetics (Fig. 8). Complexes of
ER
with LXXLL-containing peptides or cofactors were considerably
more resistant to disruption by ICI 182,780 or raloxifene than
complexes that did not involve LXXLL interactions, demonstrating that
specific ligand-dependent interactions uniquely changed the nature of
the complexes. The LXXLL-dependent reduction in the dissociation
kinetics of the preformed complexes may be a consequence of the
decreased off rate of ligand upon LXXLL binding to ER as recently
reported in vitro (39). Alternatively, translocation of the
LXXLL-containing peptide or GRIP1 from ER to another protein that
resides in the same intranuclear position as ER could explain why the
LXXLL-containing peptides and GRIP1 remain localized for prolonged
periods of time after subsequent antagonist challenge.
The ligand specificity and temporal characteristics of intranuclear
recruitment of ER with each ligand indicated that, in the cellular
environment, distinct conformations of ER are formed in response to
E2 and each SERM (7, 10). Recruitment was
measured as a function of the percentage of cells responding to each
ligand. The underlying basis for the cell-to-cell variability in
recruitment remains to be described but may be responsible for the
previous observation that dose-dependent transcriptional activation by
a nuclear receptor ligand arises through an increase in the proportion
of cells responding to the ligand rather than an equivalent,
incremental increase in all cells (40).
The different abilities of E2, tamoxifen, and
raloxifene to promote ER colocalization of the
/ßV peptide, the
class I LXXLL peptide, and GRIP1 provided dramatic evidence for the
differing cellular and molecular properties of these clinically useful
ligands. The E2- and tamoxifen-induced
recruitment of GRIP1 to the intranuclear location of ER
(Figs. 6
and 7
) also contrasted with recruitment of related cofactor SRC-1a, which
responded only to E2, and not tamoxifen (27).
This difference may be attributable to cell type or other experimental
differences between laboratories, or to different ligand specificities
for related cofactors in the context of the living cell. Nevertheless,
the detection of these differences in living cells may prove useful in
dissecting the differing clinical properties of
E2, tamoxifen, and raloxifene in different
tissues (16, 18, 41, 42). The ability to quantify these changes on a
pixel level (Fig. 4B
) provide a first indicator that automated
equipment can be developed for the high throughput measurement of
ligand-specific effects on intranuclear recruitment and dissociation in
living cells.
Recently, it was shown that the reticular intranuclear distribution of
estrogen, SERM, and antiestrogen-bound ER paralleled the tight
binding of ER
to the nuclear matrix and that one ER-interacting
factor, SRC-1a, was corecruited to the nuclear matrix via the
ligand-bound ER (27). The results presented here suggest that other
ER-interacting complexes may be similarly recruited to the nuclear
matrix compartment upon ER binding to different ligands and that each
ligand promotes the recruitment of specific nuclear
receptor-interacting peptides and proteins (Table 1
) with unique
temporal kinetics (Figs. 4
, 5
, and 7
). The ligand-regulated association
of ER and ER-interacting complexes with the nuclear matrix is
intriguing given the historical association of transcription markers
and enhancer/promoter sequences with the nuclear matrix (43, 44). The
nuclear matrix may aid the organization of transcriptionally competent
chromosomal domains (45) but a decisive correlation of nuclear matrix
association with transcriptional activation or repression remains to be
established (46).
Thus, we demonstrated that recruitment of ER- interacting factors
to the intranuclear position of ER is differentially regulated by
the nature of the interacting sequence and the type of ligand. The
complex temporal kinetics and ligand specificities of the association
of ER
and its cofactors illustrated a variety of possible responses
of ER
to ligand addition for which the intranuclear colocalization
assay provided a direct read-out in vivo. Other methods
currently used to detect ER-peptide or cofactor interactions rely on
various in vitro binding assays or on two-hybrid assays in
cells. The advantage of the intranuclear colocalization assay is that
it is an in vivo assay in which direct and indirect
interactions of ER with specific peptide of cofactor targets are
readily measured in real time. Therefore, the intranuclear
colocalization assay allows the intracellular actions of each ligand to
be dissected in unprecedented detail. The availability of many more
ER-interacting peptides and cofactors (6, 7, 12, 13, 14, 20, 47) will
permit the detection of an even more expanded series of ER activities
and may also facilitate the identification of novel ligands that induce
specific subsets of cofactor interactions with ER or other nuclear
receptors. Such novel ligands could be used to probe for the specific
molecular events involved in nuclear receptor regulation of different
genes and may even provide a rapid means for the identification of
compounds with improved specificity for hormone replacement
therapies.
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MATERIALS AND METHODS |
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Transfection
GHFT15 cells were grown in a 1:1 mixture of phenol red-free
Hams F12-DMEM containing estrogen-free 10% newborn calf serum. The
cells were harvested and transfected by electroporation as described
previously (35, 48) with 10 µg of the cytomegalovirus (CMV)-ER-BFP
or CMV-ER
-RFP expression vector, 10 µg of pEGFP-GRIP1 or
pEGFP-GRIP1-
LXXLL, and 5 µg of pEGFP-
II or 3 µg of the
pTRE-GFP-LXXLL or pTRE-GFP-
/ßV expression vectors. pUHG171 (1.2
µg), which expresses the tetracycline repressor/VP16 activator (49)
used to regulate expression of the pTRE plasmid, was cotransfected with
the pTRE-GFP-LXXLL or pTRE-GFP-
/ßV expression vectors. The
transfected cells were plated onto coverslips and grown in
estrogen-free media. Doxycycline (5 µg/ml) was added to the media to
induce the Tet-On promoter except in Fig. 3B
in which concentrations of
doxycycline were varied from 0 to 15 µg/ml. One day after
transfection, ER ligands were added at the indicated concentrations and
imaged 24 h later (
Figs. 26
). For the E2
time course experiments (Figs. 4C
and 7
), 10- 8
M E2 was added at the indicated time
before imaging on the second day after transfection. For the ICI
182,780 antagonism time courses, cells were treated with
10-7 M tamoxifen (Fig. 8A
) or
10-9 M E2
(Fig. 8
, B and C) for 24 h followed by addition of
10-6 M ICI 182,780 at the indicated
times before imaging.
Microscopy and Image Analysis
After addition of ligand or ethanol control vehicle,
fluorescence images from the transfected cells were acquired with a
Axioplan microscope equipped with a 63x-oil immersion objective lens
(Carl Zeiss, Thornwood, NY). Single-cell recordings of
cells grown in chamber slides were obtained on an IX-70 inverted
microscope (Olympus Corp., Lake Success, NY) and analyzed
with Metamorph (Universal Imaging Corp., West Chester, PA)
colocalization software. Dual color imaging using Hoechst and
fluorescein isothiocyanate filter sets or GFP and rhodamine filter sets
(Chroma Technology Corp., Brattleboro, VT) selectively distinguished
blue from green fluorescence and green from red fluorescence,
respectively. Appropriate controls in which ER-BFP, ER
-RFP, or
each GFP-peptide or GFP-GRIP were expressed individually ensured a lack
of fluorescence bleedthrough between the channels. Grayscale images of
the cells were obtained using a Xillix microscope (Carl Zeiss) or Hamamatsu ORCA microscope (Olympus Corp.)
cooled CCD cameras. The digital images were background-subtracted and
then converted to red-green-blue (RGB) images by assigning the GFP
signal to the green channel, BFP signals to the blue channel, and RFP
signals to the red channel of RGB digital images. Integration times and
image processing were kept constant within each set of experiments.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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This work was supported by American Cancer Society Grant RPG-94028-TBE, NIH Grant DK-54345, and the UCSF Academic Senate Committee on Research to F.S., by NIH Grant DK-48807 to D.P.M., and by US Army Grant DAMD1799-19173 to C.-Y.C.
1 J.D.B. has propietary interests in, and serves as a consultant and
Deputy Director to, Karo Bio AB, which has commercial interests in this
area of research.
Received for publication June 8, 2000. Revision received August 30, 2000. Accepted for publication September 11, 2000.
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REFERENCES |
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