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Gene Regulation, Bone, and Inflammation Research Lilly Research Laboratories Lilly Corporate Center Indianapolis, Indiana 46285
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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) and ERß in
the presence of 17ß-estradiol, diethylstilbestrol, and genestein. A
time-resolved fluorescence-based binding assay was used to determine
the dissociation constants for the 10 NR boxes derived from the three
p160 coactivators for both ER subtypes in the presence of the each of
the agonists. While the affinity of some NR boxes for ER was
independent of the agonist, we identified several NR boxes that had
significantly different affinities for ER depending on which agonist
was bound to the receptor. Therefore, an agonist may specify the
affinity of an NR for various NR boxes and thus regulate the
coactivator selectivity of the receptor. |
INTRODUCTION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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The conformational change in the ligand binding domain (LBD) of the NR
in response to binding of hormone is responsible for recruitment of
coactivator proteins that are required for transcriptional activation
by the receptor (5, 6, 7). Members of the p160-steroid receptor
coactivator (SRC) family of coactivators were originally characterized
by biochemical methods in which proteins were identified that bound the
estrogen receptor (ER) in a hormone-dependent fashion (8). Steroid
receptor coactivator-1 (SRC-1) was cloned using the progesterone
receptor LBD as bait in a yeast two-hybrid screen and was subsequently
shown to be a general coactivator for the NR superfamily (9). Three
proteins have been identified in the p160 coactivator family and
include: 1) SRC-1/NCoA-1, 2) SRC-2/TIF-2/NCoA-2, and 3)
SRC-3/p/CIP/AIB-1/TRAM-1/RAC-3/ACTR (10, 11, 12, 13, 14, 15, 16, 17, 18). The p160 proteins contain
a short conserved NR interaction motif (NR box), which has the core
sequence LXXLL (L = leucine and X = any amino acid) (17, 19).
All of the p160s have a core NR interaction domain that contains three
of these NR boxes arranged in tandem. A unique fourth NR box has been
characterized at the extreme carboxy terminus of an alternatively
spliced variant of SRC-1, SRC-1a (20, 21). NR boxes have also been
identified in other classes of coactivator molecules including
p300/CBP, TRAP 220/DRIP 205/PBP, PGC-1, and TRBP/ASC-2/RAP250 (22, 23, 24, 25, 26, 27, 28).
Crystal structures of agonist-bound NRs complexed to the NR boxes have
yielded considerable information about the mechanism of recognition.
The tertiary structure of the LBD of NRs is well conserved and has been
described as a three-layered sandwich composed of 12 -helices with
the ligand-binding pocket buried inside the globular structure
(29, 30, 31). Ligand binding results in a conformational change with the
most significant alteration occurring in the carboxy-terminal helix
(helix 12; H12) that contains the AF-2 transactivation domain. H12
rotates from an exposed position directed away from the LBD of the
apo-receptor to a position in which it is folded back along the surface
of the receptor where it serves to seal the ligand-binding cavity of
the receptor (29, 30, 31). This conformational change creates a groove on
the LBD that serves as the surface for the -helical LXXLL core of
the NR box to recognize the agonist-bound receptor (32, 33, 34, 35, 36). The
hydrophobic face of the LXXLL helical domain of the NR box makes direct
contact with the nonpolar groove on the LBD that is created after
binding of the ligand. The side chains of two highly conserved residues
within the LBD, a glutamic acid residue within H12 and a lysine residue
near the carboxy-terminus of H3, make specific contacts with the
backbone amides and carbonyls of amino acids within the LXXLL helix and
serve to form a charge clamp that specifies the acceptable size of the
LXXLL -helix and orientation by which it binds within the
hydrophobic groove of the LBD (33, 34, 35, 36). The hydrophobic side chains of
the leucine residues of the LXXLL helix face the hydrophobic groove
while the side chains of the X residues are solvent exposed; thus
little if any specificity is directed by the small LXXLL region.
However, residues flanking this sequence on both the amino and carboxy
termini also make significant contacts with the surface of the LBD
(33, 34, 35) and have been shown to be important for determining
preferential binding of various NR boxes to particular NRs, which may
lead to NR preference for certain coactivators (17, 18, 35, 36, 37, 38, 39, 40, 41, 42, 43).
Different ligands for a particular NR may also alter specificity for
coactivator binding (43, 44), due to conformational differences in the
LBD. These differences in the tertiary structure of the LBD due to the
binding of different ligands are exemplified by a number of structures
that have been solved with estrogen receptor (ER) isoforms bound to
agonists such as 17ß-estradiol (E2),
diethylstilbestrol (DES), genestein (GEN), and selective ER modulators
(SERMs) such as tamoxifen and raloxifene (34, 45, 46, 47). Both of the
agonists, E2 and DES, produce a similar
conformational change in the LBD of ER resulting in the formation of
the hydrophobic groove that is available for recognition by the NR box
(34). Binding of the SERMs results in unique positioning of H12 so that
this helix actually obstructs the groove by mimicking the LXXLL domain
with the sequence LXXML from H12 of the LBD, which is conserved in all
NRs (34, 45). Thus, the groove is unavailable for docking of the
coactivator consistent with the SERMs functioning as AF-2 antagonists.
GEN, a partial agonist, induces novel positioning of H12 that also
obstructs the hydrophobic groove (46) suggesting that there may be some
interference with NR box binding.
To investigate the ability of various ligands to specify the affinity
of NR boxes for ligand-bound receptor, we compared the capacity of p160
coactivator NR boxes to be recruited to ER and ERß in the presence
of E2, DES, and GEN. Using a time-resolved
fluorescence-based binding assay, we determined the dissociation
constants for several natural NR boxes for both ER isoforms in the
presence of the three agonists. The affinities of several NR boxes were
specified by the ligand, while others remained constant independent of
the nature of the ligand. In addition, we identified several NR boxes
that had significant ER isoform preference, and, interestingly, the box
that demonstrated one of the highest degrees of isoform preference is
the extreme carboxy-terminal box of SRC-1, which is localized to a
region that is present in an alternatively spliced variant of this
coactivator.
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RESULTS |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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). Although the core LXXLL
sequence is conserved, the amino acid residues flanking this sequence
are not conserved and are believed to play a role in NR box recognition
of selective NRs (17, 18, 35, 36, 37, 38, 39, 40, 41, 42, 43). Thus, the sequences immediately
flanking the core LXXLL sequence may specify a coactivator’s affinity
for a particular NR. It has been demonstrated that various ligands for
an NR such as ER induce novel conformations within the LBD (34, 45, 46, 47, 48); therefore, we sought to determine whether distinct ligands had
the ability to specify the affinity of various NR boxes for a
particular NR. Using both ER and ERß, we examined both receptor
subtype selectivity and ligand selectivity in terms of the receptors’
ability to bind to NR boxes from the p160 coactivator family. We used a
time-resolved fluorescent technique to detect binding of NR box
peptides (Fig. 1B) to recombinant human ER or ERß.
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, E2 dose-dependently induced SRC-1 NR box II
peptide binding to both ER and ERß. The potency of
E2 was similar for both receptors with an
EC50 of 3.1 nM for ER and 3.0
nM for ERß. As expected, the antiestrogen, ICI 182,780,
blocked the binding of the NR box to both ER subtypes. Interestingly,
ERß, but not ER, exhibited significant ligand-independent binding
to the SRC-1 NR II peptide. The ligand-independent portion of the
binding could be blocked by the ER antagonist ICI 182,780 (Fig. 2B) and
by unlabeled SRC-1 NR box II peptide (data not shown), indicating that
in the unliganded state, ERß retains an active conformation.
Examination of greater than 20 different NR boxes confirmed that ERß
retains a basal active conformation while ER does not (data not
shown).
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-helix created by the leucine side chains make van der Waals
contacts with the hydrophobic coactivator-binding groove in the LBD
surface. Mutation of the leucines within the LXXLL core sequence has
been shown to have a detrimental effect on the binding of the NR box or
the entire coactivator protein to an NR (17, 19, 35, 38, 39, 41, 43, 49). Unlabeled wild-type SRC-1 NR II peptide (LXXLL) dose-dependently
competed with labeled SRC-1 NR box II peptide for binding to
E2-bound ER and ERß (Fig. 2
, C and D).
Unlabeled mutant SRC-1 NR II peptide (AXXLA) was unable to compete for
labeled SRC-1 NR box II peptide binding to
E2-bound ER; however, weak dose-dependent
competition was detected with E2-bound ERß,
indicating that ERß is more tolerant of these mutations in the LXXLL
core sequence than ER. The IC50 values for the
14-amino acid SRC-1 NR II peptide (LTERHKILHRLLQE) for both ER and
ERß were in the 1–4 µM range; however, we subsequently
determined that NR box peptides with additional carboxy-terminal
residues displayed a significant increase (10-fold) in affinity and
were subsequently used in additional experiments (Fig. 1B).
Differential Recruitment of p160 Coactivator NR Boxes by ER
Subtypes
To characterize the NR box preferences for ER subtypes, we first
examined E2-dependent recruitment of each of the
10 NR boxes from the p160 coactivator family (Fig. 1B). ER and ERß
displayed unambiguous NR box preferences, and, interestingly, the two
ER subtypes exhibited significant differences in their NR box
selectivity (Fig. 3). For the SRC-1 NR
boxes, ER had a clear preference for SRC-1 NR box II with some
recruitment detected for SRC-1 NR box IV. Recruitment of SRC-1 NR box I
and SRC-1 NR box III by ER was not detected. ERß had a much
different pattern of selectivity with a very strong preference for
SRC-1 NR box IV followed by SRC-1 NR box II with SRC-1 NR boxes I and
III showing minimal recruitment. The selectivity of the ER subtypes for
SRC-1 NR box IV may be significant since the fourth box is only
expressed in an alternatively spliced variant of SRC-1, SRC-1a.
Differences between ER and ERß were also detected with respect to
their selectivity for SRC-2 NR boxes. ER displayed a clear
preference for SRC-2 NR box II followed by SRC-2 NR box I and no
detectable binding to SRC-2 NR box III. In contrast, ERß recruited
all three SRC-2 NR boxes with nearly equal efficacy. The rank order of
recruitment of SRC-3 NR boxes was identical for both ER and ERß
(NR box I>NR box 2>NR box 3), with the only difference being that
ERß displayed some binding to SRC-3 NR box III while ER did not.
Examination of the average E2
EC50 values for peptides that were significantly
recruited revealed an approximate 6-fold selectivity for ER
vs. ERß (3 ± 0.2 nM
vs. 18 ± 1 nM). This degree of
selectivity has been previously reported for E2
in both radioligand binding assays and in cell lines either transiently
or stably expressing ER subtypes (50, 51, 52). Since the NR box interaction
assay is a functional assay as it detects the recruitment of an NR
interaction domain of a coactivator in response to a ligand, we would
expect that the behavior of ligands would be similar to a cell-based
assay rather than a ligand-binding assay that typically only detects
displacement of a radioligand. The potency of E2
appears to be lower in our NR box interaction assay than according
to previously reported binding affinity data
(Kd = 0.2–0.6 nM) (50, 53, 54, 55) and some cell-based reporter assays (51, 56); however, the
potency of E2 in our assay is comparable to
reported transient cotransfection assays (50, 52) and to values that we
currently obtain in cell-based assays that assess endogenously
expressed ER. Since we are detecting the binding of an NR box peptide
to ligand-bound receptor, the potencies may not necessarily correlate
with the ability of a particular ligand to displace a radioligand or
activate transcription in a cell-based reporter assay. This is
especially true for the cell-based reporter assays since they assess
the transcriptional activity of the receptor, combining the activities
of both AF-1 and AF-2 and all of the coactivators that they may
recruit, whereas the current study examined only the ability of the
receptors to bind to NR boxes from the p160 coactivators.
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and ERß and
ERß. Saturation binding assays were conducted in which receptor was
treated with a dose of E2 that would allow
maximal NR box binding followed by titration of labeled NR box until
saturation. With few exceptions, the preferences of the ER subtypes for
the NR boxes based on the Kd values matched well
with the selectivity described above based on the
E2-dependent recruitment. The exceptions are
attributable to differences in maximal binding (efficacy) of various NR
box peptides that we detected in our saturation binding assays and are
reflected as the maximal efficacy in ligand-dependent NR box
recruitment assays described above (Fig. 3). Alterations in the
conformation of the NR box binding groove of the LBD may not only
affect the affinity of the NR box, but also the relative binding
capacity of the site. Thus, discrepancies between selectivity based on
affinity vs. efficacy may be due to the creation of a
high-affinity, low-capacity binding groove vs. a
low-affinity, high-capacity binding groove for a particular NR box. Of
the SRC-1 NR boxes, SRC-1 NR box II has the highest affinity for ER
(Kd = 155 ± 21 nM)
followed by SRC-1 NR box IV (Kd = 934 ± 259
nM) (Table 1).
SRC-1 NR box I and NR box III displayed no detectable binding to ER.
Both SRC-1 NR box II and NR box IV display similar affinities to ERß
(Kd = 204 ± 27 nM and
Kd = 261 ± 75 nM,
respectively) in contrast to the large degree of selectivity shown for
NR box IV in the ligand-dependent recruitment assay (Table 2). Unlike ER, both SRC-1 NR box I and
NR box III showed weak but detectable affinity for ERß
(Kd = 1,025 ± 190 nM
and Kd = 2,060 ± 214
nM, respectively). The significant ER subtype
selectivity for SRC-1 NR box IV identified above is also apparent when
examining the dissociation constants (ER, Kd =
934 ± 259 nM vs. ERß,
Kd = 261 ± 75 nM);
however, differences in the maximal binding of this NR box for the
receptors amplifies the differential selectivity. The dissociation
constants for the SRC-2 NR boxes retain the identical rank order as
described above for ER (NR box II>NR box I, NR box III no binding),
but ERß shows some preference for SRC-2 NR box I above SRC-2 NR boxes
II and III. SRC-3 NR boxes I and II have similar high affinities for
ER (Kd = 216 ± 26
nM and 182 ± 23 nM,
respectively) while SRC-3 NR box III does not bind. In contrast, SRC-3
NR box III does bind to ERß (Kd = 849 ±
147 nM); however, both SRC-3 NR box I and NR box
II display greater affinities (Kd = 136 ±
26 nM and 443 ± 97
nM, respectively). Northrop et al.
(42) have also examined the relative affinity of p160 NR boxes for
ERß using a competition scintillation proximity assay (42). In terms
of the rank order of potency, our results for ERß compare favorably
with this study with a few exceptions. We detected weaker binding of
several boxes, including NR boxes II and III from SRC-2 and NR box III
of SRC-3, than reported by Northrop et al. (42). These
discrepancies may be due to differences in the techniques used to
quantitate NR box affinity. We measured the dissociation constant of
each individually labeled NR box peptide to ERß, while Northrop
et al. (42) assessed the ability of these NR box peptides
fused to the maltose binding protein (MBP) to compete for binding of a
radiolabeled MBP-SRC-1 NR interaction domain fusion consisting of the
three central NR boxes to ERß.
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). The SRC-1 NR box IV
box displays a high degree of selectivity for ERß over ER
(
3.6-fold), which may be an underestimate of the degree of
functional selectivity since there is a large difference in the maximal
binding of this NR box to the two receptors (Fig. 3). Several other NR
boxes also appear to have a preference for ERß over ER including
SRC-2 NR box I, SRC-2 NR box III, and SRC-3 NR box III (Table 3). While
SRC-2 NR box I has a 2.4-fold selectivity for ERß, both SRC-2 NR box
III and SRC-3 NR box III bind reasonably well to ERß, but have no
detectable affinity for ER. Only two NR boxes were identified with
ER selectivity: SRC-2 NR II was 4.4-fold selective and SRC-3 NR II
was 2.4-fold selective.
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and ERß when bound to
E2, DES, or GEN. These three ligands were chosen
for several reasons. They display structural diversity with
E2 representing a steroid, while both DES and GEN
are nonsteroidal compounds (Fig. 4).
While E2 and DES display a slight ER subtype
preference, GEN has been characterized as having some degree of ERß
specificity in both receptor binding and cell-based assays (50, 51, 52, 57). All three ligands display full agonist activity for ER;
however, GEN is only a partial agonist for ERß while both
E2 and DES are full agonists (51). Thus, these
ligands exhibit several distinct pharmacological properties when bound
to ER that may be mediated by selectivity in NR box recruitment.
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), both DES and GEN
recruited a subset of NR box peptides to both ER and ERß in a
dose-dependent manner (data not shown). A comparison of the effect of
ligand on the maximal binding efficacies for each of the NR box
peptides to ER and ERß is shown in Fig. 5. Although the general pattern of NR box
preference for E2 and DES was very similar to
that for ER, significant differences were noted with GEN. Both NR
box I and II of SRC-3 are significantly recruited to
E2- and DES-bound ER; in fact, SRC-3 NR box I
has the highest degree of efficacy for all of the NR boxes bound to
ER for both E2 and DES. In contrast, GEN-bound
ER recruits SRC-3 NR box I with less than half of the efficacy of
either E2- or DES-bound ER, and SRC-3 NR box
II is not recruited at all (Fig. 5A). More subtle differences are noted
for SRC-2 NR boxes, where NR box I has a low but significant level of
recruitment to E2- and DES-bound ER, but no
binding to GEN-bound ER. SRC-2 NR box II also displays reduced
maximal binding to GEN-bound ER relative to both
E2- and DES-bound receptor (Fig. 5A). For ERß,
the major differences between the ligands were again within the SRC-3
NR boxes. Binding of SRC-3 NR box I to ERß showed a great deal of
dependence on the identity of the ligand (Fig. 5B). DES induced the
greatest amount of binding of ERß to SRC-3 NR box I, which was
approximately 1.8-fold greater than GEN-bound receptor and 3-fold
greater than E2-bound receptor (Fig. 5B). These
data clearly indicate that ligands can specify which NR boxes are
recruited to the receptor as is the case with SRC-3 NR box II or the
degree to which a NR box is recruited to the receptor.
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and 122 ±
53 nM for ERß (51, 57). DES also showed selectivity for
ERß with an EC50 of 43.0 ± 18.5
nM for ER and 7.1 ± 1.6 nM for ERß.
This is in contrast to other studies that demonstrate that DES has a
slight preference for ER over ERß in radioligand binding and
cell-based reporter assays (51, 57). The number of NR boxes recruited
by agonist-bound receptor was also different when comparing either the
receptor subtype or the agonist. For example, GEN-bound ERß was more
efficient in recruiting a greater number of NR boxes than GEN-bound
ER (Fig. 5). In terms of agonist specificity, DES- and
E2-bound ER recruited more NR boxes than
GEN-bound ER.
Although we cannot make direct comparisons of maximal binding of NR
boxes between ER and ERß due to differences in specific activity
of the receptors, some conclusions can be made based on the rank order
of preference of the various NR boxes between the two receptors. The
most interesting difference occurs in SRC-1 where NR box IV recruitment
by ER is not impressive for any of the ligands; however, all three
ligands induce very strong binding of NR box IV to ERß (Fig. 5). With
the SRC-2 NR boxes, ER prefers NR box II when bound to all three of
the ligands, while ERß binds to each of the SRC-2 NR boxes with no
overt preference. The rank order preference for binding to the SRC-3 NR
boxes (NR box I>NR box II>NR box III) is conserved for all three
ligands bound to both ER subtypes with the exception of the inability
of GEN-bound ER to recruit SRC-3 NR box II.
Ligands Alter ER Affinity for p160 NR Boxes
It is possible that different ER agonists may cause distinct
conformations in the region of the LBD that interacts with the
coactivator NR box, thus altering affinity for the receptor. To examine
this possibility, we determined the dissociation constants for each of
the NR boxes bound to both ER and ERß in the presence of
E2, DES, and GEN. The affinities of the NR boxes
to ER are shown in Table 1. Similar to efficacy data, SRC-1 NR boxes
I and III did not have detectable affinity for ER when bound to any
of the agonists. SRC-1 NR box II, however, bound well to ER with the
highest affinity in the presence of E2. DES- and
GEN-bound ER also had affinity for this box, but with reduced
relative binding affinity (27, 28, 29, 30, 31, 32, 33, 34, 35). Interestingly, whereas
E2-bound ER had detectable affinity for SRC-2
NR box I, we could not detect significant binding to DES- or GEN-bound
ER. Selectivity for E2-bound ER was also
apparent for NR box II from both SRC-2 and SRC-3. On the other hand,
SRC-3 NR I had similar affinities to E2- and
DES-bound ER with reduced affinity to GEN-bound receptor.
Whereas many of the NR boxes were selective for
E2-bound receptor when evaluating ER, the
opposite appears to be the case with ERß with most of the NR boxes
having greater affinity for DES- and GEN-bound receptor (Table 2). NR
boxes II and III from both SRC-2 and SRC-3 have greater affinities for
DES- and GEN-bound ERß than E2-bound receptor.
SRC-1 NR box IV and SRC-3 NR box I have similar affinities for ERß
regardless of the identity of the agonist. SRC-1 NR box II appears to
be somewhat preferential to GEN-bound ERß, while NR box I from both
SRC-1 and SRC-2 prefer E2- and DES-bound receptor
over GEN-bound receptor. These data illustrate that the nature of the
agonist bound to ER can play an important role in specifying the
affinity of the LBD for the NR box.
Determination of the dissociation constants for each of the NR boxes
for both ER and ERß allows for direct comparison of the affinities
of the boxes between the two receptors. Table 3 shows the degree of
receptor specificity for each of the p160 NR boxes with each agonist.
As described above, considerable receptor selectivity was noted when
E2 was used as the agonist. Interestingly, the
identity of the agonist also appears to play a significant role in ER
subtype selectivity of particular NR boxes. For instance, SRC-2 NR box
II has a preference for ER (4.39-fold) when E2
is the agonist, but this selectivity reverses when DES is used
(4.2-fold ERß selective). For this particular box, GEN shows only a
slight preference for ERß (1.8-fold). SRC-1 NR box II and SRC-3 NR
box I showed no subtype selectivity with E2 but
were ERß selective when either DES or GEN were used. Several NR boxes
had preference for ERß independent of the identity of the agonists
such as SRC-1 NR box IV and SRC-3 NR box III. None of the p160
coactivator boxes were ER selective with all three agonists. Thus,
depending on the agonist there may be selectivity for ER or ERß or
relative nonselectivity even when examining a single NR box.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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has been shown to require an intact NR box II of SRC-1 for
efficient coactivation, while the retinoic acid receptor required both
NR boxes II and III for normal function (17, 36, 37, 43). In contrast,
the peroxisome proliferator-activated receptor 
(PPAR) and
progesterone receptor required NR boxes I and II of SRC-1 (43). In the
current study, we examined the ability of the NR boxes of the p160
coactivator family to be recruited to both ER and ERß in a
ligand-dependent manner. We also directly measured the dissociation
constants for each of these NR boxes to both receptors. Consistent with
previous studies indicating that NR box II of SRC-1 was required for
ER, our data demonstrate that ER has very high affinity for this
box relative to the other NR boxes of SRC-1. NR box II of SRC-2 has
also been shown to be necessary for efficient interaction with ER
(41), which is also in agreement with the high affinity of this NR box
we detected. Our data predict that for SRC-3, NR box I and
NR box II will play an important role for coactivation of ER.
Interestingly, the NR box selectivity changes significantly for ERß.
For SRC-1, NR box IV is dominant, but NR box II still retains
significant activity. All of the SRC-2 NR boxes have a similar profile
with none of the NR boxes predominating while the NR boxes of SRC-3
retain a similar rank order as they did for ER. These NR box
preferences may underlie the ability of various NRs to exhibit some
degree of coactivator selectivity.
In addition, the differences in NR box preferences that we noted
between ER subtypes suggest that coactivator selectivity may play a
role in the differential actions of these receptors. One of the more
significant differences we noted was the selectivity of SRC-1 NR box IV
for ERß. Since NR box IV is only contained in the longer splice
variant of SRC-1 (SRC-1a), ERß may preferentially interact with
SRC-1a over SRC-1e. Thus, in tissues that express both ER subtypes and
greater amounts of SRC-1a than SRC-1e, ERß may display dominance.
ERß has been shown to modulate the transcriptional activity of ER,
and it was proposed that the relative expression levels of these ER
isoforms may regulate the level of transcriptional response to ER
ligands (65). Based on this observation, our data suggest that the
SRC-1a/SRC-1e expression ratio may also be important for determining
the level of transcriptional response to ER ligands by modulation of
the transactivation potential of ERß; however, additional
investigation will be required in the context of the full-length
coactivators. Differential expression of the SRC-1 splice variants has
been reported in the brain (66), and interestingly, regions that
predominately express the SRC-1a isoform, such as the paraventricular
nucleus of the hypothalamus, also predominately express the ERß
subtype (67, 68). Variation in the ability of the SRC-1 isoforms to
potentiate ER activity has been previously reported; however, the
differences were due to suppression of an activation domain in SRC-1a
(22).
Various ligands for a particular NR are believed to have the ability to
induce novel conformations within the LBD of the receptor, which was
exemplified by recent reports of the crystal structure of ER bound to
different ligands. The role that the identity of the ligand plays in
the conformation of the LBD of ER is particularly apparent when one
examines the position of H12. Whereas agonist binding, such as
E2 or DES, places H12 on the surface of the LBD
capping the binding pocket with the coactivator binding groove exposed,
SERMs such as tamoxifen and raloxifene do not allow H12 to cap the
binding pocket due to steric hindrance, and H12 blocks the hydrophobic
groove by mimicking the NR box LXXLL motif (45). Binding of GEN, a
partial ERß agonist, to the receptor results in suboptimal
positioning of H12 along the NR-box binding hydrophobic groove similar
to that found in the SERM structures (46). In this study, we examined
the ability of E2-, DES-, and GEN-bound ER and
ERß to interact with various NR boxes from the p160 proteins. Our
data indicate that the identity of the agonist plays an essential role
in determining the relative binding efficacy and potency of the p160
family NR boxes to ER and ERß. Due to these ligand-dependent
alterations in the NR box binding activity, an agonist is able to
determine the relative selectivity of several of the p160 NR boxes for
the ER isoforms. It is interesting to note that although the crystal
structure of ERß with GEN indicates that H12 binds along the NR box
binding groove on the surface of the LBD (46), our data indicate that
GEN-bound ERß binds very well to the majority of the p160 NR boxes. A
SERM such as raloxifene sterically restricts positioning of H12 in the
antagonist position due to the protrusion of the piperidine ring of
the drug from the ligand binding cavity (45); however, GEN does not
sterically hinder positioning of H12 possibly allowing for more
flexibility in its positioning and possible displacement by an NR box.
Recent two-hybrid data confirm that GEN-bound ERß binds to both p160
and nonnatural phage display-identified NR boxes (69). Similar to our
data with the p160 NR boxes, these investigators demonstrate that ERß
displays differences in its ability to recruit several of the
nonnatural NR boxes depending on whether it is bound to
E2 or GEN (69).
Coactivation is more complex than simple NR box peptide binding to the NR. NR boxes often exist in tandem copies within the coactivator polypeptide sequence, and it has been demonstrated that a polypeptide containing multiple NR boxes exhibits higher affinity for an NR dimer than a single NR box presumably due to cooperative binding (33, 42). We sought to characterize the variations in the conformation of the coactivator binding groove specified by various ER ligands as detected by selective NR box binding, and since the cooperative binding of the multimerized NR boxes could potentially interfere with this examination, we chose to characterize the binding of single NR box peptides. Our studies also do not address coactivation that is independent of NR box binding such as AF-1-dependent coactivation. However, our methods allow for sensitive characterization of a very specific event in the activation of ER, which is the creation of a NR box binding surface on the LBD in response to a ligand.
Consistent with our data indicating that a ligand can specify the
affinity of the NR for an NR box, McInerney et al. (43)
demonstrated that the identity of the agonist subtly altered the
requirement for NR box I within SRC-1 for efficient coactivation of
PPAR . These data suggest that NR box selectivity designated by
particular ligands may allow NRs to preferentially bind specific
coactivators. Indeed, a role for ligand in coactivator specificity has
been shown for both PPAR and the vitamin D receptor (VDR).
While the natural PPAR agonist, 15-deoxy-
12,14 prostaglandin
J2 (15d-PGJ2) induced binding of several classes
of NR box-containing coactivators including TRAP 220/DRIP 205, p300,
and all three p160s, the synthetic agonist,
troglitazone, was unable to induce recruitment of
any of these proteins (70). Consistent with this finding, SRC-1 and
SRC-2 coactivated PPAR when 15d-PGJ2 was used as a ligand, but not
when troglitazone was used (70). The ability of a ligand
to specify selective recruitment of a particular p160 family member has
been demonstrated for VDR. While the natural hormone,
1,25-dihydroxyvitamin D3, mediated recruitment
of all three SRC coactivator family members to VDR, the synthetic
analog OCT (22-oxa-1, 25-dihydroxyvitamin
D3) induced interaction of VDR with only SRC-2
(44). Interestingly, OCT displays a tissue-selective profile retaining
the antiproliferative activity of 1,25-dihydroxyvitamin
D3, but lacking the hypercalcemic effects (71).
Thus, selective coactivator recruitment may be an underlying
mechanism for tissue/cell/promoter specificity of NR ligands.
|
MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
|---|
.
Time-Resolved Fluorescence
Full-length recombinant baculovirus-expressed human ER or
ERß was purchased from PanVera (Madison, WI). White FluoroNunc
MaxiSorp 96-well plates (Fisher Scientific, Pittsburgh,
PA) were coated overnight at 4 C with 100 µl/well of 40 pmol/ml ER
protein that had been diluted in 0.1 M
NaHCO3. Receptor-coated plates were then blocked
with 100 µl/well 7.5% BSA in TBS (0.1 M Tris HCl, 0.15
M NaCl) containing 20 µM
diethylenetriamine-pentaacetic acid (DTPA-Sigma, St.
Louis, MO) as a stabilizer. Blocking was carried out at room
temperature for at least 1 h.
A peptide-europium (Eu) conjugate was prepared by incubation of 0.8 µl 1 mM biotin-labeled NR box peptide with 120 µl 0.1 mg/ml Eu-labeled streptavidin (Wallac, Inc./Perkin-Elmer Corp., Norton, OH) on ice for 30 min. After blocking, coated 96-well plates were washed three times with TBST (0.1 M Tris HCl, 0.15 M NaCl, 0.1% Tween) again containing 20 µM DTPA for signal stabilization. The peptide-Eu conjugate prepared previously was made up to 10 ml with DELFIA Assay Buffer (Wallac, Inc./Perkin-Elmer Corp.) and added to the receptor-coated 96-well plate at 90 µl/well. The E2 was diluted to 10x concentration in DELFIA assay buffer and added to appropriate wells at 10 µl/well. Plates were incubated with ligand and peptide conjugate for 1.5 h at room temperature and then washed five times with TBST + 20 µM DTPA. DELFIA Enhancement Solution (Wallac, Inc./Perkin-Elmer Corp.) was then added to the plate (100 µl/well) and incubated at room temperature with gentle shaking for 5 min. Plates were then read using a Wallac, Inc. Victor II plate reader (Wallac, Inc./Perkin-Elmer Corp.).
Relative fluorescence was determined by subtracting the fluorescence
value obtained in the absence of ligand from the value obtained with
ligand. This method yields negative relative fluorescence values if
hormone-independent binding occurs that is displaced by an antagonist
as illustrated in Fig. 2. Dose-response experiments were performed a
minimum of three times. Representative experiments are shown in the
figures, and the relative fluorescence values reported are derived from
the mean of three individual experiments ± SEM.
To determine the dissociation constant (Kd) for each NR box, the time-resolved fluorescence technique was used with a fixed amount of E2, DES, or GEN (1 µM), and the NR box concentration was varied. For this assay the peptide-Eu conjugate was prepared by incubation of 2.1 µl 1 mM peptide with 17 µl Eu-labeled streptavidin on ice for 30 min. The volume of the peptide conjugate was then made up to 700 µl with DELFIA assay buffer containing 1 µM E2. Eight-point (in triplicate) dose-response curves were generated, and the results from three independent experiments were normalized and pooled to determine the dissociation constant. Data were analyzed using Prism 3.0 (GraphPad Software, Inc., San Diego, CA) and the Kd values were determined with a one-site binding model. Nonspecific binding was typically less than 1% of total binding.
|
FOOTNOTES |
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Received for publication January 22, 2001. Accepted for publication March 2, 2001.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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