(Received for publication, March 7, 1995; and in revised form, June 13, 1995)
From the
We have recently identified a small region (amino acids
405-419) within the ligand binding domain of a truncated human
retinoic acid receptor (
419) that is required for binding of
9-cis-retinoic acid (RA), but not all-trans-retinoic
acid (t-RA). To probe the structural determinants of this high
affinity 9-cis-RA binding site, a series of
419 mutants
were prepared whereby an individual alanine residue was substituted for
each amino acid within this region. These modified receptors were
expressed in mammalian COS-1 cells and assayed for their ability to
bind 9-cis-RA as well as t-RA. Only two of the
mutants, M406A (mutation of methionine 406 to alanine), and I410A
(mutation of isoleucine 410 to alanine) exhibit no detectable binding
of 9-cis-RA when analyzed using saturation binding kinetics.
Substitution of methionine 406 with the amino acids leucine,
isoleucine, and valine yields mutant receptors that exhibit decreased
binding for 9-cis-RA as the length or hydrophobicity of the R
group decreases. Further substitution of methionine 406 with the small
polar amino acid, threonine, results in a loss of detectable
9-cis-RA binding. Since amino acids 405-419 on a human
RAR
(hRAR
) are predicted to form a short amphipathic
-helix, modeling of this structure into a helical wheel indicates
that these two amino acids, methionine 406 and isoleucine 410, are
actually positioned proximal to each other. Data presented here suggest
that high affinity 9-cis-RA binding to a hRAR
depends on
an interaction with the two amino acids methionine 406 and isoleucine
410.
The retinoic acid receptors (RARs) ()(1, 2, 3, 4) and the
retinoid X receptors (RXRs, 5-8) have a prominent role in
cellular differentiation and vertebrate development through their
interaction with retinoic acids (RA), which are natural derivatives of
vitamin A (retinol, (9) and (10) ). The RARs and the
RXRs are members of a superfamily of ligand-dependent transcriptional
regulators which also include the steroid hormone, thyroid hormone, and
vitamin D receptors(11, 12) . Based on amino acid
homologies and functional similarities, six regions of identity (A-F)
have been ascribed to members of this superfamily, including the RARs
and RXRs, through which modulation of transcription can
occur(11, 12) . Regions A and B contain a
ligand-independent transcription function (AF-1) while region C
contains a highly conserved zinc finger DNA-binding domain. The
functions of regions D and F are still relatively unknown, however,
region E is known to contain a multiplicity of functions, namely, those
of heterodimerization, ligand-dependent transactivation (AF-2) and
ligand binding(13) .
Both the RARs and RXRs bind endogenous
stereoisomers of RA within the ligand binding region and undergo a
conformational change prior to their activation as transcriptional
regulators(14, 15, 16, 17) . The
RXRs show a binding preference for 9-cis-RA (18, 19, 20, 21) while the RARs bind
9-cis-RA as well as the stereoisomer, all-trans-RA (t-RA), with equal affinity(18) . An analysis of
ligand binding to mouse RARs using saturation kinetics and Scatchard
analyses gave dissociation constants (Kvalues) in the range of 0.2-0.7 nM for both
9-cis-RA and t-RA(22) . Competition of
[
H]t-RA or
[
H]9-cis-RA by 9-cis-RA and t-RA indicates that the two ligands can compete with one
another for binding to the RARs(22) . These binding analyses
led to the seemingly obvious conclusion that a common binding pocket on
an RAR exists for both t-RA and its configurational isomer,
9-cis-RA.
Recently, however, we found that there are actual
differences in the binding pocket for these two ligands and that a
distinct region is required for the binding of 9-cis-RA, but
not t-RA, to a hRAR(17) . Truncation of 43 amino
acids to position 419 (removal of the F domain) on a hRAR
produced
a mutant which had a binding affinity for both t-RA and
9-cis-RA with normal K
's
of 0.2-0.3 nM for both ligands. Truncation of 58 amino
acids to position 404, however, produced a mutant which had a normal
binding affinity for t-RA (K
= 0.3 nM), but which demonstrated no
detectable binding of 9-cis-RA. We concluded that the binding
sites for t-RA and 9-cis-RA on a hRAR
were
overlapping but distinct and that the region from amino acids 405 to
419 defined a high affinity binding determinant for 9-cis-RA.
Interestingly, this region also corresponded with the highly conserved
AF-2 domain found in the majority of receptors of the steroid hormone,
thyroid hormone, and vitamin D superfamily(23) . Therefore,
this same mutant which lost the ability to bind 9-cis-RA
(
404) also failed to initiate transcription on a retinoic
acid-responsive element after binding t-RA. Addition of these
15 amino acids (405-419) to a
404 resulted in a recovery of
both 9-cis-RA binding and transactivation ability and yielded
a fully functional receptor (
419, (17) ).
Since we had
isolated a high affinity 9-cis-RA binding determinant to
within 15 amino acids, we were then able to analyze those motif(s)
responsible for this high affinity binding. Therefore, we have further
characterized the region from amino acids 405 to 419 using alanine
scanning mutagenesis on a truncated hRAR and have found that the
integrity of two amino acids, methionine 406 and isoleucine 410, appear
critical for high affinity 9-cis-RA binding. Based on a Chou
and Fasman (24) algorithm which predicts that this region forms
a short
-helical structure, these two amino acids actually exist
in close proximity to each other. From our data we propose a model of
9-cis-RA binding to a truncated hRAR
that may involve a
contact with these two amino acids, methionine 406 and isoleucine 410.
Figure 3:
Saturation binding analysis of
[H]t-RA or
[
H]9-cis-RA binding to the
404,
M406A, and E415,418A mutant receptors. Nucleosol from COS-1 cells
transiently transfected with the indicated mutant receptors was
incubated with the indicated concentrations of
[
H]t-RA (A, B, and D)
or [
H]9-cis-RA (E) in the
absence or presence of a 100-fold molar excess of unlabeled t-RA (A, B, and D) or 9-cis-RA (E). Nonspecific binding activity (open circle) was
subtracted from total binding activity (open square) to
generate specific binding activity (triangle). Scatchard
analysis (C) revealed that M406A (closed triangles, K
= 4.2 nM) had a 14-fold
lower binding affinity for [
H]t-RA than
404 (open triangles, K
= 0.3 nM). Neither mutant bound
[
H]9-cis-RA with any apparent affinity.
Scatchard analysis (F) also revealed that the double mutant,
E415,418A, bound both [
H]t-RA (open
triangles) and [
H]9-cis-RA (closed triangles) with apparent equal affinity (K
values = 0.8 and 0.9
nM, respectively).
Figure 1:
Schematic of the truncated, mutant
human retinoic acid receptor sequences. A schematic
representation of the hRAR
receptor truncated to amino acid
position 419 (
419) is indicated at the top. The dark
region corresponds to the ligand binding domain and the black
lines indicate the region containing amino acids that have been
replaced by alanine (from amino acid 405 to 419). The amino acid
sequences are shown below. Deletions are symbolized by
and the numbers refer to the positions in the amino acid
sequence. The hRAR
mutants were made as described under
``Experimental Procedures'' by ligation of synthetic,
double-stranded oligonucleotides.
For each mutant receptor depicted in Fig. 1, nucleosol was isolated from mammalian COS-1 cells
transiently transfected with the corresponding receptor expression
vector. [H]t-RA or
[
H]9-cis-RA was then incubated with
these nucleosol fractions for 4 h at 4 °C since little
isomerization has been shown to occur under these
conditions(22) . The results are shown in Fig. 2and
9-cis-RA binding is calculated as %t-RA binding
activity, where total binding of t-RA has been normalized to
yield between 300 and 500 fmol of transfected receptor binding sites
for each assay (from 15 to 25 µl of nucleosol).
Figure 2:
Percent t-RA binding activities
of hRAR
419 alanine mutants. The mutant receptors were
transiently expressed in COS-1 cells and nucleosol was prepared as
described under ``Experimental Procedures.'' Nucleosol was
incubated with [
H]t-RA or
[
H]9-cis-RA and total binding was
calculated in the presence of 100-fold molar excess cold ligand.
%t-RA binding was then reported as the total binding with
[
H]9-cis-RA/total binding with
[
H]t-RA (normalized to 300-500
fmol)
100. The truncation mutants
404 and
419 are
also indicated.
The alanine
substitutions that most strikingly disrupt 9-cis-RA binding
are those at methionine 406, isoleucine 410, and proline 407. These
substitutions result in only 5.6, 4.0, and 14% of 9-cis-RA
binding activity, respectively, relative to that of t-RA. The
parent receptor, 419, exhibits 89% 9-cis-RA/t-RA
binding activity (Fig. 2). Alanine substitution of other amino
acids proximal to methionine 406 also reduce 9-cis-RA binding
but not as severely. For example, P408A and L409A bind
9-cis-RA at 43 and 53% of t-RA binding capacity,
respectively. Interestingly, even though serine 405 is adjacent to
methionine 406, S405A has a normal capacity for 9-cis-RA. This
may be due to the fact that serine 405 lies N-terminal to the start of
the putative amphipathic
-helix.
Using saturation kinetics and
Scatchard analyses (27) we also determined the dissociation
constants (K values) of select mutant
receptors for binding 9-cis-RA and t-RA. The results
are summarized in Table 1. Mutant receptors with changes in the
amino acids of interest from Fig. 2as well as mutants with
changes in each part of the
-helical structure were analyzed. The K
values for 9-cis-RA binding to
the mutant receptors emulate those results seen in Fig. 2where
substitutions between amino acids 406 and 410, the amino-terminal
portion of the
-helix, most affect 9-cis-RA binding (Table 1). Both M406A and I410A exhibit no detectable binding
(NB), while P407A, P408A, and L409A exhibit K
values of 14, 7.9, and 2.7 nM, respectively (Table 1). Receptors with mutations in the carboxyl-terminal
portion of this region exhibit K
values
more in the normal range, 0.9 nM for both the deletion mutant
ML413,414
and the double mutant E415,418A.
Representative saturation curves and Scatchard plots shown in Fig. 3lend further support to these interpretations. Both
404 (Fig. 3A) and M406A (Fig. 3B)
exhibit saturable binding for [
H]t-RA
yielding a K
of 0.3 nM for
404, similar to wild-type hRAR
(18) , and a K
of 4.2 nM for M406A (Fig. 3C). Scatchard analysis of E415,418A, however,
shows a K
of 0.9 for
[
H]9-cis-RA and a similar K
of 0.8 for
[
H]t-RA (Fig. 3, D-F).
Figure 4:
Percent t-RA binding capacities
and R group structures of different amino acids at position 406.
Percent t-RA activity was calculated for each mutant receptor
containing the indicated amino acid at position 406 as described for Fig. 2. The amino acid R group is indicated to the right of each bar, and the percent t-RA binding activity for a
wild-type methionine (419) at position 406 has been normalized to
100% as described under ``Experimental
Procedures.''
A number of the functions that allow the RARs to act as hormone-dependent transcription factors are contained within the ligand binding domain, namely, heterodimerization, hormone-dependent transactivation, and ligand binding (reviewed in (13) ). All of these functions are dependent on the ability of the RARs to bind their cognate ligands. While it is well established that the RARs have a high affinity for the ligands, t-RA and 9-cis-RA(18) , little is actually known about critical amino acid interactions that may distinguish between the binding of these two stereoisomers.
Recently, we reported that a truncated
hRAR contains unique binding determinants for 9-cis-RA
within the carboxyl-terminal region of the ligand binding
domain(17) . In this study, we have determined the specific
amino acids within this region (amino acids 405-419) that
constitute this high affinity binding of 9-cis-RA by using
alanine scanning mutagenesis(28) . This region in RAR
is
predicted to form an amphipathic
-helix that is well conserved
among nuclear receptors(23) . In fact, analyses of the majority
of substituted mutants shown, the exceptions being P407A and P408A,
using a Chou and Fasman algorithm (24) indicates that the
predicted secondary structure of this
-helix is not perturbed
(data not shown). Overall, alanine scanning of amino acids 405 to 419
on a truncated hRAR
demonstrates that the amino-terminal end of
this region appears most important for high affinity 9-cis-RA
binding. Specifically, amino acids 406, 407, 410, and to a lesser
extent, 408, are critical as their replacement with alanine
significantly alters the ability of the resulting receptors to bind
9-cis-RA.
When we modeled this putative amphipathic
-helical region between amino acids 404 and 420 of the hRAR
into a helical wheel(29, 30) , the importance of
methionine 406 and isoleucine 410 becomes clear. Since one turn in an
-helix is approximately 3.6 amino acid residues long, these two
amino acids actually lie in close proximity to one another (Fig. 5). In addition, with the ability of the proline residues
to initiate and stabilize the formation of the
-helix(24) , a decrease in binding capacity for
9-cis-RA with the mutation of prolines at positions 407 and
408 is not surprising.
Figure 5:
Helical wheel analysis of a potential
amphipathic -helix on a hRAR
(amino acids 406-419). The
amino acid sequence of the carboxyl-terminal end of the ligand binding
domain of the hRAR
is projected showing the
-helical
structure. The helix, as modeled here using a Chou and Fasman algorithm (24) and software from IntelliGenetics, Inc., is amphipathic
with charged residues to the lower right and hydrophobic
residues to the upper left. Those residues found to be most
critical for 9-cis-RA binding as well as for maintaining the
integrity of the t-RA binding pocket are shown in boxes (methionine 406 and isoleucine 410).
Interestingly, both M406A and I410A bind t-RA with lower affinity than 404, K
values of 4.2 and 1.6 nM, respectively. We
previously showed that
404 bound t-RA as well as the
wild-type receptor which led us to the conclusion that amino acids up
to and including 404 of a hRAR
formed the t-RA binding
pocket. A 10-fold decrease in t-RA binding affinity with the
removal of just one additional amino acid to position 403 indicated to
us that this was also a sensitive region for t-RA
binding(17) . An incorrect three-dimensional interaction of
amino acids 405-419 with the rest of the receptor may occur with
the substitution of alanine at amino acid positions 406 or 410. This
perturbation in three-dimensional structure could then
``block'' the t-RA binding pocket and result in a
decreased affinity for t-RA.
We also examined the ability
of some of the mutant receptors to activate gene transcription on an
RAR retinoic acid responsive element in the presence of t-RA using transient transactivation assays. We found that
although mutations in the carboxyl-terminal portion of this
405-419 region appear to have little effect on either
9-cis-RA or t-RA binding, they do have a profound
effect on transactivation when measured in the presence of up to 10
µMt-RA. Similar to
404(17) , the
mutants L409A, I410A, and ML413,414
all exhibit a dominant
negative phenotype when compared to wild-type hRAR
. M406A, on the
other hand, yields results similar to wild-type when measured with
concentrations of t-RA greater than or equal to 100
nM. (
)These type of results have been seen
previously with the estrogen and glucocorticoid receptors (23) and further emphasize the importance of this AF-2 region
for members of the superfamily of ligand-dependent transcriptional
regulators.
Our observations suggesting that the interaction of a
methionine at amino acid position 406 with 9-cis-RA may be due
to the length or hydrophobicity of the R group, led us to do the
converse experiment where we modified the ligand, 9-cis-RA.
This type of experiment has been done before with the visual pigment,
rhodopsin, a member of the large family of G protein-linked receptors.
Rhodopsin binds its ligand, 11-cis-retinal, by means of a
protonated Schiff base linkage with the -amino group of lysine 296
on the receptor. Zhukovsky et al.(31) showed that
when they made a rhodopsin mutant which had alanine in place of lysine
at position 296, the receptor would no longer bind
11-cis-retinal. However, this mutant K296A would readily bind
11-cis-retinal when it was presented as a Schiff base with n-alkylamine(31) . We also found that the loss of
9-cis-RA binding by a methionine to alanine substitution could
be overcome by a ligand modification which substitutes a hexyl for a
methyl group on the 9 position of 9-cis-RA. It appears that an
extension of the hydrocarbon chain on the ligand at this particular
position can compensate for the decrease in length or hydrophobicity of
the R group at amino acid position 406. In fact, that the truncated
hRAR
receptor yields close to wild type activities when a
functional alkyl group is swapped from the receptor to the ligand
suggests that the methyl group at the 9 position of 9-cis-RA
is likely, but not necessarily, involved in a hydrophobic interaction
with the methionine side chain at position 406.
There are other
reports of a methionine interacting with a steroidal ligand, for
example, a methionine to arginine substitution in a naturally occurring
androgen-insensitive mutant caused a decrease in ligand binding
activity to 5% of wild type(32) . Although this androgen
mutation was found in the middle of the ligand binding domain of a
human androgen receptor (codon 807), it was still in a region that is
well conserved among the glucocorticoid, progesterone, estrogen, and
mineralocorticoid receptors. This region was also predicted to form an
-helix. Methionine residues have also been shown to be important
in the ligand binding domains of the glucocorticoid and progesterone
receptors. Carlstedt-Duke et al.(33) used affinity
labeling to determine that methionine 622, cysteine 754, and cysteine
656 of the rat glucocorticoid receptor interact with steroid
ligands(33) . Their data indicated that methionine 622 and
cysteine 754 are in close proximity when the protein folds. These
investigators also showed that methionine 759 in the progesterone
receptor corresponded to methionine 622 in the rat glucocorticoid
receptor and played a similar role in ligand binding(34) .
Strömstedt et al.(34) found that
methionine 909 in the human progesterone receptor was labeled after the
receptor was covalently charged with
[
H]promegestone. This methionine 909 lies within
the same region of the receptor as methionine 406 in the hRAR
, i.e. the conserved
-helix.
A recent report by Tairis et al.(35) showed that two positively charged amino
acids, arginine 269 and lysine 220, are crucial for the binding of t-RA to a mouse RAR. Tairis et al.(35) postulated that these two amino acids may interact
with the negatively charged carboxyl group on RA. Considering that
protease mapping experiments imply the involvement of the entire ligand
binding domain in the binding of ligands to
RARs(14, 15, 16, 17) , the
involvement of lysine 220, arginine 269, methionine 406, and isoleucine
410 in the binding of 9-cis-RA is not difficult to envision.
In fact, all four amino acids are conserved within the RAR family. A
ligand-induced three-dimensional conformation may occur upon binding of
RA to an RAR such that the ligand binding domain ``folds''
around the ligand leaving lysine 220 and arginine 269 to interact with
the carboxylate tail and methionine 406 and, possibly, isoleucine 410,
to interact with the methyl group located at the 9 position on
9-cis-RA. We suggest that this interaction of
9-cis-RA with methionine 406 and isoleucine 410 gives this
ligand a distinct binding domain compared to t-RA. The
-ionone ring of both 9-cis-RA and t-RA would
then likely fit into an overlapping hydrophobic pocket composed of a
number of hydrophobic residues. While the complete structural details
of this region await x-ray crystallographic determinations, clearly the
results we describe here can provide insight into the nature of the
interaction between the ligand binding pocket on a truncated hRAR
and two different ligands.