From the Bristol-Myers Squibb Pharmaceutical Research Institute,
Department of Cell and Molecular Biology, Buffalo, New York 14213, the Department of Chemistry, Candiac, Quebec J5R1J1,
Canada and the § Department of Central Chemistry,
Wallingford, Connecticut 06492
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ABSTRACT |
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The transcriptional response mediated by retinoic
acid involves a complex series of events beginning with ligand
recognition by a nuclear receptor. To dissect the amino acid contacts
important for receptor-specific ligand recognition, a series of
retinoic acid receptor (RAR) mutants were constructed. Transcriptional studies revealed that serine 232 (Ser232) in RAR
and methionine 272 (Met272) in RAR
are critical residues
for the recognition of their respective receptor-selective analogs.
The identification of these key amino acids in the ligand binding
pocket is confirmed by the reported crystal structure of RAR.
Interestingly, the serine at position 232 in RAR
gives an
explanation for the observed differences in the affinity of the
naturally occurring ligand, all-trans-retinoic acid (t-RA), in this receptor compared with that for the other receptors, since hydrogen bonding would not be permitted between the hydroxyl of serine
and the hydrophobic linker of t-RA. Using this model, a molecular
mechanism for the transcriptional antagonism of a synthetic analog is
suggested that involves an alteration in the structure of the receptor
protein in the region around the AF2 domain in helix 12.
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INTRODUCTION |
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Retinoic acid and its analogs (retinoids) regulate cellular
proliferation and differentiation in higher eukaryotes. The biological effects of these ligands are mediated by their binding to the retinoic
acid receptors (RARs),1 which
are members of the superfamily of steroid-thyroid hormone nuclear
receptors. These receptors act as transcriptional enhancers that bind
in a sequence-specific manner to their response elements (retinoic acid
response elements) located within the promoter region of distinct
retinoid-responsive genes. RARs activate transcription of those genes
after ligand bound to the receptor induces conformational changes
leading to activation (1-4). A crystal structure of the ligand binding
domain (LBD) of the closely related human retinoid-X receptor revealed a novel protein fold, an antiparallel
-helical sandwich,
common to the members of this superfamily. Examination of this
structure for a potential binding pocket for its ligand, 9-cis-retinoic acid, unveiled two large hydrophobic cavities
within the N-terminal portion of the LBD in the vicinity of the s1,s2
-hairpin and
-helix H5 (5). These findings are supported by
results from ligand-photoaffinity binding experiments, which identified
important residues for receptor-ligand binding in the LDBs of the
glucocorticoid receptor (6) and RAR
(7, 8). The amino acid sequence
alignment of the nuclear receptor LBDs indicates that most of the
residues identified by photoaffinity labeling or site-directed
mutagenesis correspond to the 9-cis-retinoic acid receptor
residues surrounding the putative 9-cis-retinoic acid
binding pocket (9).
Recently, the 2.0-Å structure of the LBD of human RAR bound to
all-trans-retinoic acid (t-RA) was reported showing details of ligand-receptor interactions (10). Several amino acids from
-helices H1, H3, H5, H11, H12, loops 6-7 and 11-12, and serine 289 (Ser289) from the
-sheet s1 were proposed as contact
residues between the receptor and ligand. Amino acid sequence alignment
of RAR
, -
, and -
revealed that among all of these residues,
only three positions were variable: serine 232 (Ser232),
alanine 225 (Ala225), and alanine 234 (Ala234)
in H3, isoleucine 270 (Ile270), isoleucine 263 (Ile263), and methionine 272 (Met272) in H5 and
valine 395 (Val395), valine 388 (Val388), and
alanine 397 (Ala397) in H11. These amino acid residues
therefore became obvious candidates as crucial amino acids responsible
for determining RAR selectivity to certain synthetic analogs of
t-RA.
Utilizing site-directed mutagenesis and RAR-selective retinoids,
we previously identified serine 232 (Ser232) and threonine
239 (Thr239) from the N-terminal portion of the LBD of
RAR and the corresponding alanine 225 (Ala225) and
isoleucine 232 (Ile225) from RAR
to be essential for the
recognition of retinoic acid and various analogs (11). In the present
work, the amide linker region of Am-580 and the oxime linker region of
BMS-185354 were used to precisely identify serine 232 (Ser232) in RAR
and methionine 272 (Met272)
in RAR
as critical for the specific interaction of the receptor and
retinoid. Experiments with an RAR
antagonist, BMS-185411, showed
that Ser232 is also involved in the transcriptional
antagonist activity of this compound. In RAR
, an alanine
(Ala225) at the position corresponding to
Ser232 in RAR
was shown to allow BMS-185411 to behave as
an RAR
-specific agonist. Analysis of several additional RAR
- or
RAR
-specific point mutants introduced along
-helix H3, H4/H5 and
the
loop of RAR
detected a decrease in the transactivation
activity of BMS-185411. The amino acids mutated in these experiments,
according to the crystal model of RAR
LBD, do not directly interact
with t-RA. This suggests that the effect of these residues on the
potency and selectivity of BMS-185411 could be due to intramolecular
interactions within the receptor itself. These findings support the
conclusion that the transcriptionally active ligand-receptor complex is
a result of a series of direct and indirect interactions between the
receptor and its selective ligand, which reflect the dynamic nature of
both components.
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MATERIALS AND METHODS |
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Plasmids--
Plasmids pRAR0 and pRAR
0 (12), which
contained full-length human RAR
and -
, respectively, were gifts
from Dr. P. Chambon (IGBMC, Strasbourg, France). The chloramphenicol
acetyltransferase reporter plasmid containing a 46-base pair retinoic
acid response elements of the laminin B1 gene was provided by Dr. L. Gudas (13). All-trans-retinoic acid was purchased from
Sigma. Am-580 (14), BMS-185411, and BMS-185354 (15) were synthesized by
Bristol-Myers Squibb Central Chemistry, Wallingford, CT.
[3H]RA was purchased from NEN Life Science Products.
Retinoid Transactivation and Competition
Analysis--
Transfection of HeLa cells with DNA encoding wild type
RAR, RAR
, and the various chimeric receptors was performed as
described (11, 12). Retinoid efficacy was measured by the concentration of induced chloramphenicol acetyltransferase gene product obtained from
transfected cells using the chloramphenicol acetyltransferase enzyme-linked immunosorbent assays kit (5 Prime
3 Prime, Inc., Boulder, CO). Activity was routinely normalized for transfection efficiency by
-galactosidase activity. Results (EC50)
are presented as the dose at which chloramphenicol acetyltransferase
induction was half of the level observed for t-RA at 10
6
M.
DNA and Protein Analysis-- DNA sequence analysis and protein structure predictions were performed using GeneWorks 2.3 DNA-protein analysis software from IntelliGenetics, Inc.
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RESULTS |
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Characterization of Retinoic Acid Receptor -,
-, and
-Specific Activities Using Receptor-selective Retinoids--
Wild
type RAR
, -
, and -
transiently transfected into HeLa cells
were used to establish the profile of receptor-specific transactivation
responses utilizing t-RA and the receptor-selective synthetic retinoids
Am-580, BMS-185411, and BMS-185354 (Fig.
1). In these experiments, Am-580 was
found to be an RAR
-selective agonist, displaying an EC50
of 3.2 nM for this receptor. BMS-185411 showed specific
agonist activity for RAR
with an EC50 of 34 nM, and BMS-185354 selectively activated RAR
with an
EC50 of 28 nM (Table
I).
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Effects of Serine 232 in RAR on Specific Interactions with
Receptor-selective Ligands--
Utilizing the chimeric RAR,
RAR
(nE)
, where the N-terminal portion of RAR
domain E was
subcloned into an RAR
background by polymerase chain
reaction-assisted site-directed mutagenesis, two sets of residues,
serine 232 (Ser232)/alanine 225 (Ala225) and
threonine 239 (Thr239)/isoleucine 232 (Ile232)
in RAR
and RAR
were found to be essential for
receptor-ligand-specific interactions (11).
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Methionine 272 Is Responsible for RAR-specific Transactivation
Activity in Response to RAR
-selective Retinoids--
Examination of
amino acid differences among RAR
, -
, and -
within the first
100 residues of the N-terminal portion of domain E revealed only a
limited number of amino acid differences (Fig. 2). These suggested that
the receptor chimera, RAR
(nE)
, could be used as a host to create
five constructs targeted to identify the amino acids involved in
specific RAR
-ligand interactions.
RAR-selective Transcriptional Antagonists Interact with Serine 232 in the Ligand Binding Pocket--
Analysis of the results of
transactivation experiments revealed that BMS-185411 acts as an
RAR-specific agonist with an EC50 of 34 and 54 nM for the wild type and chimeric RAR
(nE)
receptors, respectively (Table I). In competition assays, however, this retinoid
behaved as a specific antagonist for RAR
, displaying an
IC50 of 400 nM (Table
II). Furthermore, BMS-185411 is an
antagonist for the mutants RAR
/
(nE)
and
RAR
(nE-S225/T232)
, as well as for the chimeric receptors
RAR
(nE-S225)
and RAR
(S225) with IC50 values of
233, 220, 217, and 433 nM, respectively (Table II). The
antagonist activity seen with RAR
(nE-S225)
and RAR
(S225) suggested that the Ser232 residue in RAR
must also be
involved in antagonist activity and selectivity. This conclusion is
further supported by the results of competition experiments
using RAR
(nE-T232)
in which BMS-185411 did not act as an
antagonist (Table II). Based on these results, we conclude that
Ser232 in RAR
and Ala225 in RAR
are both
selective contact amino acids which are responsible for both agonist
and antagonist selectivity for the synthetic retinoids used in this
report.
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DISCUSSION |
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The retinoic acid receptors are ligand-dependent transcription factors that regulate the expression of genes involved in cell growth, differentiation, and development. Yet, despite the large number of biological activities mediated by the receptors, relatively little is known about the precise interaction of the receptor protein and its naturally occurring ligand, t-RA. This study was undertaken to clarify the role of some of the amino acid contacts that were described in a previous report (11) and identified in the x-ray crystal structure (10).
In our previous report, two amino acid residues from the N-terminal
region of the LBD, Ser232 and Thr239 in RAR,
and the corresponding Ala225 and Ile232 in
RAR
, were shown to be critical for receptor-specific interactions with t-RA and various receptor-selective retinoids (11). Here, further
analysis of these key amino acids using site-directed mutagenesis
revealed that Ser232 in RAR
and Met272 in
RAR
(Fig. 2) are critical for receptor-selective ligand
interactions. The serine residue interacts with the amide linker
portion of Am-580 in RAR
, and the methionine residue interacts with
the oxime linker in an RAR
-selective retinoid (Fig. 3).
Defined in this way, these
receptor-selective contact amino acids are located in the ligand
binding pocket and must modulate the binding of naturally occurring
retinoids as well.
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Using the results generated here for synthetic ligands,
Ser232 in RAR may participate in a hydrogen bond with
the amide linker of Am-580 and its analogs, which links the compound to
-helix H3 (Fig. 3). In RAR
and RAR
, alanine residues at
positions 225 and 234, respectively, correspond to Ser232
in RAR
. The presence of a hydrophilic linker, as seen in Am-580, in
close proximity to a hydrophobic amino acid (Ala225 or
Ala234) may result in negative interactions between the
receptor and retinoid manifested as decreased affinity of the compound
for both RAR
and
(Fig. 3 and Table
III). In contrast, mutant receptors containing Thr239 alone were found to have no effect on the
activity of the RAR
-selective agonist, Am-580, and its analogs
(Table I). This finding is supported by the results of experiments with
the chimeric receptor RAR
(nE-S232)
, where the Thr239
was substituted by serine.
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The strategy outlined above was also used for the identification of
RAR-selective amino acid contacts with receptor-selective ligands.
Several mutant receptors were made, particularly an isoleucine 263 substitution for methionine in RAR
(nE)
, which produced
RAR
(nE-M263)
. This mutation was found to be completely sufficient
to convert the RAR
type of response of RAR
(nE)
with BMS-185354
into the RAR
type. Therefore, it is likely that the methionine
residue at position 272 is exclusively responsible for the RAR
selectivity of BMS-185354 (Table I). Methionine is generally considered
to be a hydrophobic amino acid. However, the presence of a sulfur atom
in the side chain of this amino acid could account for weak hydrogen
bonding (18) between this amino acid and the hydroxyl substituent of
BMS-185354 (Fig. 1 and compound 2 in Ref. 11). On the other hand, the
overall strong hydrophobic properties of methionine can also explain
why there is only a moderate negative effect of RAR
-selective
retinoids on RAR
, which contains an isoleucine residue at a position
corresponding to Met272 (Figs. 2 and 3). These observations
are supported by conclusions from the crystal structure of RAR
,
where interactions between Met272 and t-RA were proposed
(10). The Met272 is located on
-helix H5 (Fig. 2),
which, according to the model from x-ray analysis, lies above the
ligand (10). This arrangement also explains the results of experiments
where the racemate (R,S)1 of
6-(5,5,8,8-tetramethyl-5,6,7,8-tetrahydro-2-naphthyl)hydroxymethyl-2-naphthalene carboxylic acid and both its purified S-1 and R-1
enantiomers were used (19). All three compounds showed RAR
selectivity with the S-1 enantiomer being 10-fold more
potent than R-1. In this enantiomer, the hydroxyl group is
more favorably positioned with respect to Met272 and, thus,
Met272 appears to be the only amino acid required for
receptor-specific recognition.
Recently, another group of synthetic retinoids were discovered that act
to reverse the effect of retinoic acid in transactivation assays.
BMS-185411 is an example of such a transcriptional antagonist, which is
shown here to interact with Ser232 (Table II). We propose
that the hydrogen bond between the hydroxyl group of Ser232
and the amide linker of BMS-185411 positions this compound adjacent to
-helix H3 and may also force the phenyl group of this compound into
close proximity to
-helix H12, which is important for
transactivation activity (Fig. 3) (21, 22). The crystal structure of
RAR
suggests that when t-RA binds to the receptor,
-helix-H12
closes the entry opening by swinging upward and interacting with
-helix-H4 (10, 17, 20). The
-helix-H12 is then stabilized in this position by a salt bridge between glutamic acid 412 (Glu412) from H12 and lysine 264 (Lys264) from
-helix-H4 (10). Modeling studies of the receptor with this ligand
suggest that the phenyl ring of BMS-185411 may interfere with the
proper alignment of
-helix-H12 leading to the inactivation of this
receptor, possibly by interfering with the function of the AF-2
transactivation domain. In RAR
, the lack of the hydrogen bond
between alanine 225 and the amide linker of BMS-185411 may permit
flexibility of the compound in the ligand binding pocket of this
receptor thus avoiding interference with
-helix-H12 and leading to
selective agonist activity (Fig. 3 and Table I).
A similar explanation could also be applied to RAR, which contains
an alanine residue in a position corresponding to
RAR
-Ala225 and a methionine at position 272. Using the
RAR
(nE-M263)
mutant, BMS-185411 was approximately 10-15-fold
less potent than the same compound with RAR
(nE)
or wild type
RAR
(Table I). This suggests that when BMS-185411 moves within the
ligand binding pocket to avoid conflict with the
-helix-H12, the
dimethyl groups from the tetramethyltetrahydoxynaphthalene portion of
the compound interact with
-helix-H5 at position Met272
(Fig. 3). The corresponding Ile263 in RAR
is smaller and
more hydrophobic than methionine; therefore, it can better accommodate
hydrophobic dimethyl groups in its vicinity allowing for more potent
agonist activity with RAR
(Table I).
The results obtained here for synthetic ligands can be used to explain
the pattern of binding observed for the naturally occurring ligand of
these receptors, t-RA. When bound to RAR, t-RA encounters an
entirely hydrophobic environment (Fig.
4), which is reflected in the low
apparent Kd with Ile263 and
Ala225 at the two key positions (Table III). In RAR
,
t-RA encounters Met272, a partial hydrophobic residue, and
Ala234 at these two positions giving an intermediate
apparent Kd. Finally, in RAR
, an
Ile270 and Ser232 generates hydrophilic
character in the binding pocket and increases the apparent
Kd accordingly.
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In summary, these results suggest that ligands interact with the amino
acids in the ligand binding pocket in a manner that involves subtle
positioning between the top of the pocket, represented by the
methionine at position 272, and the bottom of the pocket. Our studies
with both receptor-selective agonists and antagonists suggest that the
interaction between RARs and their selective retinoids has a dynamic
nature where both protein as well as ligand affect the final
conformation to form a transcriptionally active complex. The functional
analysis detailed here provides experimental confirmation of the RAR
LBD x-ray crystal structure. In addition, the identification of
specific amino acid contacts within the ligand binding pocket can be
exploited for the design of compounds of pharmacologic importance aimed
at increasing the biological response and eliminating unwanted side
effects.
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ACKNOWLEDGEMENTS |
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We thank Dr. Pierre Chambon for providing
plasmids containing hRAR, -
, and -
and Dr. Lorraine Gudas for
the chloramphenicol acetyltransferase reporter plasmid with the
retinoic acid-responsive element from the laminin B1 gene. We thank Dr.
Pierre Chambon for critical discussion of the manuscript. Finally, we
gratefully acknowledge Jeanette Rhone for superb technical
assistance.
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FOOTNOTES |
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* The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
¶ To whom correspondence should be addressed: Bristol-Myers Squibb Pharmaceutical Research Inst., 100 Forest Ave., Buffalo, NY 14213. Tel.: 716-887-3717; Fax: 716-887-7661.
1 The abbreviation used are: RAR, retinoic acid receptor; t-RA, all-trans-retinoic acid; LBD, ligand binding domain; AM-580, 4-[(5,6,7,8-tetrahydro-5,5,8,8-tetramethyl-2-naphthyl)carboxamide]benzoic acid; BMS-185411, 4[[[5,6-dihydro-5,5-dimethyl-8-phenyl-2-naphthalenyl]-carbonyl]amino]benzoic acid; BMS-185354, (E,Z)-6[(5,6,7,8-tetrahydro5,5,8,8-tetramethyl-2-naphthalenyl)hydroxyiminomethyl]-2-naphthalenecarboxylic acid.
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REFERENCES |
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