(Received for publication, December 11, 1996, and in revised form, February 11, 1997)
From the Departments of Biochemistry and
** Microbiology and Immunology and
Fels Institute for Cancer
Research and Molecular Biology, Temple University School of Medicine,
Philadelphia, Pennsylvania 19140
The diverse biological actions of retinoic acid
(RA) are mediated by retinoic acid receptors (RARs) and retinoid X
receptors. Although it has been suggested that the ligand binding
domains (LBDs) of RARs share the same novel folding pattern, many RAR subtype-specific agonists and antagonists have been synthesized demonstrating that the LBD of each RAR subtype has unique features. We
have examined the role of several positively charged amino acid
residues located in the LBD of RAR in RA binding. These results are
compared with previously published data for the homologous mutations in
RAR
. Lys227 of RAR
does not appear to be
important for RA binding or RA-dependent transactivation,
whereas the homologous residue in RAR
, Lys220, plays an
important synergistic role with Arg269 in these two
activities. In addition, Arg276 of RAR
, like its
homologous residue Arg269 of RAR
, was found to play an
important role in the binding of RA most likely by interacting with the
carboxylate group of RA. However, the orientation of and electronic
environment associated with Arg276 in RAR
appears to be
different from that of Arg269 in RAR
, thus contributing
to the uniqueness of the ligand binding pocket of each receptor.
Retinoic acid (RA),1 a vitamin A
metabolite, is a potent regulator of a diverse group of biological
processes, including growth, differentiation, and morphogenesis (for
review, see Ref. 1). These actions of RA are mediated by a group of
nuclear proteins, which belong to the multigene family of steroid and
thyroid hormone receptors, termed retinoic acid receptors (RARs) and
retinoid X receptors (RXRs) (for review, see Ref 2). In dimeric form, the RARs and RXRs function as ligand-inducible transcriptional regulatory factors by binding to DNA sequences called retinoic acid-responsive elements (RAREs) and retinoid X-responsive elements, which are located in the promoter region of target genes. Three subtypes, termed ,
, and
, of both RAR and RXR have been
identified along with several isoforms of each subtype (3-11).
In vitro binding assays have demonstrated that only
9-cis-RA is a ligand for the RXRs, whereas both
all-trans-RA and 9-cis-RA have been shown to be
ligands for the RARs (12, 13).
RARs, like other members of the steroid and thyroid hormone superfamily, have a modular structure consisting of six domains (A-F), each of which has been assigned specific functions (2). The C domain, which contains two zinc fingers, is important for both DNA binding and dimerization. The A and B domains have been demonstrated to have ligand-independent transcriptional transactivation activity (AF-1), whereas ligand-dependent transcriptional transactivation activity (AF-2) is associated with the E domain. The E domain, in addition, also contains all the information necessary for high affinity ligand binding and accessory dimerization sequences.
Recently, the x-ray crystal structures of the ligand binding domains of
apo-RXR and holo-RAR
were reported (14, 15). Analysis of these
two crystal structures has led to the suggestion that the novel folding
pattern observed in these two receptors, an antiparallel
-helical
sandwich, may be shared by all members of the steroid and thyroid
hormone superfamily (16). In addition, RAR subtype-specific
site-directed mutagenesis studies have identified amino acid residues
that are functionally important for the binding of RA to a given RAR
(17-22).
The similarities and differences between the ligand binding domains of
the nuclear RARs have been the topic of many articles in the current
literature. The experimentally determined differential retinoid
specificity of the three RAR subtypes has been the driving force behind
the successful efforts of several medicinal chemists to synthesize RAR
subtype-selective agonists and antagonists (23-30). On the other hand,
as described above, protein structural chemists suggest a common
folding pattern for the ligand binding domains of all RAR subtypes
(16). In an effort to help establish the structural requirements for
ligand specificity of RAR subtypes, we have examined the relative
importance of RA binding of several homologous, positively charged
amino acid residues located within the ligand binding domains of RAR
and RAR
. Lys227 of RAR
, unlike its homologous residue
Lys220 in RAR
, does not appear to be important for RA
binding. On the other hand, Arg276 in RAR
, like its
homologous residue Arg269 in RAR
, was found to play an
important role in the binding of RA most likely by interacting with the
carboxylate group of RA. However, the orientation of and electronic
environment associated with Arg276 in RAR
appears to be
different from that of Arg269 in RAR
, thus contributing
to the uniqueness of the ligand binding site of each receptor.
Mutants
were created according to the site-directed mutagenesis technique
described by Higuchi et al. (31). pSG5-mouse RAR1 and
pSG5-mouse RAR
2, generous gifts from Prof. Pierre Chambon (Institut
de Génétique et de Biologie Moléculaire et
Cellulaire, Strasbourg, France), linearized with BamHI and
XbaI, respectively, were used as templates for the
preparation of the mutants. Both sense (s) and antisense (as)
oligonucleotide primers were purchased from Ransom Hill BioScience (La
Jolla, CA). The GCT codon and the CAG codon were used to encode the
mutant Ala residues and mutant Gln residues, respectively, indicated in
bold and underlined in the mutagenic primers.
For the preparation of R276A RAR, two separate polymerase chain
reaction fragments were prepared using the primer pairs RAR
5
-s
(5
-GAGGGGGATCCATGGCCAGCAATAGCAG-3
) plus R276A-as
(5
-CTCAGGCGTGTACGTGCAG-3
) and RAR
3
-as
(5
-GAGGGAAGCTTTCATGGGGATTGGGTGG-3
) plus R276A-s (5
-CTGCACGTACACGCCTGAG-3
), respectively.
The two polymerase chain reaction fragments were purified, annealed,
and amplified in a second polymerase chain reaction using the RAR
5
-s and RAR
3
-as primers. Likewise, the K227A, R272A, R272Q, and
R276Q mutants were constructed using the RAR
5
-s and RAR
3
-as
primers and the following mutagenic primers: K227A-s
(5
-CTCTGGGACTTCAGTGAAC-3
) and K227A-as
(5
-GTTCACTGAACTCCCAGAG-3
), R272A-s
(5
-CCTGATTCTGATCTGCACG-3
) and R272A-as
(5
-CGTGCAGATCAGAATCAGG-3
), R272Q-s
(5
-CCTGATTCTGATCTGCACG-3
) and R272Q-as
(5
-CGTGCAGATCAGAATCAGG-3
), and R276Q-s (5
-CTGCACGTACACGCCTGAG-3
) and R276Q-as
(5
-CTCAGGCGTGTACGTGCAG-3
). The 527-base
pair SacI-BspEI fragment that contained the
desired mutation was exchanged with that of pSG5-RAR
wild type to
create each of the mutant DNA constructs. For pSG5-K227A/R276A, the
364-base pair PstI-EcoRV fragment of the
pSG5-R276A single mutant was exchanged with that of pSG5-K227A. The
pSG5-R265Q RAR
mutant was created in the same fashion, except that
the following primers were used: RAR
5
-s
(5
-GGGAGGGATCCATCGAGGGTAGATTTGACTGTATGGAT-3
), RAR
3
-as
(5
-GAAGGAAGCTTTCACTGCAGCAGTGGTGA-3
), R265Q-s
(5
-CTTGATTCTCATTTGTACC-3
), and R265Q-as
(5
-GGTACAAATGAGAATCAAG-3
), and the
EcoRV-BstXI fragment was exchanged between the
wild type and the mutant. In all cases, the presence of the specific
mutation and the lack of random mutations were verified by DNA sequence
analysis (32).
To make the pET-RAR prokaryotic expression constructs, the
full-length RAR
wild type protein coding region was synthesized by
polymerase chain reaction using the primers RAR
5
-s and RAR
3
-as and cloned into the BamHI and HindIII
restriction sites of pGem-3. The various mutants were prepared by
fragment exchange between the wild type construct in pGem-3 and the
given mutant via the PstI and BspEI sites. The
entire sequence of the wild type and the site of each mutation were
confirmed by DNA sequence analysis. Once constructed in pGem-3, each
RAR
cDNA was subcloned in frame in the BamHI and
HindIII restriction sites of pET-29a. For the pET-RAR
constructs, the MscI-StuI fragment containing the
desired mutation was exchanged with that of full-length wild type
RAR
previously cloned in frame into the NotI restriction site of pET29a (22).
Transactivation assays were performed as described previously (17, 18, 33). The EC50 value reported represents the concentration of retinoid that resulted in 50% of the maximal relative CAT activity determined by extrapolation from the plotted points.
To prepare recombinant protein for the Kd measurements, each pET-29a-RAR expression construct was transformed into Escherichia coli K12 strain BL21(DE3) cells (Novagen) (36). The expression of each S-Tag RAR protein and the preparation of the receptor extracts was performed as described previously (22). The production of the recombinant S-Tag wild type and mutant RAR fusion proteins in the receptor extracts was monitored using the S-Tag Western blot kit (Novagen). The Western blot analysis of the wild type and all mutant receptor extracts demonstrated a major band that migrated at the same position (approximate molecular mass, 55 kDa) along with several minor, smaller molecular mass bands, which were of similar size.
Retinoid binding assays were performed as described previously (22, 37) with the following exceptions: the total protein concentration in each assay was 6-12 µg, and both 9-cis-RA and all-trans-retinol binding were determined exactly as described for all-trans-RA using either [3H]9-cis-RA (1.74 TBq/mmol (47 Ci/mmol); Amersham Corp.) or [3H]all-trans-retinol (1.75 TBq/mmol (47.2 Ci/mmol); DuPont NEN).
Western Blot AnalysisThe levels of wild type and mutant
RAR protein were determined by Western blot analysis using CV-1
cells transfected with the indicated expression construct essentially
as described previously (17, 39).
The wild type
RAR, selected mutant RAR
proteins, wild type RXR
, and
-galactosidase used in the EMSA were all recombinant S-Tag fusion
proteins prepared in BL21 cells as described above. BL21 cell extract
(25 µg total protein) containing the indicated S-Tag proteins were
incubated in 25 mM Tris, pH 7.9, 125 mM NaCl, 2.5 mM EDTA, 25 mM dithiothreitol, 12.5 mM MgCl2, 12.5% sucrose, 12.5% glucose, 0.5%
Nonidet P-40, and 2.6 µg salmon sperm DNA (Sigma) containing a
32P-labeled RARE probe. The RARE probe was obtained by
annealing two complementary single-stranded oligonucleotides
(5
-TCGAGGGTAGGGTTCACCGAAAGTTCAC-3
and
(5
-CGAGTGAACTTTCGGTGAACCCTACCCT-3
), which contain the RARE in the
RAR
2 promoter (positions
63 to
33 relative to the start site of
transcription) (42). The resulting double-stranded RARE DNA was filled
in with Klenow polymerase (Promega) in the presence of
[32P]dCTP (111 TBq/mmol (3000 Ci/mmol); DuPont NEN).
Unlabeled cold RARE DNA was used in some assays as a competitor at a
100-fold excess. The RAR·RXR complexes were resolved by
electrophoresis through a 6% polyacrylamide gel containing 2.5%
glycerol in 0.5 × TBE (0.09 M Tris borate, pH 8.2, 0.002 M EDTA) at 200 V for 3 h. The gels were dried
and exposed to Kodak XRP x-ray film at
70 °C.
Initially
we examined the roles of Lys227 and Arg276 of
RAR in the binding of RA and RA-dependent
transactivation. These are the homologous amino acids to
Lys220 and Arg269 of RAR
(see Fig.
1), which we have previously demonstrated to act
together synergistically in the binding of RA (17, 18). Figs.
2 and 3 show representative
transactivation assays and saturation binding curves, respectively, for
wild type RAR
and selected RAR
mutants. Table I
lists the EC50 values and the apparent Kd values for the wild type and all mutant proteins. K227A displayed an EC50 value for all-trans-RA
and a Kd for both all-trans-RA and
9-cis-RA comparable to those of wild type RAR
. On the
other hand, R276A displayed low activity, with EC50 and
Kd values for both isomers of RA that were elevated approximately 100- and 50-fold, respectively, when compared with those
of wild type RAR
. Interestingly, mutation of Arg276 to
an Ala (R276A) had a more dramatic effect than the corresponding mutation to Gln (R276Q), with R276A displaying an approximately 3-fold
greater increase in both EC50 and Kd
values compared with R276Q. Finally, although the K227A/R276A double
mutant displayed very low activity in the RA-dependent
transactivation assay, the EC50 value was only increased
3-fold when compared with that of the single R276A mutant.
|
We next examined the role of Arg272 of RAR in RA binding
and RA-dependent transactivation, because its homologous
amino acid residue in RAR
(Fig. 1) has been implicated from its
x-ray crystal structure to be one of the positively charged amino acid
residues forming the electrostatic field gradient in the ligand binding pocket (15). Mutation of Arg272 to either Ala or Gln had a
negligible effect on the Kd for RA when compared
with that of the wild type receptor. On the other hand, the
EC50 value of R272A and R272Q in RA-dependent transactivation assays was increased approximately 25-fold when compared with that of the wild type receptor. Since we had observed this lack of correlation between RA binding and
RA-dependent transactivation activity when
Arg272 of RAR
was mutated, we examined the homologous
residue in RAR
(Fig. 1). Interestingly, R265Q RAR
displayed near
wild type activity in both RA-dependent transactivation
assays and RA binding studies.
Since R269Q RAR displays high affinity for and
transactivation activity with retinol (Ref. 18; see Table
II), we examined the activity of R276Q RAR
in similar
assays. Table II shows that, as expected, wild type RAR
does not
bind retinol within the limits of the binding assay and has low
activity in transactivation assays with retinol (EC50, 1 µM). Unlike R269Q RAR
, R276Q RAR
displayed no
detectable binding of retinol or measurable activity in the retinol-dependent transactivation assays. As a positive
control for the retinol binding assays, we measured the
Kd for retinol of recombinant R269Q RAR
and
obtained a value of 28 nM, which is quite comparable to our
previously measured 18 nM using nuclear extracts prepared
from COS cells transfected with R269Q DNA (18). Interestingly, R276Q
displayed at least 10-fold lower activity in the
retinol-dependent transactivation assays when compared with
that of wild type RAR
. The small amount of activity observed in the
retinol-dependent transactivation assays is likely to be
due to low levels of RA formed within the cells because of the
oxidation of retinol. If the RA formed from the oxidation of retinol is
indeed responsible for the observed activity, it is not unexpected that
R276Q would display a higher EC50 value than that of the
wild type in retinol-dependent transactivation assays,
because R276Q has an approximately 40-fold higher EC50 value with RA compared with that of the wild type receptor (300 compared with 8 nM).
|
Fig. 4 is a Western blot showing wild type
and selected RAR mutant protein levels in nuclear extracts isolated
from transfected CV-1 cells. A similar level of RAR
protein was
detected in the nuclear extracts of cells transfected with the wild
type and all the mutant DNAs. Furthermore, Fig. 5 shows
an EMSA using wild type and selected RAR
mutant S-Tag recombinant
proteins. All mutant RAR
proteins dimerized with RXR
and bound a
RARE, resulting in a gel shift pattern comparable to that of wild type
RAR
. This demonstrates that the differences between the
EC50 and Kd values of the wild type
RAR
and the mutant receptors (R272A, R272Q, R276A, R276Q, and
K227A/R276A) are not likely to be due to any gross conformational
changes in these receptors, since they behave normally with respect to
dimerization, DNA binding, and expression pattern in transfected CV-1
cells.
In this report we have examined the effect of mutation of several
positively charged amino acid residues of RAR on retinoid binding
and retinoid-dependent transactivation activity.
Arg276 was found to play a major role in RA binding and
RA-dependent transactivation, whereas Lys227
does not appear to be important for either of these two activities in
RAR
. In addition, Arg272 does not appear to be important
for RA binding but may be important in the determination of the final
active conformation of holo-RAR
, since the R272A mutant displayed a
significant reduction in RA-dependent transactivation
activity. It is unlikely that global conformational changes are
responsible for the reduced activity in the RA binding and
RA-dependent transactivation assays observed with these
RAR
mutants, since all the RAR
mutants examined displayed similar levels of expression in transfected CV-1 cells and similar activity in
the EMSA.
Table III presents a comparison of the -fold increase in
EC50 and Kd values for
all-trans-RA of several of the RAR mutants described in
this report compared with the homologous RAR
mutants that have
previously been reported (17, 18). In both RAR
and RAR
, the
homologous Arg (Arg276 and Arg269,
respectively) plays an important role in the binding of RA most likely
by interacting with the carboxylate group of RA. Site-specific mutation
of this Arg in both receptors results in a significant reduction in RA
binding and RA-dependent trans-activation activity. This is
consistent with the RAR
crystal structure, in which the homologous
amino acid residue, Arg278, has been shown to form a salt
bridge with the carboxylate O22 of RA (15). However, we observed
several important differences in the response of Arg276 in
RAR
compared with that of Arg269 in RAR
when mutated
to either Ala or Gln. Mutation of this Arg to Ala causes a significant
reduction in RA binding and RA-dependent transactivation
activity in RAR
and has a minor effect in RAR
(approximately 75- versus 8-fold). On the other hand, mutation of this Arg to
Gln results in a much less dramatic reduction in RA binding and
transactivation activity of RAR
than that of RAR
(approximately
30- versus 1000-fold). Finally, R269Q RAR
is a very
efficient retinol receptor, whereas R276Q RAR
has no detectable retinol binding or retinol-dependent transactivation
activity (Table II). Taken together these data suggest that the
orientation of Arg276 and Arg269 in the ligand
binding site and the electronic environment associated with this Arg is
different in RAR
compared with RAR
, contributing to the
uniqueness of the ligand binding site of each receptor.
|
In RAR, Lys220 was found to act synergistically with
Arg269 in the binding of RA, since the simultaneous
mutation of both residues to Ala resulted in an effect much larger than
the additive effect of each single mutation both in RA binding and
RA-dependent transactivation activity. This synergistic
effect was not observed with the K227A/R276A RAR
mutant, suggesting
that Lys227 in RAR
does not appear to be involved
directly in the binding of RA; however, it may function as part of the
electronic guidance force, proposed to guide RA into the binding site,
described in the crystal structure of RAR
(15). Since the single
mutation of Arg276 to an Ala in RAR
has such a dramatic
effect on RA binding and RA-dependent trans-activation
activity, it is possible that this single amino acid residue may act
more independently in RA binding in RAR
than the homologous Arg in
RAR
. It is interesting to note that the crystal structure of RAR
shows that Lys236 and Arg278 directly interact
with the carboxylate group of RA, whereas Lys229 does not
appear to be sufficiently close to the carboxylate group of RA to be
directly involved in its binding. It is possible that different
positively charged amino acid residues may act synergistically with the
RAR
Arg276 homologous position in each of the three RAR
subtypes, further contributing to the unique nature of each of these
three retinoid binding sites.
Based on the crystal structure of RAR, Renaud et al. (15)
have reported that there are 24 amino acid residues distributed over
eight structural elements in the ligand binding domain that are
positioned within 4.5 Å of RA and therefore delineate the ligand
binding pocket. These eight structural elements include H1, H3, H5,
-turn, loop 6-7, H11, loop 11-12, and H12. Furthermore, Wurtz
et al. (16) have suggested that the ligand binding pockets of all nuclear receptor holo-ligand binding domains have a similar architecture involving these structural elements. When the amino acid
sequences of the three RAR subtypes are compared, only 3 of these 24 amino acid residues lining the ligand binding pocket are variable
(Ser232, Ile270, and Val395 in
RAR
). All three of these divergent residues in RAR
are associated with
-helices, which form the hydrophobic portion of the ligand binding pocket and interact with the
-ionone ring and/or the isoprenoid side chain of RA. These three divergent residues have been
suggested to play a role in the determination of the ligand specificity
of the three RAR subtypes (15, 16).
Our data demonstrate that Lys227 and Arg276 of
RAR, when mutated to Ala and Gln, behave differently in both
retinoid binding assays and retinoid-dependent
transactivation assays when compared with the homologous mutations in
RAR
. These data suggest that there are other structural features of
the ligand binding pocket besides the three divergent residues
described above that are unique to each receptor subtype. Of the eight
structural elements demonstrated to form the ligand binding pocket of
RAR
, four of these (H1, H3, C-terminal portion of H5 including
Arg278, and
turn) contribute to defining that portion
of the pocket involved in the interaction with the carboxylate group of
RA. All of the amino acid residues of these four structural elements demonstrated to be within 4.5 Å of RA in RAR
are conserved among the three RAR subtypes. However, when one considers the conservation of
all amino acids that constitute these four structural elements of
RAR
and RAR
, only H1 and H3 contain residues that are not conserved. Six of the 18 residues in H1 (Ala182,
Val184, Gly185, Glu186,
Ile188, and Val190 of RAR
) and 4 of the 23 residues in H3 (Ile222, Asp223,
Ser232, and Thr237 of RAR
) differ between
RAR
and RAR
. In addition, 3 of the 5 positively charged amino
acids reported to be part of the positively charged electrostatic
guidance field in RAR
are located on H3, whereas the other 2 are
located on H5. Therefore, it is likely that these divergent residues in
H1 and H3 may play an important role in defining the orientation of and
electronic environment associated with Arg269 in RAR
compared with Arg276 in RAR
. Thus, consideration of
these divergent residues in H1 and H3 and their effect on the
positioning of the positively charged amino acid residues in H3 and H5
may be useful in the construction of subtype-selective retinoids.
We thank Erin Fitzgibbons, Sijie Zhang, Zeng-ping Zhang, Susan Horvath, and Gladys Yumet for their technical assistance. In addition, we thank Prof. Pierre Chambon for the pSG5-RAR constructs, Dr. Ronald Evans for the RARE-CAT reporter construct, and F. Hoffmann-LaRoche for the retinoids used in these studies.