(Received for publication, September 28, 1994; and in revised form, April 18, 1995)
From the
Retinoic acid receptor-
Retinoic acid (RA), ( Three RARs, termed RAR- RARs
are composed of six structurally distinct domains referred to as
domains A-F(2) . Domain C (DNA binding domain) and domain
E (ligand binding domain) of RARs are highly conserved, demonstrating
greater than 90 and 75% amino acid sequence identity, respectively.
Structural analysis of the DNA binding domains of RARs using NMR
spectroscopy has demonstrated them to contain two putative zinc fingers
formed by the first eight conserved cysteine residues(19) .
Little structural information is available concerning the ligand
binding domains of RARs; however, several retinoid analogues have been
designed, which display quite different specificities for each of the
RAR
subtypes(20, 21, 22, 23, 24, 25, 26) .
This suggests that the three-dimensional conformation of the retinoid
binding site of each RAR is somewhat unique despite the high degree of
amino acid sequence homology. Recently, we have reported that
Arg In
this report, we have further examined the role of Arg
The three Gln mutants were prepared by PCR site-directed
mutagenesis(28) . All oligonucleotides were purchased from the
Oligonucleotide Synthesis Laboratory at Temple University School of
Medicine or Ransom Hill Biosciences. Sense primers are indicated as s and antisense primers as as. XbaI-linearized pSG5-RAR
For the
dominant negative mutant experiments, the transactivation assays were
performed essentially as described above with the following exceptions.
CV-1 cells were transfected with a total of 20 µg of DNA (3 µg
of wild type pSG5-RAR
To determine the K For those
mutants in which we could not determine the K
Fig. 1shows the results of
transactivation assays from cells transfected with each of these DNA
constructs and treated with various concentrations of
all-trans-RA. K220Q, with an EC
Figure 1:
The effect of mutation of Lys
Figure 2:
Retinoid binding properties of selected
mutants. A comparison of the binding of all-trans-RA (panelsA-C), all-trans-retinol (ROH) (panelsD-F), and
all-trans-retinal (RAL) (panelG)
to wild type (WT), R269Q, and K220Q is shown. Specific
[
Since we have previously observed that K220A/R269A functioned as a
RA concentration-dependent dominant negative mutant(27) , we
wished to next examine the ability of each of the Gln mutants to
function as dominant negative factors. These experiments were performed
at two different concentrations of all-trans-RA
(10
Figure 3:
The dominant negative effect of RAR-
Fig. 4shows the results of retinol
transactivation assays, and Fig. 2, D-F shows the
results of representative retinol binding assays. A comparison of the
results of retinol transactivation assays and retinol binding assays is
shown in Table 2. In the transactivation assays, cells were
transfected with each of the wild type and mutant RAR-
Figure 4:
The effect of mutation of Lys
Fig. 2G, Fig. 5, and Table 3show the results of similar experiments in which we
determined the ability of the wild type and mutant RAR-
Figure 5:
The
effect of mutation of Lys
Figure 6:
The dominant negative effect of RAR-
In similar
experiments, cells were transfected with the wild type RAR-
Figure 7:
The dominant negative effect of RAR-
This work confirms and extends our previous report in which
Arg Each of the single
and the double Gln mutants of Arg It is unlikely that the loss of RA activity of the Gln mutants
results from a large global conformational change in the structure of
the mutant RARs. Each of the Gln mutants, like our previously described
Ala mutants(27) , can function in vivo as dominant
negative mutants when transfected with wild type RAR- Mutation of either Arg The
exact reason for these changes in retinoid specificity and the
understanding of how Arg This is
the first report documenting that the ligand specificity, with respect
to the functional group of a retinoid, of any RAR or RXR can be
altered. However, several investigators have reported the ability to
alter the specificity of a number of cellular retinoid-binding proteins
and fatty acid-binding proteins by site-directed mutagenesis. For
example, mutation of Arg Like our previously described
K220A/R269A mutant(27) , each of the Gln mutants function as
efficient dominant negative mutants in a RA concentration-dependent
fashion. In addition, the dominant negative effect exhibited by those
mutants, which have acquired significant retinol and/or retinal
activity and binding, was also relieved by retinol and/or retinal in a
concentration-dependent manner. This indicates that once the retinoid
concentration added to the culture media is in the range of the
EC Among the mutant receptors that we have created with
altered retinoid specificity, two (R269Q and K220Q) appear to be
particularly interesting. K220Q displays similar transactivation
activity for all three retinoids (all-trans-RA,
all-trans-retinal, and all-trans-retinol) with
EC Although there is no experimental evidence
supporting that the three-dimensional structure of the ligand binding
pocket of the nuclear RAR/RXR family of receptors is the same as that
of the well studied serum and cellular retinoid-binding proteins, it is
interesting to compare the ligand binding properties of our mutant
receptors (K220Q and R269Q) to that of these other proteins, which very
specifically bind retinol. The similar moderate binding and
transactivating activity of K220Q with retinol, retinal, and RA is
reminiscent of that of retinol-binding protein. Retinol-binding protein
has a similar, moderate affinity (K
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
(RAR-
) specifically binds
retinoic acid (RA) and functions as a RA-inducible transcriptional
regulatory factor. Simultaneous mutation of Arg
and
Lys
of RAR-
to Ala results in a dramatic reduction
in both transactivation and affinity for RA along with creating a RA
concentration-dependent dominant negative mutant. In this report, we
found that mutation of these two amino acid residues singly and
simultaneously to Gln results in mutant RAR-
s, each displaying a
more dramatic reduction in transactivation and affinity for RA than
their corresponding Ala mutant, with the R269Q more profoundly affected
than K220Q. Furthermore, we examined both the Ala and Gln mutants for
their ability to transactivate and bind two other retinoids with
different functional end groups (all-trans-retinol and
all-trans-retinal). Mutation of Lys
to either an
Ala or a Gln favors transactivation and binding of retinal, while
mutation of either Lys
or Arg
to Gln favors
retinol transactivation and binding. Taken together, these results
suggest that Arg
and Lys
lie within the
ligand binding pocket of RAR-
and that these two amino acid
residues play an important role in determining retinoid specificity
most likely by directly interacting with the carboxylate group of RA.
)a vitamin A metabolite, is
necessary for a diverse group of biological processes including growth,
differentiation, and morphogenesis (for review, see (1) ).
These actions of RA are mediated by a group of nuclear proteins, which
belong to the multigene family of steroid/thyroid hormone receptors,
termed retinoic acid receptors (RARs) and retinoid X receptors (RXRs)
(for review, see (2) ). RARs and RXRs are RA-inducible
transcriptional regulatory factors that transduce the RA signal at the
level of gene expression via retinoic acid response elements (RAREs)
and retinoid X response elements. Both all-trans-RA and
9-cis-RA have been demonstrated to be ligands for the RARs,
whereas only 9-cis-RA has been shown to be a ligand for
RXRs(3, 4) .
,
RAR-
, and RAR-
, have been described (2, 5, 6, 7, 8, 9, 10, 11) .
In addition, seven isoforms of RAR-
and RAR-
and four
isoforms of RAR-
are produced as a result of a combination of
differential promoter usage and alternative
splicing(10, 12, 13, 14) . The
isoforms of each RAR contain a common DNA binding and ligand binding
domain with unique amino-terminal sequences. These unique
amino-terminal sequences have been proposed to modulate the
transcriptional activities of each isoform(14) . Furthermore,
each of the RAR subtypes and their isoforms display a distinctive
spatial and temporal pattern of expression during development, implying
specific nonoverlapping
functions(15, 16, 17, 18) .
and Lys
of RAR-
together play an
important role in binding of RA, possibly by interacting with the
negatively charged carboxylate group of RA(27) . In these
studies, simultaneous mutation of Arg
and Lys
of RAR-
to Ala (K220A/R269A) resulted in a 500-fold
elevation in the EC
value for all-trans-RA in
transactivation assays and a 580-fold increase in the apparent K
for all-trans-RA. In addition,
K220A/R269A acted as a dominant negative mutant when transfected with
the three RAR subtypes in a RA concentration-dependent fashion.
and
Lys
of RAR-
in the binding of ligand by preparing
and assaying mutants of RAR-
, in which either singly (K220Q and
R269Q) or simultaneously (K220Q/R269Q) these two positively charged
amino acids have been changed to Gln. Each of the Gln mutants displayed
a greater decrease in RA-dependent functional activity and RA affinity
when compared with their corresponding Ala mutant, with R269Q more
profoundly affected than K220Q. In addition, mutation of either of
these two amino acid residues individually to Ala or Gln resulted in
the creation of a number of unique mutant receptors (K220Q, R269Q, and
K220A), which display an increased transactivation activity and
affinity for all-trans-retinol and/or
all-trans-retinal compared with that of the wild type
receptor. Taken together, these data suggest that Arg
and
Lys
lie within the ligand binding pocket of RAR-
and
that these two amino acid residues play an important role in
determining retinoid specificity of RAR-
most likely by directly
interacting with the carboxylate group of RA.
Plasmid Constructs and Site-directed
Mutagenesis
The entire coding sequences of mouse RAR-2
cDNA cloned into the mammalian expression vector, pSG5 (pSG5-RAR
),
was a generous gift from Prof. Pierre Chambon (Strasbourg, France) (11) . The wild type RAR-
and the mutants K220A, R269A,
and K220A/R269A were previously described(27) . Three
additional mutants of wild type RAR-
were prepared in which
Lys
was replaced with Gln (K220Q), Arg
was
replaced with Gln (R269Q) and a double mutant in which Lys
and Arg
were each replaced with Gln (K220Q/R269Q).
In each case, the CAG codon was used to encode the mutant Gln indicated
in bold and underlined in the mutagenic primers shown
below.
was used as the template for the
single mutants. For K220Q, two PCR fragments were synthesized using the
primer pairs RAR
-5s (5`-GGGAGGGATCCATCGAGGGTAGATTTGACTGTATGGAT-3`) plus K220Q-as (5`-CTCACTGAACTGGTCCCAGAG-3`) and RAR
-3as (5`-GAAGGAAGCTTTCACTGCAGCAGTGGTGA-3`) plus K220Q-s (5`-CTCTGGGACCAGTTCAGTGAG-3`), respectively. The two PCR
fragments were purified, annealed, and amplified in a second PCR
reaction using the primers RAR
-5s and
RAR
-3as. The SacI-BstXI restriction
fragment that contained the mutation was exchanged with that of the
wild type pSG5-RAR
to create the pSG5-RAR
-K220Q mutant
expression vector. The pSG5-RAR
-R269Q mutant was prepared in a
similar manner except that (i) the two mutagenic primers were
R269Q-as (5`-TGGGGTATACTGGGTACAAT-3`) and R269Q-s (5`-ATTTGTACCCAGTATACCCCA-3`) and (ii) the EcoRV-BstXI restriction fragment of the final PCR
product was exchanged with that of the wild type pSG5-RAR
. Finally
the double mutant pSG5-RAR
-K220Q/R269Q expression vector was
prepared exactly the same as the K220Q mutant, except that the template
DNA was XbaI-linearized pSG5-RAR
-R269Q DNA. All clones
were verified by DNA sequence analysis using the Sanger methodology (29) and Sequenase version II. No codon mutations were found in
the entire RAR-
2 coding sequences except for the desired
mutations. The ability of each mutant protein to bind DNA and dimerize
was determined by electrophoretic mobility shift assay using nuclear
extracts prepared from cells transfected with each of the mutant
constructs as previously described(27) .
Transactivation Assays
Transactivation
assays were performed essentially as previously
described(20, 30) . Briefly, CV-1 cells were plated at
500,000 cells/60-mm dish. The next day, the cells were transfected with
a total of 12 µg of DNA (4 µg of wild type or mutant
pSG5-RAR expression construct, 4 µg of RARE-CAT reporter
construct obtained as a generous gift from Dr. Ronald Evans (Salk
Institute, La Jolla, CA), and 4 µg of pRSV-
-gal) by
Ca
phosphate (Promega) according to the
manufacturer's protocol. 24 h later, the cells were treated with
various concentrations of all-trans-RA,
all-trans-retinol, or all-trans-retinal ranging from
10
to 10
M prepared in
ethanol. Control cells were treated with identical volumes of ethanol.
After an additional 24 h, the cells were harvested and assayed for
chloramphenicol acetyltransferase (CAT) activity (31) and
-galactosidase (
-gal) activity(32) . CAT activity was
normalized with respect to
-gal activity to control for
transfection efficiency and expressed as a percentage of relative CAT
activity. The normalized CAT activity of wild type RAR-
at
10
M all-trans-RA was chosen as
100% relative CAT activity. The EC
values for the wild
type and each of the mutants represent the concentration of retinoid
that resulted in 50% of the maximal activity of wild type RAR-
determined by extrapolation from the plotted points.
expression construct, 4 µg of RARE-CAT
reporter construct, 4 µg of pRSV-
-gal, and 9 µg of the
RAR-
mutant construct or pSG5 carrier DNA). This resulted in a 1:3
molar ratio of wild type RAR-
DNA to mutant RAR-
DNA. In some
experiments, the cells were treated with either 10
or 10
M all-trans-RA. In
other experiments, the cells were treated with a combination of
10
M all-trans-RA and either
all-trans-retinol (10
or 10
M) or all-trans-retinal (10
or 10
M). Control cells were treated
with ethanol carrier. Normalized CAT activity of wild type RAR for each
treatment group was chosen as 100% relative CAT activity.
Retinoid Binding Assays
Nuclear extracts
of COS cells transfected as described above with the wild type or the
mutant pSG5-RAR DNA constructs were prepared as previously
reported by Jetten et al.(33) . For the RA binding
assays, 10-60 µg of total nuclear protein were added in an
Eppendorf tube containing various concentrations of
[
H]all-trans-RA (DuPont NEN, 47.5
Ci/mmol) ranging from 0.5 to 25 nM for K220Q and from 50 to
330 nM for R269Q and K220Q/R269Q in buffer B (10 mM Tris, pH 8.0, 1.5 mM EDTA, 2 mM dithiothreitol,
10% glycerol, 1 mM phenylmethylsulfonyl fluoride, 0.8 M KCl, 50 µg/ml aprotinin, 50 µg/ml leupeptin) to the final
volume of 200 µl. Nonspecific binding was measured in the presence
of 200-fold excess unlabeled all-trans-RA. After 4 h
incubation at 4 °C, bound
[
H]all-trans-RA was separated from free
radioactivity by applying the mixture to a PD-10 gel filtration column
(Sephadex G-25, Pharmacia Biotech Inc.) equilibrated with equilibration
buffer (5 mM sodium phosphate, pH 7.4, 10% glycerol, 1 mM phenylmethylsulfonyl fluoride, 1 mM dithiothreitol, and
0.4 M KCl). The column was eluted with equilibration buffer,
0.5-ml fractions were collected, and the radioactivity was determined
using a Beckman LS 6000BC liquid scintillation counter. For the
RAR-
mutants, the specific binding was further corrected for the
low endogenous specific binding (endogenous wild type protein in the
cells) of the mock-transfected cells. The K
values were determined by Scatchard plot analysis (34) using Cricket graphics.
for all-trans-retinol, the
retinol binding assays were performed exactly the same as described for
RA except that 24-36 µg of nuclear protein were incubated in
buffer B with various concentrations of
[
H]all-trans-retinol (DuPont NEN, 37.3
Ci/mmol) ranging from 3 to 150 nM for K220Q, from 1 to 50
nM for R269Q; and up to 270 nM for wild type, R269A,
K220Q/R269Q, and K220A/R269A. Nonspecific binding was determined with a
200-fold excess unlabeled all-trans-retinol.
for all-trans-retinol and/or
all-trans-retinal, we calculated the concentration of the
retinoid that inhibited 50% of all-trans-RA binding
(IC
). In these experiments, 18-36 µg of nuclear
protein were incubated in a total volume of 200 µl of buffer B with
a saturating concentration of
[
H]all-trans-RA (30 nM for wild
type, K220A, R269A; 100 nM for K220Q) and various
concentrations of unlabeled all-trans-retinol (5-1000
nM) or unlabeled all-trans-retinal (50-1000
nM) for 4 h. Following incubations, the samples were processed
as described above for the K
determinations. IC
values were calculated by
determining the concentration of all-trans-retinol or
all-trans-retinal, which inhibited 50% of
all-trans-RA binding from a plot of relative binding versus the log of the all-trans-retinol or
all-trans-retinal concentrations.
The Effect of Site-specific Mutations on
RA-dependent Transactivation and RA Binding
Initially, we
wished to examine the effect of mutation of Lys and
Arg
of RAR-
singly (K220Q and R269Q) and
simultaneously to Gln (K220Q/R269Q) on RA-dependent transactivation
activity and RA binding. All three of these mutant RAR-
s contained
functional DNA binding and dimerization domains. This was demonstrated
in an in vitro electrophoretic mobility assay (data not shown)
in which each mutant receptor bound an RARE and dimerized comparable to
both wild type RAR-
and our previously described complementary Ala
mutant RAR-
s (27) .
value of 200
nM, displayed the least reduction (20-fold) in activity
compared with that of the wild type receptor. On the other hand, both
R269Q and K220Q/R269Q displayed extremely low transactivation activity
with an EC
value greater than 10,000 nM, which is
more than 1000-fold higher than that of the wild type receptor (see Table 1). In addition, we also measured the apparent K
of each of the Gln mutant receptors for
all-trans-RA. As can be seen in Fig. 2, A-C (which contain representative RA binding data) and Table 1,
the K
value for all-trans-RA of
K220Q was 13 nM (34-fold higher than wild type) while the K
values for R269Q and K220Q/R269Q were
278 nM and greater than 1000 nM, respectively.
Comparison of the EC
and K
values of each of the mutant receptors with that of wild
type RAR-
demonstrates a similar relationship between the fold
reduction in functional activity and all-trans-RA binding.
and Arg
of RAR-
to Gln on transactivation by
all-trans-RA. CV-1 cells were cotransfected with one of the
RAR-
expression vectors (wild type (WT), K220Q, R269Q, or
K220Q/R269Q), RARE-CAT reporter construct, and pRSV-
-gal. 24 h
after transfection, the indicated concentrations of
all-trans-RA were added to the cells. 24 h after addition of
the RA, the cells were harvested for CAT and
-gal assays. CAT
activity was normalized for efficiency of transfection using
-gal
activity and expressed as relative CAT activity. The percent relative
CAT activity was calculated using the CAT activity of the wild type
RAR-
at 10
M all-trans-RA as
100%. Each point represents the mean of four to six
independent transfection assays ±
S.E.
H]RA and
[
H]all-trans-retinol binding data from
representative experiments for wild type is shown in panelsA and D, for K220Q in panelsB and E, and for R269Q in panelsC and F. Note that the [
H]RA or
[
H]all-trans-retinol concentrations
indicated in each panel are different. It should also be noted
that the binding data were not corrected for transfection efficiency,
resulting in differences in the maximum observed cpms of bound
retinoid. Shown in the inset for each panel is the
Scatchard plot used to calculate the K
values. PanelG is a representative
competitive inhibition experiment in which wild type and K220Q were
saturated with [
H]RA, the specific binding was
competed with the indicated concentrations of
all-trans-retinal, and the IC
values were
determined. This experiment was not possible for R269Q due to an
inability to saturate this mutant with commercially available
[
H]RA. K
and
IC
values are presented in Tables I-III as the mean
± S.E. for at least three independent
determinations.
and 10
M) to
determine if the dominant negative repression of wild type activity by
the mutant receptors could be relieved by increasing the concentration
of RA. Fig. 3shows that each of the Gln mutants were effective
dominant negative mutants when transfected with wild type RAR-
. At
a concentration of all-trans-RA below the EC
value for all the mutants (10
M),
only 40, 25, and 15% of the wild type activity was detectable when
cells were transfected with K220Q, R269Q, and K220Q/R269Q,
respectively. At 10
M all-trans-RA, the dominant negative effect of each of the
mutant constructs was reduced to a level consistent with each of the
mutant receptors' transactivation activity and ability to bind
all-trans-RA. Thus, each of the Gln mutants (K220Q, R269Q, and
K220Q/R269Q), like our previously described RAR-
2 mutants (K220A,
R269A, and K220A/R269A), function as ligand-dependent dominant negative
factors with their potency inversely proportional to their affinity for
all-trans-RA.
Gln mutants at different concentrations of all-trans-RA. CV-1
cells were transfected with a total of 20 µg of DNA, which included
3 µg of pSG5-RAR
, 4 µg of RARE-CAT reporter construct, 4
µg of pRSV-
-gal, and 9 µg of either one of the mutant
expression constructs (K220Q, R269Q, and K220Q/R269Q) or pSG5 vector.
24 h after transfection, the cells were treated with either
10
or 10
M
all-trans-RA. Cells were harvested 24 h later for CAT and
-gal assays. Normalized CAT activity of the wild type RAR-
plus pSG5 at each RA concentration was set to 100% relative activity.
Values are mean ± S.E. of three to five independent
experiments.
The Effect of Site-specific Mutations on Retinoid
Specificity
Examination of the amino acids demonstrated to
be required for binding of retinoids and fatty acids to the cellular
retinoid-binding proteins and fatty acid-binding proteins reveals that
those proteins that bind molecules which contain an alcohol (retinol)
or aldehyde (retinal) functional group have two conserved Glns in the
binding site, and those that bind ligands with a carboxylate functional
group (RA or fatty acids) have two conserved Args at the homologous
positions in the binding pocket (for review, see (35) ). We
therefore wished to determine if our mutant receptors displayed an
enhanced affinity for either retinol or retinal compared to the wild
type receptor. DNA
constructs and treated with various concentrations of
all-trans-retinol. Retinol binding was measured by determining
either the apparent K
or IC
values for all-trans-retinol. Wild type RAR-
displayed low all-trans-retinol transactivation activity with
an EC
value of approximately 800-fold greater than that
observed with all-trans-RA. Wild type RAR-
also displayed
low affinity for all-trans-retinol with no detectable specific
binding of [
H]all-trans-retinol up to
270 nM (Fig. 2D) and an IC
value
greater than 1000 nM, comparable to other
reports(5, 6, 8, 22) .
Interestingly, three of the mutant constructs displayed higher
transactivation activity and affinity for all-trans-retinol
than that of the wild type receptor. R269Q had the highest
all-trans-retinol-dependent transactivation activity with an
EC
value 120-fold lower than that of the wild type and the
greatest affinity for all-trans-retinol (K
of 18 nM) (Fig. 2E). K220Q had
intermediate activity with an EC
value 50-fold lower than
that of the wild type and a K
value for
all-trans-retinol of 127 nM (Fig. 2F). Finally, K220A was the least active of
the three with an EC
value 10-fold lower than that of the
wild type and an IC
value for all-trans-retinol
of 407 nM. The other mutants were found to have activities in
the transactivation assay and retinol binding assays equal to or less
than that of wild type RAR-
. Interestingly, the two most active
mutants (K220Q and R269Q) had relative transactivation activities at
the highest all-trans-retinol concentration examined of 200%,
2-fold greater than the maximal activity of the wild type receptor with
10
M all-trans-RA in this same
assay (Fig. 4). There are many possible explanations for this
high rate of transactivation activity with retinol compared with that
of RA; however, one possibility is that retinol has an increased
uptake, stability, or storage (esterification) in the cells compared to
that of RA.
and Arg
of RAR-
to either Ala or Gln on
transactivation by all-trans-retinol. CV-1 cells were
cotransfected with one of the RAR-
expression vectors (wild type (WT), K220A, K220Q, R269A, R269Q, K220A/R269A, and
K220Q/R269Q), RARE-CAT reporter construct, and pRSV-
-gal. 24 h
after transfection, the indicated concentrations of
all-trans-retinol were added to the cells. Additional plates
of cells transfected with wild type RAR-
expression construct were
treated with 10
M all-trans-RA (ATRA). 24 h after addition of the retinoid, the cells were
harvested for CAT and
-gal assays. CAT activity was normalized for
the efficiency of transfection using
-gal activity and expressed
as relative CAT activity. The relative activity of the wild type and
each mutant construct with all-trans-retinol was calculated as
a percent of the wild type activity at 10
M
all-trans-RA, which was set at 100%. Each point represents the
mean of four to ten independent transfection assays ±
S.E.
receptors
to transactivate with all-trans-retinal and bind
all-trans-retinal. Two of the mutant receptors (K220A and
K220Q) displayed higher all-trans-retinal transactivation
activity compared with that of the wild type receptor. K220A, with an
EC
of 150 nM, was greater than six times more
active than the wild type receptor; K220Q, with an EC
value of 350 nM, was greater than three times more
active than the wild type receptor. In addition, these two mutant
receptors have higher affinity (greater than 4-fold) for
all-trans-retinal than the wild type receptor (IC
values are approximately 250 nM for K220A and K220Q (Fig. 2G) compared to greater than 1000 nM for
the wild type). The remainder of the mutant receptors displayed
EC
values and IC
values for
all-trans-retinal similar to or larger than that of wild type
RAR-
.
and Arg
of
RAR-
to either Ala or Gln on transactivation by
all-trans-retinal. See Fig. 4legend, except that
all-trans-retinal was used instead of
all-trans-retinol.
Release of Dominant Negative Effect of RAR-
Since several of the
mutants displayed high functional activity in transactivation assays
with retinol and/or retinal, which correlates well with their affinity
for these retinoids, we next wished to explore the effect of these two
retinoids on release of the dominant negative repression of wild type
activity displayed by these mutants ( Fig. 3and (27) ).
This information would further demonstrate in a physiological context
if the mutants that display retinoid specificity change function
efficiently with either retinol and/or retinal. Fig. 6shows
dominant negative experiments in which wild type RAR- Mutants by Retinol and/or Retinal
was
cotransfected with each of the mutant constructs. To measure the
dominant negative effect of the mutant constructs, it was necessary to
activate the wild type receptor by treating the cells with
10
M all-trans-RA. This
concentration of all-trans-RA is equal to the EC
value of the wild type receptor and well below that of each of
the mutant receptors. In addition, the cells were treated with either
10
M (approximately the EC
value of K220Q and R269Q) or 10
M all-trans-retinol (greater than the EC
value
for all single mutants). Transfection of cells with the two mutants
that display the highest all-trans-retinol affinity and
functional activity (K220Q and R269Q) and treatment with
10
M all-trans-retinol resulted in
only a 50% inhibition of the wild type activity. The remaining mutant
proteins, which have a much lower functional activity and affinity for
all-trans-retinol, displayed a greater reduction in wild type
activity to levels ranging from 5 to 20% of that of the wild type
receptor. Increase of the all-trans-retinol concentration to
10
M resulted in a relief of the dominant
negative repression of wild type activity by each mutant receptor in a
fashion that was proportional to the functional activity and affinity
of each receptor for all-trans-retinol.
mutants at different concentrations of all-trans-retinol. CV-1
cells were transfected with a total of 20 µg of DNA (3 µg of
pSG5-RAR
, 4 µg of RARE-CAT reporter construct, 4 µg of
pSV-
-gal, and 9 µg of either one of the mutant expression
constructs (K220A, K220Q, R269A, R269Q, K220A/R269A, and K220Q/R269Q)
or pSG5 vector. 24 h after transfection, the cells were treated with
10
M all-trans-RA along with
either 10
or 10
M all-trans-retinol. Normalized CAT activity of the wild
type RAR-
plus pSG5 at each retinol concentration was set to 100%
relative activity. Values are mean ± S.E. of three to five
independent experiments.
along
with one of each of the mutant receptors and treated with
10
M all-trans-RA along with
either 10
or 10
M all-trans-retinal (Fig. 7). Again, those mutant
receptors that displayed the highest functional activity and affinity
for all-trans-retinal (K220A and K220Q) showed the least
amount of dominant negative effect on the wild type activity at an
all-trans-retinal concentration of 10
M. All the other mutant receptors displayed a high
degree of inhibition of wild type activity in the range of 5-20%.
Again, when the concentration of all-trans-retinal was
increased to 10
M, there was a relief of
the dominant negative effect to a level proportional to the functional
activity of each receptor for all-trans-retinal.
mutants at different concentrations of all-trans-retinal. See Fig. 6legend, except that all-trans-retinal was used
at a concentration of either 10
or 10
M instead of
all-trans-retinol.
and Lys
of RAR-
were found to be
critical for high affinity binding of RA(27) . In addition, we
have demonstrated here that mutation of either of these two amino acid
residues results in the creation of several mutant RAR-
s that
display greatly increased affinity for and functional activity with
retinol and/or retinal compared to that of the wild type protein. Thus,
changing the chemical nature of amino acid residues at positions 220
and 269 of RAR-
from positively charged to either neutral or polar
residues results not only in a large reduction in RA binding and
transactivation activity but also dramatically changes the retinoid
specificity of the receptor. This strongly suggests that Arg
and Lys
of RAR-
may interact with the
carboxylate group of RA and that the nature of this interaction
determines specific, high affinity binding of RA.
and Lys
displays a dramatic reduction (20- to
1000-fold) in
RA-dependent transactivation activity and RA binding affinity when
compared with that of the wild type RAR-
receptor. It should be
noted that the toxicity of RA limits the concentrations that could be
used in the transactivation assay. Thus, the EC
data
presented for the low affinity mutants are obtained from plots that do
not show saturation and should be considered as estimates of the actual
EC
values. In addition, each of these mutants functioned
as dominant negative mutants with their potency inversely proportional
to their affinity for RA. Comparison of the EC
and K
values of these Gln mutants to our
previously published mutants involving mutation of the same amino acid
residues to Ala (27) demonstrates two important points (Table 1). First, the mutation of Arg
and/or
Lys
to the polar amino acid, Gln, compared with the
neutral amino acid, Ala, results in a much more dramatic reduction in
all-trans-RA-dependent functional activity and
all-trans-RA binding for all mutants. This is consistent with
our previous suggestion that the negatively charged carboxylate group
of RA lies in close proximity to Arg
and
Lys
. Second, R269Q and K220Q receptors have markedly
different EC
and K
values
for all-trans-RA in comparison to both R269A and K220A
receptors, which have similar EC
and K
values. This suggests that Arg
, and its
neighboring electronic environment, is much more sensitive to the
change to a polar amino acid, Gln, than that of Lys
.
in a
RA-concentration dependent fashion (Fig. 3). In addition, each
mutant exhibits the ability to dimerize and bind an RARE in an in
vitro electrophoretic mobility shift experiment to an extent which
is comparable with that of the wild type receptor (see (27) and data not shown). Finally, all of the mutants
demonstrated measurable transactivation activity albeit at very high
retinoid concentrations. Taken together, these experiments clearly
demonstrate that functional DNA binding, dimerization, and
transactivation domains are present in the mutant receptors.
or Lys
to either
Ala or Gln results in the creation of a family of mutant RAR-
receptors with altered retinoid specificity. This was demonstrated by
comparing the activity of each of the mutants in both transactivation
assays and as dominant negative mutants with RA, retinol, and retinal
along with directly measuring the affinity of each mutant for all three
retinoids. As previously discussed, the reported EC
values
are extrapolated from non-saturated plots and should be considered as
estimates of the actual EC
values. Wild type RAR-
is
highly specific for all-trans-RA displaying a much higher
functional activity and affinity for all-trans-RA compared
with all-trans-retinol and all-trans-retinal.
However, each of the mutant receptors has lost differing degrees of
affinity for all-trans-RA while acquiring to a variable extent
the ability to bind and transactivate with all-trans-retinol
and/or all-trans-retinal. The single mutation of either
Arg
or Lys
to Gln highly favored
transactivation and binding of all-trans-retinol, while the
single mutation of Lys
to either Ala or Gln resulted in
receptors with the highest functional activity and affinity for
all-trans-retinal. Interestingly, mutation of both sites
simultaneously, to either Ala or Gln, did not result in the acquisition
of enhanced retinol or retinal binding or functional activity.
and Lys
are
jointly involved in ligand recognition will have to await the
availability of three-dimensional structural information. It is
interesting to note that in a recent report by Ostrowski et
al.(36) , Lys
and Arg
of
RAR-
were predicted to be located on one side of an amphipathic
helix opposite Ala
and Ile
. These two
hydrophobic residues have been suggested to be important in
receptor-specific retinoid selectivity. In the lipid binding protein
family, which includes several cellular retinoid-binding proteins and
fatty acid-binding proteins, two of the critical residues for ligand
binding to each protein are separated by approximately 20 amino acids
(for review, see (35) ). Furthermore, Newcomer et al.(37) has suggested that since retinoic acid-binding
protein from rat epididymis (E-RABP) and RARs both bind
all-trans-RA and 9-cis-RA, they may both contain a
hydrophobic pocket with a similar shape formed by a number of amino
acid side chains in the context of unique structural motifs. In the
case of E-RABP, crystallographic data demonstrates that the side chains
lining the RA-binding cavity include residues from Phe
to
Tyr
, suggesting that distinct regions of protein, which
are quite distant in the linear sequence of the protein, may fold to
form the three-dimensional ligand binding pocket. In addition, the
changes that we have observed in retinoid specificity of RAR-
upon
mutation of Arg
and Lys
will most likely
involve both the nature of the amino acid charge and the surrounding
electronic environment of residues at positions 220 and 269.
of intestinal fatty acid-binding
protein to Gln greatly reduces the binding of fatty acids and allows
binding of retinol and retinal(38) . In addition, mutation of
Gln
of cellular retinol-binding protein, type I (CRBP-I)
to Arg creates a mutant CRBP-I that now binds not only retinol but
retinal and RA as well(39) . Finally, mutation of Gln
of cellular retinol-binding protein, type II (CRBP-II) to Arg
creates a mutant protein that now binds fatty acids with high affinity
instead of retinol and retinal(40) . In each of the cases
described above, the amino acid that was mutated has been demonstrated
by crystal structure analysis to be critically involved in interacting
with the functional end group (alcohol, aldehyde, or carboxylic acid)
of the ligand. Therefore, since we have created marked changes in
retinoid specificity of RAR-
by mutation of either Arg
or Lys
, it is very likely that these two amino
acids are directly involved in interacting with the carboxylate group
of RA. This information will ultimately lead to the elucidation of the
exact nature of the ligand binding site of the RARs leading to the
rational design of new receptor-specific retinoid analogues for
pharmacological applications.
value of the mutant of interest, sufficient binding of
ligand by the mutant will occur, which then results in the mutant
displaying wild type activity and release of the dominant negative
activity. To our knowledge, this is the first report of RAR dominant
negative mutants whose activity can be relieved by a retinoid other
than RA.
values in the range of 150-350 nM. Thus,
K220Q has lost the ability to discriminate between the functional end
groups of these three different retinoids while still binding and
transactivating with moderate activity. However, the most interesting
mutant receptor is R269Q. R269Q is a very specific, high affinity
all-trans-retinol receptor with a K
value for all-trans-retinol of 18 nM and an
EC
of 70 nM while exhibiting extremely low
functional activity and affinity for all-trans-retinal and
all-trans-RA. In addition, this mutant receptor is 120 times
more active with all-trans-retinol than the wild type
RAR-
receptor. In other words, R269Q functions as a nuclear
retinol receptor.
of
190 nM) for retinol, retinal, and RA in
vitro(41) . On the other hand, comparison of the K
of R269Q for all-trans-retinol
(18 nM) with other proteins that specifically bind retinol
demonstrates that R269Q has a similar high affinity for
all-trans-retinol. The K
of both
CRBP-I and CRBP-II for retinol is in the range of 10-50
nM(42, 43, 44, 45) . These
mutants (R269Q and K220Q) will be extremely useful in future
experiments aimed at elucidating the distinct targets and mechanisms of
RAR-
.
-gal,
-galactosidase; CAT, chloramphenicol acetyltransferase.
We thank Prof. Pierre Chambon for the wild type
RAR- expression construct, Dr. Ronald Evans for the RARE-CAT
reporter construct, and F. Hoffmann-LaRoche and Co. (Nutley, NJ) for
the retinoids that were used in these studies.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.