(Received for publication, July 9, 1996, and in revised form, September 25, 1996)
From the Department of Biochemistry,
¶ Fels Institute for Cancer Research and Molecular
Biology and
Department of Microbiology and
Immunology, Temple University School of Medicine,
Philadelphia, Pennsylvania 19140
The diverse biological functions of retinoic acid
(RA) are mediated through retinoic acid receptors (RARs) and retinoid X receptors. RARs contain a high affinity binding site for RA which is
sensitive to treatment with sulfhydryl modification reagents. In an
attempt to identify which Cys residues are important for this loss of
binding, we created three site-specific RAR mutants: C228A, C258A,
and C267A. The affinity for RA of all three mutant receptors was in the
range of that of the wild type protein, suggesting that none of these
Cys residues are critical for RA binding. Rather, these modified Cys
residue(s) function to sterically hinder RA binding; however, the
modified Cys residues critical for the inhibition of binding differ
depending on the reagent employed. Only modification of
Cys228 is necessary to inhibit RA binding when RAR
is
modified by reagents which transfer large bulky groups while both
Cys228 and Cys267 must be modified when a small
functional group is transferred. These data suggest that both
Cys228 and Cys267 but not Cys258
lie in the ligand binding pocket of RAR
. However, Cys228
lies closer to the opening of the RAR
ligand binding pocket whereas
Cys267 lies more deeply buried.
Retinoic acid is the potent mediator of the biological effects of
vitamin A that include growth, differentiation, and morphogenesis (for
review, see Ref 1). These actions of retinoic acid are mediated by two
evolutionarily distinct groups of nuclear receptors that belong to the
multigene family of steroid/thyroid hormone receptors called retinoic
acid receptors (RARs)1 and retinoid X
receptors (RXRs) (for review, see Ref. 2). The RARs and RXRs are each
made up of three receptor types, designated ,
, and
(2,
5, 6, 7, 8, 9, 10, 11). In dimeric form, these proteins function as
ligand-dependent transcriptional regulatory factors by
binding to DNA sequences located in the promoter of target genes called
retinoic acid-responsive elements (RAREs) or retinoid X-responsive
elements (RXREs). In vitro binding studies have demonstrated
that both all-trans-RA (RA) and 9-cis-RA are
ligands for the RARs, whereas only 9-cis-RA has been shown to be a ligand for the RXRs (12, 13).
Like other members of the steroid/thyroid hormone superfamily, RARs and RXRs consist of six functionally distinct domains designated A-F (2). Unique functions have been described for several of these domains. The A and B domains are important for the ligand-independent transactivation function (AF-1). The C domain, which contains two zinc fingers, is important for both DNA binding and receptor dimerization. The E domain is functionally complex. In addition to containing all the information necessary for high affinity ligand binding, it also contains a ligand-dependent transactivation function (AF-2) and accessory dimerization sequences.
Recently there have been major advances in understanding the nature of
the ligand binding domain of RARs and RXRs. High resolution crystal
structures of the ligand binding domains of apo-RXR and holo-RAR
have demonstrated that these receptors share a similar overall fold
(14, 15). Furthermore, several amino acids within the ligand binding
domains of RARs have been reported to be functionally important for
high affinity binding of RA. Previous studies from this laboratory have
demonstrated that the positively charged amino acid residues,
Arg269 and Lys220, are critical for the high
affinity binding of RA and retinoid specificity of RAR
(16, 17).
Ostrowski et al. (18) have reported that
Ala225/Ile232 of RAR
and the homologous
amino acid residues Ser232/Thr239 of RAR
are
important for discrimination of subtype-specific synthetic ligands, and
Lupisella et al. (19) have demonstrated using fluorescence
quenching techniques that Trp227 is within the ligand
binding site of RAR
. Finally, Dallery et al. (20) and
Sani et al. (21) have demonstrated a loss of RA binding
after treatment of the EF domain of RAR
and chicken skin RARs,
respectively, with several sulfhydryl-modifying reagents suggesting
that Cys residue(s) may be located in the ligand binding pocket of
these receptors. The goal of this work was to identify which Cys
residue(s) are important for this loss of RA binding and to address the
role that this Cys residue(s) plays in the binding of RA.
In the current report we have shown that, similar to RAR, RA binding
to both RAR
and RAR
is inhibited by the sulfhydryl-specific modifying reagents DTNB and MMTS and that the sulfhydryl group of the
reactive Cys residue(s) of RAR
lies in or near the retinoid binding
pocket. These Cys residue(s) alone do not play a critical role in RA
binding. Rather, these modified Cys residue(s) of RAR
function to
sterically hinder the binding of RA. The modified Cys residues critical
for the inhibition of RA binding differ depending on the reagent
employed. Only modification of Cys228 is necessary when
RAR
is treated with reagents that transfer large bulky groups to
reactive Cys residues, whereas both Cys228 and
Cys267 must be modified when a small functional group is
transferred such as thiomethyl or thiocyanide to fully inhibit RA
binding. Taken together these data suggest that Cys228 most
likely lies closer to the opening of the ligand binding pocket of
RAR
, whereas Cys267 lies more deeply buried within the
ligand binding pocket.
Mutants
were created according to the PCR site-directed mutagenesis technique
described by Higuchi et al. (22). pSG5-RAR, a gift from
Professor Pierre Chambon, Strasbourg, France, was linearized with
XbaI and used as a template for the preparation of each
mutant. Both sense (s) and antisense (as) oligonucleotide primers were
purchased from Ransom Hill BioScience (La Jolla, CA). The GCT codon was
used to encode the mutant Ala residue indicated in bold and
underlined in the mutagenic primers.
For the preparation of C228A, two separate PCR fragments were prepared
using the primer pairs RAR 5
-s
(5
GGGAGGGATCCATCGAGGGTAGATTTGACTGTATGGAT 3
) plus C228A-as
(5
CTTAATAAT
CTTGGTGGC 3
) and RAR
3
-as
(5
GAAGGAAGCTTTCACTGCAGCAGTGGTGA 3
) plus C228A-s
(5
GCCACCAAG
ATTATTAAG 3
),
respectively. The two PCR fragments were purified, annealed, and
amplified in a second PCR reaction using the RAR
5
-s and RAR
3
-as primers. Likewise, the C258A and the C267A mutants were
constructed using the RAR
5
-s and the RAR
3
-as primers and the
following mutagenic primers: C258A-s
(5
AAAGCCGCC
TTGGATATC3
), C258A-as (5
GATATCCAA
GGCGGCTTTG3
), C267A-s
(5
CTCAGAATT
ACCAGGTATAC3
) and C267A-as (5
ATACCTGGT
AATTCTGAG3
). The
MscI-StuI restriction fragment containing each Ala mutation
was exchanged with that of full-length wild type RAR
previously
cloned in frame into the NotI restriction site of pET29a(+)
(Novagen) (pET29a-RAR
). In all cases, the presence of the specific
mutation and the lack of random mutations were verified by DNA sequence
analysis (23).
The entire coding sequences of mouse RAR and RAR
(gift from
Professor Pierre Chambon) were cloned in frame into the
BamHI/HindIII (RAR
) or NotI
(RAR
) restriction sites of pET29a(+).
Each RAR expression construct was transformed into
Escherchia coli K12 strain BL21(DE3) cells (Novagen) (24).
Ten µl from a frozen glycerol stock was used to inoculate 5 ml of LB
medium containing 30 µg/ml kanamycin and shaken at 37 °C until the
A600 was 0.6-1.0. This culture was stored
overnight at 4 °C, and the following morning 1 ml of cells was used
to inoculate 50 ml of LB medium containing 30 µg/ml kanamycin. This
culture was incubated with shaking at 37 °C until the
A600 reached 0.6 to 1.0. At this time the cells
were induced to express the recombinant S-Tag RAR fusion proteins by
the addition of isopropyl-1-thio--D-galactopyranoside to
a final concentration of 1 mM. Ninety minutes later the
cells were harvested by centrifugation at 5000 × g for
15 min.
The receptor extracts were prepared from a 50-ml culture by
resuspending the cell pellet in lysis buffer (50 mM Tris,
pH 8.0, 0.1% Triton X-100, and 200 µg/ml lysozyme) at a final
concentration of 1 ml of lysis buffer/10 ml of induced culture,
freezing at 70 °C followed by rapid thawing at 37 °C,
sonication until the solution lost its viscosity, and centrifugation at
17,000 × g for 30 min. The supernatant was aliquoted
and stored frozen at
80 °C. Total protein concentration of the
receptor extracts was determined with the Bio-Rad protein assay kit
using crystalline bovine serum albumin as the standard. 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 receptor extracts were
diluted in binding buffer (40 mM HEPES, pH 7.9, 120 mM KCl, 10% glycerol, 0.1% (w/v) gelatin, 1 mM EDTA, 4 mM dithiothreitol (DTT), and 5 µg/ml each of the protease inhibitors aprotinin and leupeptin) to a
final concentration of 10-30 µg of total protein (0.1-0.3 pmol of
wild type). Similar total protein concentrations were used for the
mutant proteins. Total RA binding was determined in the diluted protein
extracts by adding [3H]RA (1.82-1.92 TBq/mmol or
49.2-52.0 Ci/mmol, DuPont NEN) in the concentration range of 0.1-20
nM and incubating for 3 h at 27 °C. Dilutions of RA
were made in ethanol. In a duplicate set of reactions nonspecific
binding was determined in the presence of 200-fold molar excess
unlabeled RA (generous gift from Hoffmann-LaRoche). The contribution of
ethanol to the total volume was the same for each RA concentration and
was equal to 2%. Bound RA was separated from free by extraction with
3% (w/v) equal particle size charcoal-dextran prepared as described by
Dukoh et al. (25). All steps in the procedure were performed
under yellow light. Specific RA binding was determined by subtracting
the nonspecific binding from the total binding. The nonspecific binding
was always less than 12% of the total binding. No specific RA binding
was detected in receptor extracts prepared from cells containing
pET29a-RAR which were not induced to express RAR with
isopropyl-1-thio-
-D-galactopyranoside or in receptor
extracts prepared from cells containing the empty pET29a plasmid and
treated with isopropyl-1-thio-
-D-galactopyranoside. Apparent equilibrium dissociation constants (Kd)
were determined for the wild type and each mutant protein by Scatchard analysis (26).
The receptor extracts
were diluted in the same binding buffer as described above for
Kd determinations except that DTT was omitted. Stock
solutions of modifying or reducing agents were made up as 100 × stocks in the following solvents: 5,5-dithiobis(2-nitrobenzoic acid)
(DTNB) (Sigma) in ethanol; methyl methanethiosulfonate (MMTS) (Sigma)
in ethanol;
-mercaptoethanol (BME) (Fisher) in binding buffer; DTT
(Sigma) in binding buffer; sodium arsenite (Fisher) in binding buffer;
N-ethylmaleimide (NEM) (Sigma) in ethanol, p-hydroxymercuribenzoate (p-HMB) (Sigma) in binding buffer,
tri-n-butyl-phosphine (TBP) (Aldrich) in ethanol; and sodium
cyanide (NaCN) in 0.225 M potassium phosphate, pH 7.2. In
all cases, the chemical modification reactions were performed on ice
for 5 min. For the chemical reversal or termination of a reaction,
chemically modified extract was incubated with reducing agent for an
additional 5 min on ice. Total RA binding was determined in duplicate
reactions for each treatment group by incubating the sample in the
presence of a saturating concentration of 15 nM
[3H]RA as determined from the saturation binding
experiments (Fig. 3) as described above. The specific
[3H]RA binding was determined for each treatment group
and expressed as the percent of RA binding in the absence of the
chemical-modifying reagent for each protein (% of control binding).
The amount of specific bound RA at 100% relative binding was in the
range of 0.1-0.3 pmol. Nonspecific binding was always less than 12%
of the total bound RA.
Fig. 1, A-F, depicts the
results of the treatment of the full-length recombinant RAR, RAR
,
and RAR
fusion proteins with the Cys-modifying reagents DTNB and
MMTS. Similar to what has been previously reported (20, 21),
full-length RAR
exhibits a dramatic reduction in RA binding upon
treatment with both of the sulfhydryl-modifying reagents (Fig. 1,
A and B). Furthermore, this loss of RA binding is
highly specific and is not due to the disruption of structural
disulfide bonds in the protein since it is reversible to a level
comparable with that of the untreated RAR
upon the addition of the
reducing agents BME or DTT. Likewise, RA binding to RAR
and RAR
was similarly affected by both DTNB (Fig. 1, C and
E) and MMTS (Fig. 1, D and F). In all
cases this inhibition of RA binding was also highly specific since it
was reversed by reducing agents. Note that RA binding by RAR
following MMTS treatment was reversible to approximately 50% of that
of untreated RAR
using DTT. However, treatment of the MMTS modified RAR
with the more hydrophobic reducing reagent TBP (27) restored RA
binding to a level comparable with that of the untreated RAR
.
Inhibition of RA binding activity after
treatment of RARs with sulfhydryl-modifying reagents. Receptor
extracts containing RAR (A and B), RAR
(C and D), and RAR
(E and
F) were treated with the indicated concentrations of either
DTNB (A, C, and E) or MMTS (B, D, and F) for 5 min on ice. In a duplicate set of reactions the DTNB modification was reversed with 20 mM
BME, and the MMTS modification was reversed with either 10 mM DTT or 10 mM TBP. In all cases, the reducing
agent was added at the end of the 5-min incubation with the modifying
reagent, and the samples were incubated on ice for an additional 5 min.
Following treatment RA binding was determined using 15 nM
[3H]RA as described under the "Materials and
Methods." The specific RA binding was determined for each treatment
group and was expressed as the percent of RA binding in the absence of
the chemical modifying reagent for each protein (% of control
binding). Values are mean ± S.E. for at least three independent
experiments performed in duplicate.
We next wished to determine if any of these receptors contain reactive
vicinal dithiols which are responsible for this loss of RA binding. For
these experiments we used sodium arsenite which has been demonstrated
to cross-link two thiol groups that are spatially close (vicinal
dithiols) (28, 29). Treatment of the recombinant RAR, RAR
, and
RAR
fusion proteins with 10 mM sodium arsenite did not
result in any significant inhibition of RA binding (data not
shown).
In order to determine if the modified Cys residues following DTNB and
MMTS treatment are located within the RA binding pocket of RAR, we
tested the ability of RA to protect critical Cys residues from chemical
modification. In this experiment we used the alkylating agent NEM since
the modification reaction can be terminated but not reversed by the
addition of a reducing agent. Fig. 2 demonstrates that
similar to DTNB and MMTS, RA binding to apo-RAR
is highly sensitive
to modification by NEM. Furthermore, Fig. 2, inset, shows
that 82% of the RA binding activity was maintained when holo-RAR
was treated with 1 mM NEM. This demonstrates protection of
the critical Cys residue(s) to NEM modification when RAR
is liganded
with RA (holo-RAR) prior to NEM treatment. This result strongly
suggests that the target Cys residue(s) in RAR
, whose modification
by sulfhydryl-modifying reagents including NEM, DTNB, and MMTS results
in inhibition of RA binding, lie in or near the retinoid binding
site.
Creation of Site-specific Mutants
Chemical modification of
critical Cys residues located within the retinoid binding site of
RAR could result in loss of RA binding by either the obliteration of
a direct interaction of one or more Cys residues with RA or steric
hindrance resulting in the inability of RA to access the binding
pocket. In order to identify the critical Cys residue(s) and to
determine which of these mechanisms are important in the inhibition of
RA binding to RAR
following sulfhydryl modification, we have created
three site-specific mutants of RAR
in which Cys228,
Cys258, and Cys267 were individually replaced
with an Ala residue (C228A, C258A, and C267A). We have chosen to focus
on these residues because Dallery et al. (20) have
demonstrated that RA binding to the RAR
EF domains is sensitive to
chemical modification of sulfhydryls. The EF domains of RAR
contain
six Cys residues that are absolutely conserved among the three RARs. Of
these six residues, only three (Cys228, Cys258,
and Cys267 of RAR
) lie within the
NH2-terminal 100 amino acid portion of the E domain
previously demonstrated to be sufficient for high affinity binding of
RA (30, 31, 32).
Western blot analysis of receptor extracts containing the wild type and
mutant RAR fusion proteins demonstrated a major band that migrated
at the same position (approximate molecular mass of 55 kDa) along with
several smaller molecular weight degradation products also of similar
size. In addition, the wild type and mutant RAR
fusion proteins
displayed a similar level of expression (data not shown).
Fig.
3 shows representative saturation curves and
corresponding Scatchard plots for wild type and each mutant RAR
fusion protein. Average apparent Kd values were
determined for each receptor using at least three separately prepared
receptor extracts (Table I). The Kd
value of 0.6 nM RA for wild type RAR
fusion protein is
in good agreement with previous reports (16, 17). Both C258A and C267A
displayed a similar affinity for RA comparable with that of wild type
RAR
. Interestingly C228A, which has a Kd value of
4.4 nM, displayed a small decrease (approximately 7-fold)
in affinity for RA when compared with that of wild type RAR
.
|
Since the three mutant RARs have an affinity for RA
in the range of that of the wild type RAR
, it is unlikely that
sulfhydryl modification of RAR
inhibits RA binding by obliterating a
critical direct interaction between the ligand and any one of these Cys residues in the ligand binding domain of the receptor. Therefore, the
alternative mechanism of inhibition involving steric blocking of RA
entry into the binding pocket upon sulfhydryl modification of RAR
was explored. We tested this hypothesis by subjecting each of the Cys
mutants to sulfhydryl modification with the assumption that if one or
more chemically modified Cys residues mediate the inhibition of RA
binding, then replacement of the reactive Cys with an unreactive Ala
residue should result in a receptor with RA binding activity comparable
with that of the unmodified receptor.
Fig. 4A shows the results of DTNB treatment
of each of the mutants, plotted along with wild type RAR for
comparison. The profiles for inactivation of RA binding by C258A and
C267A after DTNB treatment are similar to that of wild type RAR
suggesting that Cys258 and Cys267 are either
not accessible to modification by DTNB or that modification of either
of these residues by DTNB is not sufficient to interfere with RA
binding. On the other hand, RA binding to C228A is not affected by DTNB
modification. This strongly suggests that modification of
Cys228 alone with DTNB is responsible for the inhibition of
RA binding following DTNB treatment.
In order to confirm this conclusion we have also used the
sulfhydryl-specific modifying reagent p-HMB to test each
mutant in a similar fashion. Modification of a sulfhydryl by
p-HMB involves the transfer of a substituted benzyl ring
which is similar in size and structure to the thiobis(2-nitrobenzoic
acid) moiety which is transferred during DTNB modification. As can be
seen in Fig. 4B, the profiles of inactivation of RA binding
by the wild type and mutant RARs treated with p-HMB are
essentially identical to that of their respective DTNB-modified
receptor. These results further demonstrate that modification of
Cys228 alone with either DTNB or p-HMB is
sufficient to inhibit RA binding.
In order to determine if the size of the modifying group is
important, we performed similar experiments using MMTS. Unlike DTNB and
p-HMB, MMTS treatment of a protein results in the transfer of the small thiomethyl-blocking group to reactive sulfhydryl groups
(27). Fig. 5A shows the RA binding activity
of the wild type and mutant fusion proteins after modification with
MMTS. C258A demonstrated a loss of RA binding upon MMTS treatment which was identical to that of wild type RAR further demonstrating that
Cys258 is not the Cys residue whose modification inhibits
the binding of RA. C228A modified with MMTS displayed RA binding which
was approximately 85% of that of the unmodified C228A. This is
consistent with the findings obtained with DTNB and p-HMB
which demonstrate that Cys228 is an important reactive Cys.
Interestingly the MMTS-treated C267A RAR
displayed approximately
60% of the RA binding activity of the unmodified C267A (Fig.
5A). Furthermore, this inhibition of RA binding by the
unmodified C267A was not reversible with DTT but was reversible with
the hydrophobic reducing agent TBP (Fig. 5B).
Since both C228A and C267A retained a considerable amount of RA binding
following treatment with MMTS, we next measured the apparent
Kd of both the MMTS unmodified and modified forms
(treated with 0.25 nM MMTS) of these two receptors along with C258A (Table I). No specific RA binding was observed up to a
concentration of 20 nM by the MMTS-modified C258A. This
further demonstrates that either Cys228,
Cys267, or both are the modified sulfhydryls in RAR
responsible for the loss of RA binding upon MMTS modification.
Interestingly the Kd values of the MMTS-modified
forms of both C267A and C228A were very similar to that of their
unmodified form (Table I). This suggests that the inactivation of RA
binding of RAR
by MMTS, which involves the transfer of the small
thiomethyl-blocking group, requires the modification of both
Cys228 and Cys267.
To further examine the effect of modification of only
Cys228 with a small functional group compared with a large
bulky group on RA binding, we have examined the effect of cyanolysis of
DTNB-modified C258A and C267A. As demonstrated above in Fig. 4,
inhibition of RA binding of DTNB-modified RAR involves the
modification of Cys228. Treatment of DTNB-modified Cys
residues with NaCN results in the replacement of the bulky
thiobis(2-nitrobenzoic acid) group with thiocyanide which is similar in
size to the thiomethyl group transferred upon MMTS treatment (33, 34).
It is important to note that MMTS-treated C267A and DTNB-modified C267A
treated with NaCN are virtually the same except for the presence of a thiomethyl group instead of a thiocyanide group on the modified Cys.
Treatment of DTNB-modified C258A and C267A with NaCN results in a
dose-dependent restoration of RA binding which plateaued at
greater than 70% of the wild type RA binding at a concentration of 50 nM NaCN (data not shown). It is important to note that this level of RA binding is similar to the level of RA binding observed with
MMTS-treated C228A and C267A. This suggests that modifications of
Cys228 which result in the transfer of small moieties such
as thiocyanide and thiomethyl is not sufficient to fully inhibit RA
binding. Taken together this suggests that loss of RA binding by MMTS
treatment of RAR
is the result of the modification of both
Cys228 and Cys267.
In the current report we have extended the findings of Dallery
et al. (20) to demonstrate that RA binding to all three RAR types is blocked similarly by specific sulfhydryl-modifying agents. The
concentrations of DTNB and MMTS necessary to inhibit RA binding to
RAR and RAR
are similar to those which have been reported previously for the ligand binding domain of RAR
(20). The
reversibility of this effect by reducing agents demonstrates the
specificity of these reactions to sulfhydryl groups. Interestingly,
unlike RAR
and RAR
, RA binding by RAR
following MMTS treatment
was only reversible to about 50% of control binding with the polar reducing agent DTT. However RA binding was restored to near unmodified levels using the hydrophobic reducing agent TBP. This suggests that
there is at least one critical Cys residue responsible for inhibition
of RA binding upon methanethiolation with MMTS which lies within a more
hydrophobic region of RAR
than in RAR
and RAR
. This region of
greater hydrophobicity in the ligand binding pocket of RAR
compared
with RAR
and RAR
is most likely one of several factors
responsible for the ability of synthetic retinoids to display receptor
selectivity.
The glucocorticoid receptor (GR), another member of the steroid/thyroid hormone superfamily of receptors, has also been shown to be sensitive to sulfhydryl modification (35). GR modified with MMTS exhibits a bimodal response in which inhibition of ligand binding is maximal both at low and high concentrations of the modifying reagent (36, 37). It has been determined that this response is a result of intramolecular disulfide bond formation between two closely spaced Cys residues (vicinal dithiols) located in the ligand binding pocket of GR upon treatment with low concentrations of MMTS (36, 37). We did not observe this biphasic pattern of inhibition of RA binding in any of the RARs treated with MMTS under similar conditions. In addition, our sodium arsenite results further support the conclusion that there are no reactive vicinal dithiols in any of the three RARs responsible for inhibition of RA binding.
The results of our protection-exchange experiment with RAR
demonstrating that RA binding can protect Cys residues from chemical modification suggests that these critical Cys residue(s) may be located
in or near the ligand binding pocket. Protection may result from either
the physical blocking of the modifying reagents by RA or a
conformational change in the ligand binding domain upon RA binding
which blocks the accessibility of the reactive Cys residues to the
modification reagents. The latter is more likely based on the work of
Renaud et al. (15) showing that the major difference in the
crystal structure of apo-RXR
and holo-RAR
ligand binding domains
is in the position of helix 12. In the apo-receptor the entrance to the
RA binding site is open while in the holo-receptor helix 12 collapses
over the entrance to the binding pocket blocking access to the
site.
The loss of RA binding following treatment with MMTS and DTNB cannot be
explained by the elimination of critical interactions between any of
the Cys residues examined and RA since each of the three Cys mutants
have an apparent Kd for RA in the range of that of
wild type RAR. Interestingly, the C228A mutant did display a 7-fold
decrease in affinity for RA when compared with wild type RAR
. This
is not surprising since two amino acids of RAR
, close in the primary
sequence of Cys228 (Ala225 and
Ile232), have been demonstrated to be important for
interaction with type-selective ligands (18). Furthermore, the crystal
structure of holo-RAR
indicates that Cys237, the
homologous residue to Cys228 of RAR
, lies within 4 Å of
RA (15). It is possible that substitution of Cys228 with an
Ala removes an interaction between this Cys residue and RA which
contributes to the overall stability of holo-RAR
. On the other hand,
it is also possible that this mutation has caused a small disruption of
the local topography of the ligand binding pocket sufficient to result
in this modest decrease in the affinity of C228A for RA.
Although inhibition of RA binding upon sulfhydryl modification of
RAR is a result of steric blocking of RA binding, the Cys residues
whose modification is critical for this inhibition of RA binding differ
depending on the reagent employed. Wild type RAR
displays similar
sensitivity to DTNB and MMTS treatment; however, the C228A and C267A
mutants represent novel receptors with unique sensitivity to these two
sulfhydryl modifying reagents. Our finding of a differential response
of these two mutants to reagents that transfer different size blocking
groups is not unprecedented. It has been reported that specific
sulfhydryl modification of rabbit muscle creatine kinase with blocking
groups of increasing size resulted in the incremental decrease in
enzymatic activity (38). Similar results have been found for the GR.
Modification of critical Cys residues in GR with MMTS results in a
greater amount of residual binding of ligand than modification by
iodoacetamide which transfers a relatively large blocking group
(36).
Our data demonstrate that both Cys228 and
Cys267 are located within the ligand binding pocket of
RAR such that when modified they inhibit the binding of RA. However
Cys228 lies closer to the opening of the ligand binding
pocket than Cys267 because Cys228 is accessible
to modification by both large and small reagents and upon modification
results in the inhibition of RA binding, whereas Cys267 is
only sensitive to small reagents. We have estimated the size of DTNB
and MMTS by measuring the carbon center to carbon center distances on
molecular models. The dimensions of MMTS, when viewed as a molecular
structure, are 3.0 Å in height and 4.0 Å in width, whereas those of
DTNB are 6.0 and 7.4 Å, respectively. Based on our sulfhydryl
modification results, these measurements suggest that
Cys267 is located in a region of the pocket not accessible
to molecules or side chains of molecules much larger than 4.0 Å in
diameter. Since RA is no greater than 4.0 Å at its carboxylate end and
at least 5.0 Å at the
-ionone ring, we can conclude that
Cys267 of RAR
is closest to the carboxylate end of RA.
This conclusion is supported by the recently published crystal
structure of the ligand binding domain of holo-RAR
which
demonstrates that the homologous residue of Cys228
(Cys237) is within 4 Å of carbon 13 of RA (15). In
addition, there is only one amino acid residue in the primary sequence
between Cys267 and Arg269 of RAR
(Arg278 of RAR
). We have previously shown
Arg269 of RAR
to be important for RA binding and
retinoid specificity most likely by interaction with the carboxyl group
of RA (16, 17), and the crystal structure of the ligand binding domain of RAR
places Arg278 within 4 Å of the carboxylate
oxygen 22 of RA (15).
We thank Professor Pierre Chambon for the pSG5-RAR constructs and F. Hoffmann-LaRoche for the RA used in these studies. We also thank Dr. Charles Grubmeyer for many helpful discussions.