(Received for publication, December 4, 1996, and in revised form, April 11, 1997)
From the Preclinical Research, Department of Infectious Diseases, F. Hoffmann-La Roche Ltd., CH-4070 Basel, Switzerland
Retinoids exert their pleiotropic effects on cell
differentiation and proliferation through specific nuclear receptors,
the retinoic acid receptors (RARs) and retinoid X receptors (RXRs). Two
biologically highly active natural retinoids have been identified, all-trans-retinoic acid (t-RA) and
9-cis-retinoic acid (9-cis-RA). The RXRs
exclusively bind 9-cis-RA, whereas the RARs bind both isomers of RA with comparable affinity. Recently published results suggest that RARs have the same binding site for t-RA and
9-cis-RA but with different determinants (1-3). Antagonist
binding on RAR has been suggested to induce distinct conformational
changes in comparison with agonist binding. To elucidate the region
minimally required for efficient binding of agonist (t-RA and
9-cis-RA) and antagonist Ro 41-5253 to the RAR
, we
generated N- and C-terminally truncated mutants of the receptor.
Characterization of these deletion mutant proteins using protease
mapping and ligand binding experiments revealed that different parts of
the ligand-binding domain are necessary for t-RA, 9-cis-RA,
and antagonist binding. Three distinct regions of the ligand-binding
domain of the human retinoic acid receptor-
are required for binding
of t-RA (RAR
187-402), 9-cis-RA (RAR
188-409), and
the antagonist Ro 41-5253 (RAR
226-414).
Retinoic acid (RA)1 has a broad
spectrum of biological activities in vertebrate development and
homeostasis (4-6). Due to their fundamental role in the control of
cell differentiation and proliferation, RA and synthetic retinoids are
clinically very useful, predominantly in the treatment of leukemia and
nonmalignant hyperproliferative disorders of the skin. However,
retinoids have undesirable side effects such as hypervitaminosis A
syndrome and teratogenicity (4, 7-10). Two biologically active
stereoisomers of RA have been identified, all-trans-retinoic
acid (t-RA), and 9-cis-retinoic acid (9-cis-RA)
(11-13). The direct biological effects of the retinoids are mediated
by the activation of two subfamilies of nuclear receptors, the retinoic
acid receptors (RARs) and the retinoid X receptors (RXRs) (13-16).
Both subfamilies consist of three receptor subtypes referred to as
RAR, -
, and -
(14, 15, 17-20) and RXR
, -
, and -
(13,
16, 21). RARs and RXRs belong to the nuclear hormone receptor
superfamily, whose members act as ligand-inducible transcription
enhancer factors (22-24). On the basis of homology to other nuclear
hormone receptors, the sequences of RARs are divided into six distinct
regions designated A through F. The C domain constitutes a highly
conserved DNA-binding domain, while the E domain confers the ligand
binding properties of each receptor (25). RARs seem to operate
effectively only as heterodimeric RAR·RXR complexes (26-31), but
RXR-independent transactivation by RARs has also been observed (32).
The RXRs are able to activate genes via homodimers (33, 34), but act predominantly as coregulators, enhancing the binding of RA, vitamin D3,
thyroid hormone, and peroxisome proliferator-activated receptors to
their response elements via heterodimerization (26-31, 35). t-RA and
9-cis-RA are the natural ligands for RARs (14, 36), whereas
9-cis-RA and phytanic acid are the natural ligands for RXRs
(11, 12, 37). Both RA isomers compete with each other for RAR binding
(38). A synthetic retinoid Ro 41-5253 has been identified as a
selective RAR
antagonist (39), which, when bound to the receptor,
induces a different conformational change as detected by limited
proteolysis (40). Recently, the structure of the cocrystal hRAR
-t-RA
has been determined (1), and modeling of 9-cis-RA in the
RAR
binding pocket suggests that the two isomers also compete for
the same binding site in this receptor. In addition, two residues,
Met-406 and Ile-410, have been shown to be critical, specifically for
the binding of 9-cis-RA to hRAR
(2). Binding experiments
using RA and different synthetic ligands have shown different
requirements in the C-terminal part of the hRAR
but no distinction
between t-RA and 9-cis-RA binding specificity (41). It has
also been demonstrated recently that Cys-235, Arg-217, and Arg-294 of
hRAR
play a role in antagonist binding, showing specific
requirements for efficient antagonist binding to hRAR
(3). As shown
by competitive binding experiments, t-RA competes with the antagonist
for RAR
binding (40). This strongly suggests that the antagonist
shares a common binding site with the RA isomers. To analyze the
minimal structural requirements for an efficient binding of either
t-RA, 9-cis-RA, or Ro 41-5253 to the hRAR
, we generated
N- and C-terminally truncated RAR
mutants. Characterization of these
deletion-mutant proteins using protease mapping and ligand binding
experiments revealed that different parts of the ligand-binding domain
of RAR
are necessary for t-RA, 9-cis-RA, and antagonist binding.
[3H]t-RA (50 Ci/mmol) was obtained from NEN Life Science Products. 9-cis-[3H]RA (25 Ci/mmol) was obtained from Amersham Corp. [3H]Ro 41-5253 (28 Ci/mmol), unlabeled RA isomers and analogues were synthesized at F. Hoffmann-La Roche Ltd., Basel, Switzerland. All procedures using retinoids were carried out under dimmed light. The TNT® coupled reticulocyte lysate system was purchased from Promega. [35S]Methionine (>1000 Ci/mmol), 14C-methylated protein molecular weight markers, and AmplifyTM were obtained from Amersham. Restriction enzymes were obtained from Boehringer Mannheim. Trypsin (type I: from bovine pancreas) was purchased from Sigma.
PlasmidsThe human RAR cDNA insert of pT7-RAR
(42) was subcloned as a MscI-BamHI fragment into
pSG5-MscI giving pSG5-RAR
. pSG5-MscI was
constructed by replacing the EcoRI-BamHI fragment
of pSG5 (43) with a linker containing an MscI site. RAR
deletion mutants were produced by PCR. The correctness of the sequence
was confirmed by sequencing. For N-terminal deletions: removal of the
EcoRI site of pET3a (44) was carried out by restriction with
EcoRI, fill-in reaction and religation, yielding pET3a-
E.
The linker (5
-TATGGAATTCTAGCGCTTA-3
), including a start
codon and a subsequent EcoRI site, was ligated into the
NdeI site of pET3a-
E giving pET3a-E. pSG5-M was
constructed by ligation of the linker
(5
-AATTGCTACCACCATGGAATT-3
) in the EcoRI site
of pSG5. The mutants were generated by PCR using pT7-RAR
as
template. The PCR products were restricted with
EcoRI-BamHI (located in the 5
primer and 3
primer, respectively) and cloned into pET3a-E (expression in
Escherichia coli for binding studies) or pSG5-M (in
vitro translation for limited proteolytic digestion). For
C-terminal deletions, the mutants were generated by PCR using pT7-RAR
as template, 5
primers (positions 466-486), and 3
primer containing a BamHI site. The PCR products were restricted
with SacI-BamHI and cloned into either the
full-length pSG5-RAR
restricted with
SacI-BamHI (in vitro translation for
limited proteolytic digestion) or the truncated pET3a-RAR
-
155
restricted with SacI-BamHI (expression in
E. coli for binding studies).
RAR deletion mutants were expressed in E. coli BL21(DE3)pLys (44). Expression and solubility problems
occurred with the N-terminal deletion mutants hRAR
-
188, -
189,
-
225, -
226, and -
227 expressed in E. coli. These
truncated proteins were synthesized with the TNT® coupled reticulocyte
lysate system (Promega) for the determination of the IC50
values. For these last mutant receptors, a 40-60% specific binding
was sufficient to allow the IC50 determination. Since
Kd values, for either t-RA or 9-cis-RA,
have been established for the C-terminal point mutants of hRAR
(aa
405-419) (2), we determined the binding activity of the C-terminally truncated receptors by Scatchard analysis for the same ligands. From
the N-terminally truncated hRAR
(aa 187-227), no binding activity
was determined previously, and we verified the binding activity by
IC50 determination. The binding assays
(Kd and IC50 determinations) were
performed as described elsewhere (3).
Human RAR (wt or truncated) in pSG5 was in
vitro transcribed and translated in the presence of
[35S]methionine by using rabbit reticulocyte lysates as
specified by Promega. The limited proteolytic digestions were performed as described elsewhere (40).
An important region for the t-RA binding (aa 186-198)
was defined previously in hRAR by a N-terminally truncated receptor (41). A C-terminal deletion to position 403 resulted in a moderate decrease in affinity for the same ligand (41). On the other hand, a
C-terminal deletion to position 404 of hRAR
exhibited a
Kd of 0.3 nM for t-RA (2). To determine
the precise region of the hRAR
-LBD required for an efficient t-RA
binding, we used binding assays and protease mapping to probe t-RA
binding to N- and C-terminally truncated hRAR
-LBD (Figs. 1 and
2).
A Kd of 1.1 nM for t-RA was obtained
with the hRAR-LBD (aa 155-462) (Table I). This value
is in the range of the Kd published for the
full-length hRAR
expressed in E. coli or COS cells (0.67 nM (42), 1.7 nM (45)), and also for the
hRAR
-LBD expressed in E. coli (6.2 nM (46);
0.6 nM (3)).
|
Among the N-terminally truncated receptors, the hRAR-
187
exhibited good protection against trypsin digestion when bound to t-RA.
A strongly reduced signal was observed for the digestion of the
hRAR
-
188 truncated receptor under the same conditions. At the
C-terminal end of the receptor, the same tendency was observed with the
two mutants hRAR
-
402 and hRAR
-
401. In regard to the binding
activity of these mutant receptors, the N-terminally truncated hRAR
-
187 exhibited a Kd value 4.5-fold higher
than the wild type hRAR
-LBD, whereas a deletion of a single further amino acid abolished the binding activity in such a way that neither Kd nor IC50 could be determined (Table
II).
Similar experiments were carried out using N-terminally truncated
mutants of hRAR. We found that hRAR
-
189 (hRAR
-
187) was
able to bind t-RA with an affinity comparable with the full-length hRAR
, whereas hRAR
-
190 (hRAR
-
188) showed no detectable
binding activity to this ligand (data not shown). Therefore, it appears that, while the C-terminal region of the D domain of RAR
(aa 188-199) is required for efficient binding of the t-RA, the C-terminal part of the E domain (aa 403-419) and the entire F domain can be
deleted without affecting the ability of the RAR
to bind its natural
t-RA ligand (see Fig. 3).
Binding of 9-cis-RA to the N- and C-terminally Truncated RAR
Two contradictory results have been published concerning
the binding of 9-cis-RA to C-terminal deletion mutants of
hRAR. Lefebvre et al. (41) observed that a deletion up to
position 403 of the hRAR
expressed in E. coli resulted in
a moderate decrease in affinity for 9-cis-RA (41). On the
other hand, a small region (aa 405-419) within the ligand binding
domain of a truncated hRAR
was demonstrated to be required for the
9-cis-RA binding (2). In our study, a Kd
value of 1 nM was determined for the binding of
9-cis-RA to the hRAR
-LBD. This value is in
agreement with the range of the reported data for the truncated
receptors expressed in E. coli (0.3 nM for the
hRAR
-
419 (2) and 1.2 nM for the hRAR
-LBD (3)).
After the limited trypsin digestion of the N-terminally truncated
hRAR liganded to 9-cis-RA, the corresponding protection was almost completely abolished for the mutant hRAR
-
189, and a
reduction of the signal was already observed for the mutant hRAR
-
188 (Fig. 2). At the C-terminal end of the receptor, the digestion of the hRAR
-
409 liganded to 9-cis-RA by
trypsin yielded a band of the appropriate size, whereas the truncated
hRAR
at the next residue (hRAR
-
408) exhibited only a faint
band (Fig. 1). IC50 and Kd
determinations for the binding of the 9-cis-RA to the above
mentioned truncated receptors confirmed the N- and C-terminal deletion
borders of the hRAR
concerning the RA isomer. No 9-cis-RA
binding was detectable to the hRAR
-
189, whereas a 5-fold higher
IC50 value was obtained for the binding of the t-RA isomer
to the hRAR
-
188 in comparison with the hRAR
-LBD (Table II). At
the C-terminal end, a 35-fold higher Kd value, was
obtained for the hRAR
-
409, while the hRAR
-
408 bound the
9-cis-RA more than 150-fold less efficiently than the
hRAR
-LBD (Table I). From these results, it is clear that the
C-terminal region of the D domain of RAR
(aa 189-199) is required
for an efficient binding of 9-cis-RA. Furthermore, the
C-terminal part of the E domain (up to aa 409) is also crucial for the
binding efficiency of this ligand to the hRAR
(see Fig. 3).
The antagonist Ro 41-5253 has been reported to induce a
different conformational change when bound to the hRAR (40). It has
also been shown recently that Ro 41-5253, as well as other antagonists,
exhibit distinctly different requirements for efficient binding to
hRAR
(3). In the present study, we determined an IC50
value of 16 nM for the binding activity of the antagonist Ro 41-5253 to the hRAR
-LBD (Table I). Limited trypsin proteolysis of
the N- and C-terminally truncated hRAR
bound to Ro 41-5253 led to
the definition of the N-terminal deletion border between the residue
D226 and the residue Lys-227 and the C-terminal deletion border between
the residue L414 and M413 of the E region of the hRAR
-LBD.
Concerning the binding activity of Ro 41-5253 to the truncated
hRAR
s, 24- and 15-fold higher IC50 values for
hRAR
-
227 and hRAR
-
413 have been observed, respectively, in
comparison with the hRAR
-LBD (Tables I and II). Remarkably, the
hRAR
-
226 and the hRAR
-
414 mutant exhibited only a 2- and
3.5-fold reduction in IC50, respectively, in comparison
with the hRAR
-LBD. These results evidence distinctly different
requirements of the N- and C-terminal regions of the ligand-binding
domain of the hRAR
for binding Ro 41-5253 compared with the two
other ligands t-RA and 9-cis-RA (see Fig. 3).
It has been shown that the binding of both t-RA and
9-cis-RA induces a different conformational change in
hRAR to that observed with the binding of the antagonist Ro 41-5253 (40). At present, several arguments allow one to think that, depending
on the chemical structure of the ligands, distinctly different
determinants are required for the efficient binding to the RARs.
Recently published results also showed distinct determinant
requirements for the binding of different ligands to the estrogen
receptor (47). The recently described crystal structure of the RAR
ligand-binding domain bound to t-RA has clarified the ligand binding
interactions. Modeling 9-cis-RA in the hRAR
binding
pocket revealed that the binding site of this receptor was likely to be
very similar to that of t-RA (1). In addition, three residues have been
shown to play a significant role in ligand binding (agonist and
antagonist) to hRAR
, whereas Cys-235, Arg-217, and Arg-294 have been
shown to be important residues of the hRAR
-LBD, specifically for
antagonist interactions (3). Furthermore, Arg-269 of the hRAR
(Arg-276 of hRAR
and Arg-278 of hRAR
) has been shown to be a
crucial residue for RA binding (48, 49). These findings and the highly conserved amino acid homology of the E domain of the RARs (92%) (23)
suggest that the binding conditions of t-RA to the three subtypes of
receptors RAR
, -
, and -
are very similar. It also indicates
that these ligands may compete for a unique binding site in the
RARs.
In the present study, the 30-fold higher Kd value
for 9-cis-RA binding to the C-terminal deletion
hRAR-
409 reinforces the role of the residue Ile-410 in the
9-cis-RA binding as shown by Tate and Grippo (2). We have
provided evidence by this study that the complete F domain can be
removed without disturbing the ability of the hRAR
to bind any
ligand. The C-terminal part of the E domain of the hRAR
(to aa 414)
is sufficient for the efficient binding of the antagonist Ro 41-5253, whereas further deletion of the E domain of the hRAR
to positions
409 or 402 for 9-cis-RA and t-RA, respectively, does not
disturb the ability of the receptor to bind these two ligands.
Interestingly, t-RA and 9-cis-RA require the C-terminal part
of the D domain of the hRAR
(from aa 187 and 188, respectively),
while the D domain and the N-terminal part of the E domain (aa 155 to
226) are not required for the efficient binding of the antagonist Ro
41-5253.
From the crystal structure of the hRAR-LBD bound to t-RA and the
modeled 9-cis-RA in the hRAR
binding pocket, less
distance was expected between the ligand 9-cis-RA and the
C-terminal region of the hRAR
(helix-11 and helix-12) in comparison
with the t-RA spatial disposition (1). It was also clear that the
C-terminal part of helix-1 overlaps with the C-terminal part of
helix-3. The importance of residue Cys-235 of the hRAR
in antagonist
binding (3) indicates a role of the helix-3 in antagonist binding. Remarkably, all of helix-1 can be deleted without disturbing antagonist binding, whereas this completely abolishes agonist binding. An explanation for this could be that the C-terminal part of helix-1 may
stabilize the spatial location of helix-3 required specifically for the
binding of the agonists.
Taken together, these results indicate that three different fragments
of hRAR are minimally required for the binding of t-RA, 9-cis-RA, and Ro 41-5253. The data shown here suggest that
distinct determinants of hRAR
are either directly involved in the
binding of these three ligands or, alternatively, they are needed to
maintain the binding pocket in an appropriate conformation as a
prerequisite for the binding of each of the isomers of RA as well as Ro
41-5253.
Limited trypsin digestion of either wild type or truncated receptor
yielded the same digested peptides corresponding to the potential
cleavage sites (data not shown). The undigested mutant receptors showed
positions in the gel that were in accordance with their calculated
molecular weights (Fig. 1, lanes 2-13; Fig. 2, lanes
2-10). Also, the molecular weights of the fragments as determined
from the SDS gel were in good accordance with the magnitude of the
truncation of the mutants (Figs. 1 and 2). This indicates that the
binding of either the agonists t-RA and 9-cis-RA or the antagonist Ro 41-5253 to the truncated receptors induces the same conformational change in all the mutants as that observed with the
full-length RAR. In this study, we demonstrate that with trypsin
digestion of 9-cis-RA-bound full-length hRAR
, the
resulting 30-kDa fragment is less protected against further proteolysis than the one derived from the analogous experiment with t-RA. This was
evidenced by the time course of tryptic digestion of RAR
bound to
t-RA versus 9-cis RA-bound receptor (Fig.
4). These results were further confirmed by tryptic
proteolysis with increasing protease concentrations (data not shown).
These differences in the proteolytic resistance cannot be explained by
distinct affinities of the RA isomers to the RAR
, because both
exhibit Kd values in the range of 1 nM
(Tables I). The decreased stability of the 30-kDa fragment obtained
from RAR
bound to 9-cis-RA could reflect the higher
off-rate of 9-cis-RA from RAR
in comparison with the
t-RA. Displacement assays have demonstrated that 9-cis-RA exhibits about 2-fold higher off-rates from murine RAR
than t-RA (38). Time course experiment using Ro 41-5253 as ligand yielded also a
decreased stability of the 25-KDa antagonist characteristic fragment
compared with the stable 30-kDa fragment observed with t-RA (Fig. 4).
Incubation of the receptor with Ro 41-5253 or 9-cis-RA instead of t-RA in the digestion assay could result in more unliganded receptors, or fragments, for a short period of time. During this time,
they might change to a more relaxed conformation, more accessible to
the protease.
In conclusion, we have evidenced that three distinct regions of the of
the hRAR-LBD are required for the efficient binding of t-RA,
9-cis-RA, and the antagonist Ro 41-5253. t-RA binding requires the region of the hRAR
-LBD from amino acid 187 to amino acid 402, 9-cis-RA requires the region of the hRAR
-LBD
from amino acid 188 to amino acid 409, while the antagonist requires
the region of the hRAR
-LBD from amino acid 226 to amino acid
414.
We thank M. Klaus (F. Hoffmann-La Roche Ltd., Basel, Switzerland) for synthesizing the retinoids, P. LeMotte, P. Lardelli and W. Keck for critically reading the manuscript. We acknowledge C. Lacoste, B. Rutten,and O. Partouche for their expert technical assistance.