Department of Chemistry (A.C.G., K.E.C., J.A.K.) University of
Illinois Urbana, Illinois 61801
Department of Molecular
and Integrative Physiology (B.S.K., P.G.V.M.) University of
Illinois and University of Illinois College of Medicine Urbana,
Illinois 61801
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ABSTRACT |
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INTRODUCTION |
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A variety of coactivator proteins are now known to interact with the ER in a ligand-dependent fashion and to play important roles in modulating the magnitude of transcriptional activity of the ER (5, 6, 7, 8, 9). For example, the steroid receptor coactivator-1 (SRC-1), a member of the p160 class of nuclear hormone receptor coactivators, binds to the ER when it is liganded with agonists, but not when it is unoccupied or liganded with antagonists (10). Mutational mapping studies and yeast two-hybrid and glutathione-S-transferase (GST) pull-down interaction experiments have pointed to a specific LXXLL sequence motif, termed a nuclear receptor interaction box (NR box), as an element that is important in mediating the interaction of coactivators such as SRC-1 with ER (11, 12, 13, 14, 15, 16). SRC-1 contains four NR boxes, three being located within a reasonably compact region near the middle the protein (termed the nuclear receptor domain, SRC1-NRD), and the fourth at the extreme C terminus (12, 13). Recent mutational mapping experiments have indicated that the second of these four boxes has the strongest interaction with ER (16).
The first x-ray crystal structures of the ER complexed with the agonist estradiol and the mixed antagonist raloxifene were already suggestive of the manner in which the NR box sequences in steroid receptor coactivators would interact with ER and how this might be regulated by the ligand (17). In the agonist complex, helix 12 is oriented antiparallel to helix 11, where it forms a portion of the ligand binding pocket. By contrast, in the complex with raloxifene, helix 12, apparently displaced by the extended side chain of the ligand, moves outward, rotates, and packs into a hydrophobic groove between helix 3 and helices 4 and 5. This hydrophobic grooveunoccupied and available in the agonist complex, but occupied in the antagonist complexseemed to be a natural site for the interaction of the NR box peptides of SRC-1 and related coactivators.
This mode of NR box peptide binding to agonist-liganded nuclear hormone
receptors has recently been illuminated by mutational mapping studies
(14, 15, 16, 18) and especially by recent x-ray crystal structures: an ER
complex with the agonist diethylstilbestrol cocrystallized with the
NR-2 box peptide of GRIP-1 (a mouse ortholog of SRC-1) positioned
precisely in this groove (19). The same peptide also crystallized in
this groove in an agonist complex of the thyroid hormone receptor (14)
and an 88-amino acid fragment of SRC-1 and was shown to bind to the
peroxisome proliferator-activated receptor- (PPAR
) homodimer,
with the NR-1 and NR-2 boxes from one peptide bound in the two grooves
of a protein dimer (19).
The fact that the binding of agonists and antagonists to the ER engenders distinct receptor conformations that promote and prevent, respectively, the interaction of NR box peptides raises the interesting question as to whether there is a reciprocal effect of the binding of these NR box peptides on ligand binding. One might imagine that the binding of the NR box peptide with the preformed ER-agonist complex might stabilize ligand binding in a manner that would reduce the rate of ligand dissociation. Conversely, the lack of NR box peptide binding to the ER-antagonist complex might obviate such a ligand dissociation stabilization effect. If this were, in fact, the case, then the differing levels of coactivators in different estrogen target tissues might modulate the relative potency and/or duration of action of compounds having differing agonist and antagonist characters; this could be an important determinant of the tissue-selective pharmacology of estrogens.
In this report, we have examined the effect of NR box peptides and a fragment of SRC-1 containing three NR box sequences on the rate of dissociation of agonist and antagonist ligands from the ER. These investigations have been greatly assisted by the development of a convenient fluorescence assay for measuring ligand dissociation rates from the ER. Our findings reveal that NR box peptides markedly stabilize ER agonist complexes against ligand dissociation, while having only marginal effects on the dissociation rate of antagonist ligands. We also show that the potency of estrogen in transcriptional activation by ER in cells can be enhanced by increasing the levels of a coactivator.
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RESULTS |
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The Dissociation Rate of Various Ligands from the ER Can Be
Determined Conveniently Using Fluorescence Energy Resonance Transfer
(FRET) with a Fluorescent Ligand, the THC-Ketone
The dissociation rate of ligands from ER can be followed
conveniently with the fluorescent ligand, THC-ketone. The dissociation
of THC-ketone itself from ER can be monitored directly, as ER-bound
THC-ketone emits both at longer wavelength and with a 9-fold higher
intensity than unbound THC-ketone. However, to further enhance the
sensitivity of this assay, we used FRET. Because the ER LBD contains
three tryptophan residues that are situated quite close to the
ligand-binding pocket, ER-bound THC-ketone can be selectively excited
by illumination at 285 nm, the absorbance maximum of tryptophan.
Efficient resonance energy transfer then occurs between the tryptophan
(emission maximum, 340 nm) and the nearby bound THC-ketone (broad
excitation maximum at 380390 nm). By using FRET, we can increase the
fluorescence intensity from ER-bound THC-ketone by nearly 20-fold over
that from free THC-ketone excited at 285 nm. The dissociation of
THC-ketone from ER (effected by exchange with a nonfluorescent ligand)
as well as the reverse dissociation of a nonfluorescent ligand
(effected by exchange with the THC-ketone) can be measured directly by
FRET (see Fig. 1, equations 13, and Fig. 2
). The ER is completely stable under the
temperature and illumination conditions of the experiment.
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The dissociation rate of other ligands from ER can be determined in a
reversed assay, where the THC-ketone is used as the exchanging ligand.
Because higher concentrations of THC-ketone are needed in this
protocol, ligand and receptor concentrations were carefully optimized
to provide a sufficient differential in fluorescence. In this assay,
the dissociation of the ligand is measured indirectly by following one
of two changes in fluorescence: 1) the increase in fluorescence that
results from the association of THC-ketone with the ER after the other
ligand has dissociated (Fig. 1, equation 2), or 2) the quenching of
intrinsic tryptophan fluorescence that occurs when the THC-ketone binds
to ER (Fig. 1
, equation 3). Both methods yielded nearly identical
dissociation rates.
Examples of these ligand dissociation assays with estradiol are shown
in Fig. 2B. The resulting curves show either a rising fluorescence
intensity (in the case of after THC-ketone fluorescence) or a
decreasing fluorescence intensity (in the case of after tryptophan
fluorescence). Both transients fit well to single-component exponential
equations, from which a dissociation rate constant for estradiol can by
obtained (Fig. 2B
, inset); at 23 C, the rate constant
obtained by observing tryptophan fluorescence is 0.192
min-1 (t1/2 = 3.61 min), and by
observing THC-ketone fluorescence (not shown) is essentially identical,
0.210 min-1 (t1/2 = 3.30 min). In
practice, it was easier to obtain dissociation rate data using the
second protocol (equation 3), monitoring the quenching of intrinsic
tryptophan fluorescence by the THC-ketone.
To verify that this assay is giving an appropriate measure of ligand
dissociation rates from ER, we compared the rate of dissociation of the
THC-ketone from the wild-type ER-LBD with that of the corresponding
constitutively active Y537S mutant. From radiometric assays, this
mutant is known to have a ca. 3-fold slower rate of
E2 dissociation (24). Figure 2C
, which displays the linear
semilog plots fitted to the fluorescence transients from THC-ketone
dissociation from the two ERs, shows that the dissociation rate of the
THC-ketone from Y537S ER
is ca. 5-fold slower than from
wild-type ER
. Thus, the fluorometric assay is able to detect the
change in ligand dissociation rate that results from this mutation.
One limitation that we have encountered in these THC-ketone fluorescence intensity ligand dissociation assays is that it is difficult to measure the dissociation rate of certain ligands that are either fluorescent themselves or are very strong quenchers of fluorescence. This problem was encountered with trans-hydroxytamoxifen, which is known to undergo photocyclization-oxidation to a fluorescent stilbene (26), and with raloxifene, which we found to be a very efficient fluorescence quencher (data not shown). Also, we have found it impractical to measure association rate constants by this protocol, because at the concentrations of receptor and ligand that are needed to produce good fluorescent signals, the association rate is too fast to be measured using conventional instrumentation. This rapid association rate of the THC-ketone, however, is a benefit, because it is a required element for measuring the dissociation rate of other ligands by the reversed assay protocol.
NR-2 Box Peptides and the SRC1-NRD Markedly Retard the Rate of
Dissociation of Agonist Ligands from the ER
The effect of NR peptides and the SRC1-NRD on the dissociation
rate of THC-ketone from ER is illustrated in Fig. 3
, in which linear semilog plots fitted
to the dissociation transients of THC-ketone from ER are shown. It is
clear from the changing slopes that the ligand dissociation rate is
markedly decreased by the addition of high concentrations of the NR-2
peptide; in fact, in this experiment, the THC-ketone dissociation
half-life of 3.45 min in the absence of the NR-2 peptide increases
17-fold to 60.3 min in the presence of 10 µM of NR-2
peptide (Fig. 3A
). As one would anticipate, the NR-2A peptide, which
has an alanine in place of the second leucine (cf. Fig. 1
),
has, at the same concentration, a lesser effect on the dissociation
rate of the THC-ketone than does the NR-2 peptide, increasing the
dissociation half-time only 2-fold, to 6.48 min (Fig. 3A
). It is of
note that the SRC1-NRD, even at a lower concentration (1
µM), has a greater effect on the dissociation rate of the
THC-ketone than the NR-2 peptide, slowing it to a half-life of 93.0
min.
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Effectiveness of Coactivator Peptides in Retarding Agonist Ligand
Dissociation Rate Depends on Length and Sequence
The relative potency of the three peptides in retarding the
dissociation rate of various ligands from ER is illustrated in Fig. 4, where the dissociation rate constants
are plotted as a function of peptide concentration. As anticipated from
the initial results illustrated in Fig. 3
, the NR-2 peptide is more
potent in reducing the THC-ketone dissociation rate than is the NR-2A
peptide (Fig. 4A
); the midpoint of the dissociation rate constant plot
falls at 0.9 µM for NR-2, but at 17 µM for
NR-2A. These numbers correspond favorably to estimates of the binding
of NR-2 peptides to the thyroid hormone receptor (see
Discussion).
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A plot of the percent of the maximum change in dissociation rate as a
function of the concentration ratio of SRC1-NRD to ER (Fig. 4B) has an
x-intercept close to 0.5. The data curve of dissociation rate constants
more closely parallels the line expected for a 1:2 stoichiometry for
SRC-1 to ER binding (steep line) than for a 1:1 binding
stoichiometry (shallow line). This suggests that a single
SRC1-NRD peptide is binding to two ER LBDs, presumably to both
coactivator-binding sites in the ER dimer (see Discussion).
Because the binding of divalent ligands is expected to show an affinity
amplification (28, 29, 30, 31, 32), it is not surprising that the affinity of the
multivalent SRC1-NRD to the ER is much higher than that of the
monovalent NR-2 peptide.
The effectiveness of the NR-2 peptide also appears to show some
dependence on the nature of the agonist ligand. As shown in Fig. 4C, this peptide was most potent in affecting the dissociation rate of the
ER
complex with E2, and was progressively less potent
with the DES and THC complexes. While these variations in NR box
peptide potency are small compared with that with ER
complexes with
antagonist ligands, they suggest that changes in agonist ligand
structure can affect the strength of coactivator interaction and that
coactivator interaction may differentially affect the stability of ER
complexes with different agonists.
NR-2 Box Peptides from the Coactivator SRC-1 and the SRC-1 NRD
Sequence Have Only a Modest Effect on the Dissociation Rate of
Antagonists
The effect of the NR-2 peptide on the dissociation rate of the
antiestrogen ICI 182,780 from ER is illustrated in Fig. 5
. At 10 µM, a
concentration that markedly retards the dissociation rate of
THC-ketone, E2 and DES (cf. Fig. 3
, AC), the
NR-2 peptide has only a modest retarding effect on the dissociation
rate of the antiestrogen ICI 182,780 (Fig. 5
). In the absence of the
NR-2 peptide, ICI 182,780 dissociates from the ER
-LBD with a
half-life of 2.69 min, but in the presence of 10 µM NR-2
peptide, this is extended only 2.5-fold, to 6.84 min.
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Effect of SRC Peptides and SRC Fragment on the Dissociation of the
THC-Ketone from ERß
THC-ketone was found to dissociate from ERß-LBD 5 times slower
(t1/2 = 17.6 min) than from the ER-LBD in the
absence of any coactivator peptide. As shown in Fig. 6
, the potencies of the NR-2 peptide
and SRC1-NRD in altering ligand dissociation rate from ERß-LBD were
quite similar to that measured for ER
. The midpoints of the
dissociation rate constant plot fall at 0.9 µM for NR-2
and at 25 nM for the SRC1-NRD, which are nearly identical
to those measured for ER
-LBD. In the presence of 10 µM
NR-2 peptide, the THC-ketone dissociation rate is extended 7-fold to
127 min. With 1 µM SRC1-NRD, the dissociation rate is
extended 12-fold to 211 min. The NR-2A peptide, however, had a much
different effect on the THC-ketone dissociation rate from ERß-LBD
than it did with ER
-LBD. In fact, there was no appreciable
difference in the midpoint of dissociation rate constant plot for NR2A
(at 0.9 µM) from that of NR-2. NR-2A peptide at 10
µM was found to extend the dissociation rate of
THC-ketone from ERß 5-fold to 91.0 min (see
Discussion).
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DISCUSSION |
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There is a Mutually Supportive Reciprocity in the Binding of
Agonist Ligands and NR Box Peptides to the ER
The binding of coactivators to the ER is known to be ligand
dependent, with agonists but not antagonists recruiting these proteins
(5, 6, 7, 8, 9). Recent crystallographic studies in three receptor systems now
provide a rather refined picture of how portions of these coactivator
proteins interact with receptors and how this interaction is modulated
by alternative ligand-induced shifts in receptor conformation (14, 19, 33). None of these studies, however, addressed the issue of a
reciprocal effect that coactivator binding might have on the kinetics
of ligand dissociation from the receptor. We have now shown that the
binding of peptides derived from SRC-1 does markedly affect the
kinetics of agonist ligand binding to ER, but has only a minimal effect
on the dissociation rate of antiestrogens.
Our findings are understandable in light of the recent x-ray structures of the ER LBD (19). For example, in the DES complex with the GRIP1 peptide, the binding of the hydrophobic face of the amphipathic NR box coactivator helix into the hydrophobic groove, formed by portions of receptor LBD helices 35, and helix 12, is likely to afford additional stabilization of this complex, making it more difficult for the ligand to egress. By contrast, in ER-antagonist complexes (17, 19), NR box peptide binding to this hydrophobic groove is blocked by the repositioning of helix-12, so that little retardation of the dissociation of antagonist ligands is expected.
SRC-1 Nuclear Receptor Domain Sequence Binding to the ER-Agonist
Complexes is High Affinity and Has the Stoichiometry of One Peptide per
ER Homodimer
A rough estimate of the binding affinity of coactivator
peptides to agonist-liganded ER can be obtained from the concentration
dependence of the agonist dissociation rate retardation. The
IC50 values for the NR-2 peptide of approximately 1
µM is similar to that which has been estimated by
pulldown experiments of Darimont et al. (14). Two things are
striking about the interaction of the longer SRC1-NRD that embodies all
three of the NR boxes with ER: 1) The longer peptide has a much higher
apparent affinity that cannot be accurately measured under our assay
conditions, but is likely to be in the low nanomolar range, and 2) the
longer peptide appears to bind with the stoichiometry of one SRC1-NRD
per ER dimer. Both of these results suggest that two NR boxes from a
single peptide are interacting with the two coactivator binding
grooves in a single ER homodimer. It is not surprising that the
SRC1-NRD binding to ER shows an amplification of affinity, because of
the bivalent nature of this interaction. This stoichiometry is also
consistent with the recent crystal structure of an agonist-liganded
PPAR homodimer with a somewhat shorter fragment of SRC-1 that
embodies only the NR-1 and NR-2 boxes (33). In this structure, the two
NR boxes from a single SRC-1 strand were found to be bound in the two
grooves of one homodimer.
SRC-1 Peptides Show a Different Pattern of Interaction with ER
and ERß
One unexpected finding was that the NR-2A mutant peptide was as
potent in reducing agonist dissociation rate from ERß as was the NR-2
peptide, whereas the NR-2A peptide was markedly less potent than the
NR-2 peptide on ER. At the time we began this study, no atomic level
details were known about how the NR peptides interacted with ER. Thus,
without guidance we selected the second leucine in the NR-2 box
sequence for change to alanine, hoping to perturb the strength of
coactivator peptide interaction with ER.
From the recent crystal structure of ER-LBDDESGRIP1 peptide
complex (19), we now know that this second leucine interacts with only
the surface of ER; the first and third leucines are the ones that are
more fully enveloped by the receptor. Nevertheless, the leucine to
alanine change in the NR-2 sequence had a very significant effect on
the potency of the peptide interaction with ER, but not with ERß.
Because the residues in ER
that are shown to be in contact with the
GRIP1 peptide in the ER
-LBDDES complex are identical to those in
ERß, this difference in the potency of peptide interaction with the
two ER subtypes must arise from more subtle conformational differences
between the receptor subtypes. Thus, there is clearly multifactorial
control over the details of receptor-coactivator interaction, its
strength being determined not only by the agonist vs.
antagonist nature of the ligand and the particular nature of the
agonist ligand (i.e. E2 vs. DES
vs. THC-ketone), but also by both the receptor subtype and
the NR box sequence.
Fluorescence Methods Facilitate ER-Ligand Binding Kinetic
Studies
Our investigations of coactivator peptide effects on ER ligand
interactions have been facilitated immeasurably by the availability of
the fluorescent ER ligand, THC-ketone, as well as by the intrinsic
tryptophan fluorescence of ER that can be observed in pure ER-LBD
preparations. Both of our ER-LBD constructs (ER and ERß) have
three tryptophans at identical positions; all of them are located quite
close to one another and to the ligand binding pocket, so that FRET
among them and between them and the ligand is efficient (34). We have
used this FRET to enhance the sensitivity of the ligand dissociation
assay. The ability to measure, in real time, the dissociation rate of
ligands expands the types of experiments that can be undertaken,
obviating the need for radiolabeled ligands and cumbersome radiometric
receptor assays.
Up to now, fluorescence methods have not been widely used in the nuclear hormone receptor field. Noy and colleagues (35, 36, 37, 38, 39), however, have done extensive studies using ligand quenching of intrinsic tryptophan fluorescence in the retinoid receptors to study ligand binding and receptor oligomerization. Other workers have used fluorescent-labeled oligonucleotides to study receptor-DNA interactions (40). Recently, a FRET assay for measuring receptor recruitment of coactivators has been reported (41), and a THC-ester closely related to the THC-ketone has been used in an assay for ER ligand binding affinity by fluorescence depolarization (42).
In our experiments, limits to instrument sensitivity and sample background fluorescence require that receptor and fluorescent ligand concentrations be in the range of 50100 nM. At these concentrations, rates of ligand association are too rapid to measure conveniently. Also, the rates of ligand dissociation measured by fluorescence (at these concentrations) are somewhat faster than those we have measured earlier by radiometric methods (at lower concentrations) (24). Recent studies in our laboratory have shown that the dissociation rate of E2 from ER is somewhat concentration dependent, increasing progressively at higher concentrations (our unpublished data). At present, we are unable to explain this phenomenon; perhaps differences in the state of ER-LBD oligomerization will prove to be a factor (43).
Biophysical Studies of ER-Ligand Interaction In Vitro
vs. Estrogen Action in the Whole Cell
The fluorescence assays require relatively large quantities of
purified ER and SRC-1. Thus, the peptides and the purified, recombinant
proteins through which we have demonstrated the marked effects of
coactivator binding on agonist dissociation rate from ER should be
viewed as models for the interactions of full-length ER with
full-length SRC-1. Full-length ER contains a transcriptional activating
function near the N terminus (AF-1) that is known to interact with
portions of SRC-1 outside of the sequence we have used (5), so the
interaction of full-length receptors with full-length coactivators may
well be more complex. Using radiometric assay (data not shown), we
determined that SRC-1 NRD was equally effective at the same
concentration with both purified full-length human ER or unpurified
ER in lamb uterine extracts. The NR-2 peptide at 10 µM
was only about 50% as effective with full-length ER as with the
ER-LBD.
In the intact cell, there are, of course, many other factors that could also come into play in the three-way ligand-receptor-coactivator interaction: unliganded receptors bind to heat shock proteins, and ER-agonist complexes bind to members of other coactivator classes, such as CBP/p300 and pCAF (7, 8), to form larger multiprotein coregulasome assemblies (44, 45, 46). Some of these interactions may be energy driven. At this point we do not know what effect these additional layers of protein surrounding an ER-agonist complex might have on complex stability and ligand dissociation rate.
Regardless of the details, the assembly of these large complexes still relies on agonist-receptor binding as the key initiating signal. Thus, it is significant that we have demonstrated in cells that the elevation of SRC-1 levels sensitizes cells to transcription activation by estradiol by about a factor of 20 and reduces the potency of antiestrogens in reversing this activation, although by a smaller factor. Thus, in cells, as in our simple in vitro system, we can demonstrate an effect of elevating coactivator levels that is consistent with their preferential stabilization of the ER-agonist complex.
The Tissue-Selective Pharmacology of Estrogens May Be Modulated by
Differing Cellular Concentrations of Coactivators
A remarkable feature of the action of estrogens is that their
activity can be very tissue selective, an aspect that is being
exploited in the development of SERMs (4). While this tissue
selectivity might be explained, in part, by differing distribution and
selectivity of the two ER subtypes, ER and ERß (47), it is likely
that other factors are also involved (48).
It is clear from our work that the interaction of coactivator sequence elements with ER can markedly retard the dissociation rate of estrogen agonist ligands, while having little effect on antagonist ligands, and that alterations in coactivator levels in cells can modulate cell sensitivity to estrogens. Thus, the differing potencies and agonist/antagonist balance that is shown by some estrogens and some SERMs in different tissues might be a reflection of variations in the lifetime that various ER-ligand complexes have in these tissues that arise from their differing coactivator levels.
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MATERIALS AND METHODS |
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ER Preparations
The human ER-LBD, comprising amino acids 304554 of human
ER, was expressed from a pET-15b vector in BL21(DE3)pLysS E.
coli and purified as described previously (1). A pET-15b ERß
construct (ERß-LBD) coding for the corresponding amino acids in ERß
256505 (2) was also prepared using the same cloning site, and the
ERß-LBD was expressed and purified in the same fashion as the
ER
-LBD.
Steroid Receptor Coactivator-1 (SRC-1) Preparations
A construct for the expression of the SRC-1-NRD in E.
coli was prepared as a pET-15b vector using standard methods. The
cDNA sequence encoding SRC-1 amino acids 629831 sequence from SRC-1
was amplified using 15 rounds of PCR (Expand High Fidelity PCR System;
Roche Molecular Biochemicals, Indianapolis, IN) with
primers that added an N-terminal NdeI site and a C-terminal
XhoI site for insertion into the pET-15b vector at these
restriction sites. The DNA sequences were confirmed by dideoxy sequence
analysis. The SRC1-NRD was expressed and purified in the same fashion
as was the ER-LBD.
The nuclear receptor interaction box 2 peptide, NR-2, peptide
corresponding to the NR-2 box sequence of SRC-1 (cf. Fig 1)
and the mutant NR-2A peptide were synthesized by the University of
Illinois Biotechnology Center, utilizing
9-fluorenylmethoxycarbonyl solid-phase strategy on a multiple
peptide synthesizer and purified by C18 reverse phase HPLC.
A single C-terminal cysteine residue was included in these peptides to
allow for future chemical derivatization. Peptide quality was
determined by analytical HPLC and mass spectroscopy (University of
Illinois Biotechnology Center).
Fluorescence-Based Dissociation Assay
For all fluorescence assays, purified hER-LBD was diluted into
Tris-glycerol buffer (50 mM Tris, pH 8.0, 10% glycerol).
For assays investigating the dissociation rate of THC from the hER-LBD,
the receptor was diluted to a final concentration of 50 nM,
saturated with 150 nM THC, and an appropriate volume of a
stock NR-2 peptide or SRC1-NRD solution to yield the desired
concentration of peptide or SRC1-NRD. The resultant solution was then
incubated on ice for 1 h before performing the assay, to reach
binding equilibrium. An aliquot (900 µl) of the solution was placed
into a 4 x 10 mm quartz fluorescence cuvette (the 4-mm face was
used for excitation), and the cuvette was placed into the sample
chamber of a Fluorolog II (model IIIc) fluorometer (Spex Industries,
Inc., Edison, NJ) for 3 min before beginning the assay. The sample
chamber was held at a constant 23 C, and the dissociation was initiated
by the addition of 1.35 µl of a 10 mM solution of
E2 in ethanol, to give a final concentration of 15,000
nM E2. Samples were excited every 10 sec for
110 h at 285 nm (1.25-mm slits), and the THC emission was followed at
595 nm (2.5-mm slits). A KV-370 filter was placed between the sample
chamber and the emission monochromator to filter out scattered
excitation light. The data were then fitted to a single exponential
function (y = X - ce-kt) by the sum of least
squares method, using Excel 97 (Microsoft Corp., Inc.,
Redmond, WA) or Prism 3.00 (GraphPad Software, Inc., San
Diego, CA).
For assays investigating the dissociation of ligands other than THC from hER-LBD, samples were prepared in an identical fashion to that described above, except that the receptor concentration was increased to 100 nM and was saturated with 120 nM of the ligand of interest. The sample chamber was again held at a constant 23 C. The dissociation was initiated by the addition of 1.08 µl of a 0.1 mM solution of THC-ketone in ethanol, giving a final concentration of 1200 nM THC-ketone. The sample was excited every 10 sec for 110 h at 285 nm (1.25-mm slits). Either the tryptophan fluorescence emission (at 335 nm and 2.5-mm slits) or the THC fluorescence could be followed as described above. The data were then fitted to a single exponential function (y = X + ce-kt), using Excel 97 or Prism 3.00.
In both sets of experiments, only data sets that could be fitted with
an r2 value 0.95 were used.
Cell Culture and Transfection
The pCMV5 expression vector for the wild-type human ER (WT
ER) and the estrogen response element (ERE)-containing reporter
(ERE)2-TATA-CAT have been previously described (49, 50).
The plasmid pCMVß (CLONTECH Laboratories, Inc., Palo
Alto, CA) was used as a ß-galactosidase internal control for
transfection efficiency. The expression vector, pBK-CMV-SRC-1 (10), was
kindly provided by Dr. Bert OMalley.
Chinese hamster ovary (CHO) cells were maintained in cell culture and transfected by the CaPO4 coprecipitation method exactly as previously described (49, 51). CHO cells were plated at 1.8 x 105 per 60-mm plate and were transfected 48 h later with 2 µg (ERE)2-TATA-CAT, 0.2 µg pCMVß, 5 ng pCMV5-ER expression vector, 5 µg pBK-CMV-SRC-1, and carrier DNA to 8 µg total DNA per plate. Cells were harvested 24 h after hormone treatment and cell extracts were prepared. ß-Galactosidase activity, which was measured to normalize for transfection efficiency, and CAT activity were assayed as described (49).
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FOOTNOTES |
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This research was supported by NIH Grants PHS 1R37 DK-15556 (to J.A.K.) and PHS 5R37 CA-18119 (to B.S.K.) and a postdoctoral fellowship from the Susan G. Komen Foundation (to P.G.V.M.)
Received for publication June 16, 1999. Revision received July 29, 1999. Accepted for publication August 4, 1999.
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REFERENCES |
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