Differential Role of Homologous Positively Charged Amino Acid Residues for Ligand Binding in Retinoic Acid Receptor alpha  Compared with Retinoic Acid Receptor beta *

(Received for publication, December 11, 1996, and in revised form, February 11, 1997)

Angela Scafonas Dagger §, Christopher L. Wolfgang Dagger , Jerome L. Gabriel Dagger par , Kenneth J. Soprano par ** and Dianne Robert Soprano Dagger par Dagger Dagger

From the Departments of Dagger  Biochemistry and ** Microbiology and Immunology and par  Fels Institute for Cancer Research and Molecular Biology, Temple University School of Medicine, Philadelphia, Pennsylvania 19140

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES


ABSTRACT

The diverse biological actions of retinoic acid (RA) are mediated by retinoic acid receptors (RARs) and retinoid X receptors. Although it has been suggested that the ligand binding domains (LBDs) of RARs share the same novel folding pattern, many RAR subtype-specific agonists and antagonists have been synthesized demonstrating that the LBD of each RAR subtype has unique features. We have examined the role of several positively charged amino acid residues located in the LBD of RARalpha in RA binding. These results are compared with previously published data for the homologous mutations in RARbeta . Lys227 of RARalpha does not appear to be important for RA binding or RA-dependent transactivation, whereas the homologous residue in RARbeta , Lys220, plays an important synergistic role with Arg269 in these two activities. In addition, Arg276 of RARalpha , like its homologous residue Arg269 of RARbeta , was found to play an important role in the binding of RA most likely by interacting with the carboxylate group of RA. However, the orientation of and electronic environment associated with Arg276 in RARalpha appears to be different from that of Arg269 in RARbeta , thus contributing to the uniqueness of the ligand binding pocket of each receptor.


INTRODUCTION

Retinoic acid (RA),1 a vitamin A metabolite, is a potent regulator of a diverse group of biological processes, including growth, differentiation, and morphogenesis (for review, see Ref. 1). These actions of RA are mediated by a group of nuclear proteins, which belong to the multigene family of steroid and thyroid hormone receptors, termed retinoic acid receptors (RARs) and retinoid X receptors (RXRs) (for review, see Ref 2). In dimeric form, the RARs and RXRs function as ligand-inducible transcriptional regulatory factors by binding to DNA sequences called retinoic acid-responsive elements (RAREs) and retinoid X-responsive elements, which are located in the promoter region of target genes. Three subtypes, termed alpha , beta , and gamma , of both RAR and RXR have been identified along with several isoforms of each subtype (3-11). In vitro binding assays have demonstrated that only 9-cis-RA is a ligand for the RXRs, whereas both all-trans-RA and 9-cis-RA have been shown to be ligands for the RARs (12, 13).

RARs, like other members of the steroid and thyroid hormone superfamily, have a modular structure consisting of six domains (A-F), each of which has been assigned specific functions (2). The C domain, which contains two zinc fingers, is important for both DNA binding and dimerization. The A and B domains have been demonstrated to have ligand-independent transcriptional transactivation activity (AF-1), whereas ligand-dependent transcriptional transactivation activity (AF-2) is associated with the E domain. The E domain, in addition, also contains all the information necessary for high affinity ligand binding and accessory dimerization sequences.

Recently, the x-ray crystal structures of the ligand binding domains of apo-RXRalpha and holo-RARgamma were reported (14, 15). Analysis of these two crystal structures has led to the suggestion that the novel folding pattern observed in these two receptors, an antiparallel alpha -helical sandwich, may be shared by all members of the steroid and thyroid hormone superfamily (16). In addition, RAR subtype-specific site-directed mutagenesis studies have identified amino acid residues that are functionally important for the binding of RA to a given RAR (17-22).

The similarities and differences between the ligand binding domains of the nuclear RARs have been the topic of many articles in the current literature. The experimentally determined differential retinoid specificity of the three RAR subtypes has been the driving force behind the successful efforts of several medicinal chemists to synthesize RAR subtype-selective agonists and antagonists (23-30). On the other hand, as described above, protein structural chemists suggest a common folding pattern for the ligand binding domains of all RAR subtypes (16). In an effort to help establish the structural requirements for ligand specificity of RAR subtypes, we have examined the relative importance of RA binding of several homologous, positively charged amino acid residues located within the ligand binding domains of RARalpha and RARbeta . Lys227 of RARalpha , unlike its homologous residue Lys220 in RARbeta , does not appear to be important for RA binding. On the other hand, Arg276 in RARalpha , like its homologous residue Arg269 in RARbeta , was found to play an important role in the binding of RA most likely by interacting with the carboxylate group of RA. However, the orientation of and electronic environment associated with Arg276 in RARalpha appears to be different from that of Arg269 in RARbeta , thus contributing to the uniqueness of the ligand binding site of each receptor.


MATERIALS AND METHODS

Plasmid Constructs and Site-directed Mutagenesis

Mutants were created according to the site-directed mutagenesis technique described by Higuchi et al. (31). pSG5-mouse RARalpha 1 and pSG5-mouse RARbeta 2, generous gifts from Prof. Pierre Chambon (Institut de Génétique et de Biologie Moléculaire et Cellulaire, Strasbourg, France), linearized with BamHI and XbaI, respectively, were used as templates for the preparation of the mutants. Both sense (s) and antisense (as) oligonucleotide primers were purchased from Ransom Hill BioScience (La Jolla, CA). The GCT codon and the CAG codon were used to encode the mutant Ala residues and mutant Gln residues, respectively, indicated in bold and underlined in the mutagenic primers.

For the preparation of R276A RARalpha , two separate polymerase chain reaction fragments were prepared using the primer pairs RARalpha 5'-s (5'-GAGGGGGATCCATGGCCAGCAATAGCAG-3') plus R276A-as (5'-CTCAGGCGTGTACGTGCAG-3') and RARalpha 3'-as (5'-GAGGGAAGCTTTCATGGGGATTGGGTGG-3') plus R276A-s (5'-CTGCACGTACACGCCTGAG-3'), respectively. The two polymerase chain reaction fragments were purified, annealed, and amplified in a second polymerase chain reaction using the RARalpha 5'-s and RARalpha 3'-as primers. Likewise, the K227A, R272A, R272Q, and R276Q mutants were constructed using the RARalpha 5'-s and RARalpha 3'-as primers and the following mutagenic primers: K227A-s (5'-CTCTGGGACTTCAGTGAAC-3') and K227A-as (5'-GTTCACTGAACTCCCAGAG-3'), R272A-s (5'-CCTGATTCTGATCTGCACG-3') and R272A-as (5'-CGTGCAGATCAGAATCAGG-3'), R272Q-s (5'-CCTGATTCTGATCTGCACG-3') and R272Q-as (5'-CGTGCAGATCAGAATCAGG-3'), and R276Q-s (5'-CTGCACGTACACGCCTGAG-3') and R276Q-as (5'-CTCAGGCGTGTACGTGCAG-3'). The 527-base pair SacI-BspEI fragment that contained the desired mutation was exchanged with that of pSG5-RARalpha wild type to create each of the mutant DNA constructs. For pSG5-K227A/R276A, the 364-base pair PstI-EcoRV fragment of the pSG5-R276A single mutant was exchanged with that of pSG5-K227A. The pSG5-R265Q RARbeta mutant was created in the same fashion, except that the following primers were used: RARbeta 5'-s (5'-GGGAGGGATCCATCGAGGGTAGATTTGACTGTATGGAT-3'), RARbeta 3'-as (5'-GAAGGAAGCTTTCACTGCAGCAGTGGTGA-3'), R265Q-s (5'-CTTGATTCTCATTTGTACC-3'), and R265Q-as (5'-GGTACAAATGAGAATCAAG-3'), and the EcoRV-BstXI fragment was exchanged between the wild type and the mutant. In all cases, the presence of the specific mutation and the lack of random mutations were verified by DNA sequence analysis (32).

To make the pET-RARalpha prokaryotic expression constructs, the full-length RARalpha wild type protein coding region was synthesized by polymerase chain reaction using the primers RARalpha 5'-s and RARalpha 3'-as and cloned into the BamHI and HindIII restriction sites of pGem-3. The various mutants were prepared by fragment exchange between the wild type construct in pGem-3 and the given mutant via the PstI and BspEI sites. The entire sequence of the wild type and the site of each mutation were confirmed by DNA sequence analysis. Once constructed in pGem-3, each RARalpha cDNA was subcloned in frame in the BamHI and HindIII restriction sites of pET-29a. For the pET-RARbeta constructs, the MscI-StuI fragment containing the desired mutation was exchanged with that of full-length wild type RARbeta previously cloned in frame into the NotI restriction site of pET29a (22).

Determination of EC50 and Kd Values

Transactivation assays were performed as described previously (17, 18, 33). The EC50 value reported represents the concentration of retinoid that resulted in 50% of the maximal relative CAT activity determined by extrapolation from the plotted points.

To prepare recombinant protein for the Kd measurements, each pET-29a-RAR expression construct was transformed into Escherichia coli K12 strain BL21(DE3) cells (Novagen) (36). The expression of each S-Tag RAR protein and the preparation of the receptor extracts was performed as described previously (22). The production of the recombinant S-Tag wild type and mutant RAR fusion proteins in the receptor extracts was monitored using the S-Tag Western blot kit (Novagen). The Western blot analysis of the wild type and all mutant receptor extracts demonstrated a major band that migrated at the same position (approximate molecular mass, 55 kDa) along with several minor, smaller molecular mass bands, which were of similar size.

Retinoid binding assays were performed as described previously (22, 37) with the following exceptions: the total protein concentration in each assay was 6-12 µg, and both 9-cis-RA and all-trans-retinol binding were determined exactly as described for all-trans-RA using either [3H]9-cis-RA (1.74 TBq/mmol (47 Ci/mmol); Amersham Corp.) or [3H]all-trans-retinol (1.75 TBq/mmol (47.2 Ci/mmol); DuPont NEN).

Western Blot Analysis

The levels of wild type and mutant RARalpha protein were determined by Western blot analysis using CV-1 cells transfected with the indicated expression construct essentially as described previously (17, 39).

Electrophoretic Mobility Shift Assay (EMSA)

The wild type RARalpha , selected mutant RARalpha proteins, wild type RXRalpha , and beta -galactosidase used in the EMSA were all recombinant S-Tag fusion proteins prepared in BL21 cells as described above. BL21 cell extract (25 µg total protein) containing the indicated S-Tag proteins were incubated in 25 mM Tris, pH 7.9, 125 mM NaCl, 2.5 mM EDTA, 25 mM dithiothreitol, 12.5 mM MgCl2, 12.5% sucrose, 12.5% glucose, 0.5% Nonidet P-40, and 2.6 µg salmon sperm DNA (Sigma) containing a 32P-labeled RARE probe. The RARE probe was obtained by annealing two complementary single-stranded oligonucleotides (5'-TCGAGGGTAGGGTTCACCGAAAGTTCAC-3' and (5'-CGAGTGAACTTTCGGTGAACCCTACCCT-3'), which contain the RARE in the RARbeta 2 promoter (positions -63 to -33 relative to the start site of transcription) (42). The resulting double-stranded RARE DNA was filled in with Klenow polymerase (Promega) in the presence of [32P]dCTP (111 TBq/mmol (3000 Ci/mmol); DuPont NEN). Unlabeled cold RARE DNA was used in some assays as a competitor at a 100-fold excess. The RAR·RXR complexes were resolved by electrophoresis through a 6% polyacrylamide gel containing 2.5% glycerol in 0.5 × TBE (0.09 M Tris borate, pH 8.2, 0.002 M EDTA) at 200 V for 3 h. The gels were dried and exposed to Kodak XRP x-ray film at -70 °C.


RESULTS

Effect of Site-specific Mutations of RAR-alpha on RA-dependent Transactivation and RA Binding

Initially we examined the roles of Lys227 and Arg276 of RARalpha in the binding of RA and RA-dependent transactivation. These are the homologous amino acids to Lys220 and Arg269 of RARbeta (see Fig. 1), which we have previously demonstrated to act together synergistically in the binding of RA (17, 18). Figs. 2 and 3 show representative transactivation assays and saturation binding curves, respectively, for wild type RARalpha and selected RARalpha mutants. Table I lists the EC50 values and the apparent Kd values for the wild type and all mutant proteins. K227A displayed an EC50 value for all-trans-RA and a Kd for both all-trans-RA and 9-cis-RA comparable to those of wild type RARalpha . On the other hand, R276A displayed low activity, with EC50 and Kd values for both isomers of RA that were elevated approximately 100- and 50-fold, respectively, when compared with those of wild type RARalpha . Interestingly, mutation of Arg276 to an Ala (R276A) had a more dramatic effect than the corresponding mutation to Gln (R276Q), with R276A displaying an approximately 3-fold greater increase in both EC50 and Kd values compared with R276Q. Finally, although the K227A/R276A double mutant displayed very low activity in the RA-dependent transactivation assay, the EC50 value was only increased 3-fold when compared with that of the single R276A mutant.


Fig. 1. Ligand binding domain of RARs showing homologs of RARalpha Lys227, Arg272, and Arg276 in RARbeta and RARgamma .
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Fig. 2. Transactivation activity of wild type RARalpha and representative RARalpha mutants. CV-1 cells were cotransfected with 3 µg of pSG5-RARalpha wild type (WT) or mutant expression vector DNA, 3 µg of RARE-CAT reporter DNA, and 1 µg of pCMV-beta -galactosidase DNA. Twenty-four h later, the cells were treated with one of the indicated concentrations of all-trans-RA. After an additional 24 h cells were harvested and assayed for CAT (34) and beta -galactosidase (35) activities. beta -Galactosidase activity was used to normalize CAT activity for transfection efficiency. The percentage relative CAT activity was calculated using the maximum relative CAT activity achieved with RARalpha wild type as 100%. Each data point represents the mean of two to four independent experiments performed in duplicate ± S.E. (bars).
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Fig. 3. All-trans-RA binding properties of wild type RARalpha and representative RARalpha mutants. Recombinant full-length RARalpha wild type and mutant proteins were expressed as S-Tag fusion proteins in BL21 E. coli cells and used for the binding assays. Specific binding of [3H]all-trans-RA to wild type, R272A, R276A, and R276Q from a representative experiment is shown. Note that similar amounts of bacterial extract protein were used in each assay without normalization for the level of expression of the recombinant protein, resulting in differences in the maximum bound all-trans-RA between the different mutant protein preparations. Inset for each panel, one of the three to six Scatchard plots (38) obtained from different preparations of protein that was used to calculate the apparent Kd values presented in Table I.
[View Larger Version of this Image (33K GIF file)]


Table I.

EC50 and Kd values of wild type and mutant RARs; RETINOIC ACID


RAR EC50a all-trans-RA Kdb
All-trans-RA 9-cis-RA

nM nM
RARalpha
  Wild type 8 1.7  ± 0.3 2.6  ± 0.6
  K227A 10 2.6  ± 0.5 3.6  ± 0.5
  R272A 175 4.2  ± 0.6 5.7  ± 0.4
  R272Q 200 2.0  ± 0.5 NDc
  R276A 900 86.2  ± 5.0 92.1  ± 3.0
  R276Q 300 34.5  ± 3.5 36.8  ± 0.6
  K227A/R276A 3000 >100d >100d
RARbeta
  Wild type 8 0.6  ± 0.1 NDc
  R265Q 25 1.2  ± 0.2 NDc

a Values calculated from Fig. 2.
b Mean ± S.E. calculated from Fig. 3.
c ND, not determined.
d Receptor did not saturate at concentrations of [3H]all-trans-RA or [3H]9-cis-RA up to 250 nM.

We next examined the role of Arg272 of RARalpha in RA binding and RA-dependent transactivation, because its homologous amino acid residue in RARgamma (Fig. 1) has been implicated from its x-ray crystal structure to be one of the positively charged amino acid residues forming the electrostatic field gradient in the ligand binding pocket (15). Mutation of Arg272 to either Ala or Gln had a negligible effect on the Kd for RA when compared with that of the wild type receptor. On the other hand, the EC50 value of R272A and R272Q in RA-dependent transactivation assays was increased approximately 25-fold when compared with that of the wild type receptor. Since we had observed this lack of correlation between RA binding and RA-dependent transactivation activity when Arg272 of RARalpha was mutated, we examined the homologous residue in RARbeta (Fig. 1). Interestingly, R265Q RARbeta displayed near wild type activity in both RA-dependent transactivation assays and RA binding studies.

Effect of Mutation of Arg276 of RARalpha on Retinol-dependent Transactivation and Retinol Binding

Since R269Q RARbeta displays high affinity for and transactivation activity with retinol (Ref. 18; see Table II), we examined the activity of R276Q RARalpha in similar assays. Table II shows that, as expected, wild type RARalpha does not bind retinol within the limits of the binding assay and has low activity in transactivation assays with retinol (EC50, 1 µM). Unlike R269Q RARbeta , R276Q RARalpha displayed no detectable binding of retinol or measurable activity in the retinol-dependent transactivation assays. As a positive control for the retinol binding assays, we measured the Kd for retinol of recombinant R269Q RARbeta and obtained a value of 28 nM, which is quite comparable to our previously measured 18 nM using nuclear extracts prepared from COS cells transfected with R269Q DNA (18). Interestingly, R276Q displayed at least 10-fold lower activity in the retinol-dependent transactivation assays when compared with that of wild type RARalpha . The small amount of activity observed in the retinol-dependent transactivation assays is likely to be due to low levels of RA formed within the cells because of the oxidation of retinol. If the RA formed from the oxidation of retinol is indeed responsible for the observed activity, it is not unexpected that R276Q would display a higher EC50 value than that of the wild type in retinol-dependent transactivation assays, because R276Q has an approximately 40-fold higher EC50 value with RA compared with that of the wild type receptor (300 compared with 8 nM).

Table II.

EC50 and Kd values of wild type and mutant RARs: RETINOL


RARalpha
RARbeta
EC50a Kd EC50b Kd

nM nM nM nM
Wild type 1000 NBc Wild type 8000d NBc,d
R276Q >10000 NBc R269Q 70d 28

a Relative to the maximum relative activity displayed by wild type RARalpha at 10-6 M RA.
b Relative to the maximum relative activity displayed by wild type RARbeta at 10-6 M RA.
c No specific binding up to 200 nM [3H]all-trans-retinol.
d From Tairis et al. (18).

Western Blot Analysis and EMSA of Wild Type and Mutant RARalpha Levels

Fig. 4 is a Western blot showing wild type and selected RARalpha mutant protein levels in nuclear extracts isolated from transfected CV-1 cells. A similar level of RARalpha protein was detected in the nuclear extracts of cells transfected with the wild type and all the mutant DNAs. Furthermore, Fig. 5 shows an EMSA using wild type and selected RARalpha mutant S-Tag recombinant proteins. All mutant RARalpha proteins dimerized with RXRalpha and bound a RARE, resulting in a gel shift pattern comparable to that of wild type RARalpha . This demonstrates that the differences between the EC50 and Kd values of the wild type RARalpha and the mutant receptors (R272A, R272Q, R276A, R276Q, and K227A/R276A) are not likely to be due to any gross conformational changes in these receptors, since they behave normally with respect to dimerization, DNA binding, and expression pattern in transfected CV-1 cells.


Fig. 4. Levels of wild type RARalpha and representative RARalpha mutant proteins in transfected CV-1 cells. Nuclear extracts of CV-1 cells transfected with empty pSG5 DNA, pSG5-RARalpha DNA, or the indicated pSG5-mutant DNAs were analyzed by Western blot. Twenty-five µg of total nuclear proteins were loaded per well on a discontinuous SDS-polyacrylamide gel composed of a 5% stacking gel and a 10% resolving gel. Proteins were electroblotted to a polyvinylidene difluoride membrane, and RARalpha protein was detected by immunoblotting with a rabbit anti-RARalpha polyclonal antibody followed by a goat anti-rabbit IgG-horseradish peroxidase-conjugated secondary antibody (40, 41). Proteins were visualized using the Enhance chemiluminescence kit. The standard molecular mass markers shown are phosphorylase b (97.4 kDa), serum albumin (66.2 kDa), ovalbumin (45 kDa), and carbonic anhydrase (31 kDa).
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Fig. 5. DNA binding of wild type RARalpha and representative mutant RARalpha proteins. EMSA was performed using BL21 cell extracts or BL21 cell extracts containing the following recombinant S-Tag proteins: beta -galactosidase, RXRalpha , wild type (WT) RARalpha , and a representative RARalpha mutant protein. The indicated protein mixtures (approximately 25 µg of total protein) were incubated with a 32P-labeled RARE probe. One hundred-fold molar excess unlabeled RARE was added to the wild type RARalpha /RXRalpha mixture as a competitor (100 × cold probe). After the binding reaction the products were resolved by electrophoresis through a 6% polyacrylamide gel containing 2.5% glycerol, and the retarded bands were visualized by autoradiography.
[View Larger Version of this Image (24K GIF file)]



DISCUSSION

In this report we have examined the effect of mutation of several positively charged amino acid residues of RARalpha on retinoid binding and retinoid-dependent transactivation activity. Arg276 was found to play a major role in RA binding and RA-dependent transactivation, whereas Lys227 does not appear to be important for either of these two activities in RARalpha . In addition, Arg272 does not appear to be important for RA binding but may be important in the determination of the final active conformation of holo-RARalpha , since the R272A mutant displayed a significant reduction in RA-dependent transactivation activity. It is unlikely that global conformational changes are responsible for the reduced activity in the RA binding and RA-dependent transactivation assays observed with these RARalpha mutants, since all the RARalpha mutants examined displayed similar levels of expression in transfected CV-1 cells and similar activity in the EMSA.

Table III presents a comparison of the -fold increase in EC50 and Kd values for all-trans-RA of several of the RARalpha mutants described in this report compared with the homologous RARbeta mutants that have previously been reported (17, 18). In both RARalpha and RARbeta , the homologous Arg (Arg276 and Arg269, respectively) plays an important role in the binding of RA most likely by interacting with the carboxylate group of RA. Site-specific mutation of this Arg in both receptors results in a significant reduction in RA binding and RA-dependent trans-activation activity. This is consistent with the RARgamma crystal structure, in which the homologous amino acid residue, Arg278, has been shown to form a salt bridge with the carboxylate O22 of RA (15). However, we observed several important differences in the response of Arg276 in RARalpha compared with that of Arg269 in RARbeta when mutated to either Ala or Gln. Mutation of this Arg to Ala causes a significant reduction in RA binding and RA-dependent transactivation activity in RARalpha and has a minor effect in RARbeta (approximately 75- versus 8-fold). On the other hand, mutation of this Arg to Gln results in a much less dramatic reduction in RA binding and transactivation activity of RARalpha than that of RARbeta (approximately 30- versus 1000-fold). Finally, R269Q RARbeta is a very efficient retinol receptor, whereas R276Q RARalpha has no detectable retinol binding or retinol-dependent transactivation activity (Table II). Taken together these data suggest that the orientation of Arg276 and Arg269 in the ligand binding site and the electronic environment associated with this Arg is different in RARalpha compared with RARbeta , contributing to the uniqueness of the ligand binding site of each receptor.

Table III.

Comparison of the -fold increase in EC50 and Kd values for all-trans-retinoic acid displayed by the homologous mutants of RARalpha and RARbeta


RARalpha a
RARbeta b
EC50 Kd EC50 Kd

nM nM nM nM
Wild type 1 1 Wild type 1 1
K227A 1.3 2 K220A 3 5
R272Q 25 1 R265Q 3 2
R276A 110 50 R269A 7 10
R276Q 38 21 R269Q >1000 730
K227A/R276A 375 >60 K220A/R269A 500 580

a Fold increases relative to wild type RARalpha calculated from Table 1.
b Fold increases relative to wild type RARbeta calculated from Table 1 and Tairis et al. (17, 18).

In RARbeta , Lys220 was found to act synergistically with Arg269 in the binding of RA, since the simultaneous mutation of both residues to Ala resulted in an effect much larger than the additive effect of each single mutation both in RA binding and RA-dependent transactivation activity. This synergistic effect was not observed with the K227A/R276A RARalpha mutant, suggesting that Lys227 in RARalpha does not appear to be involved directly in the binding of RA; however, it may function as part of the electronic guidance force, proposed to guide RA into the binding site, described in the crystal structure of RARgamma (15). Since the single mutation of Arg276 to an Ala in RARalpha has such a dramatic effect on RA binding and RA-dependent trans-activation activity, it is possible that this single amino acid residue may act more independently in RA binding in RARalpha than the homologous Arg in RARbeta . It is interesting to note that the crystal structure of RARgamma shows that Lys236 and Arg278 directly interact with the carboxylate group of RA, whereas Lys229 does not appear to be sufficiently close to the carboxylate group of RA to be directly involved in its binding. It is possible that different positively charged amino acid residues may act synergistically with the RARalpha Arg276 homologous position in each of the three RAR subtypes, further contributing to the unique nature of each of these three retinoid binding sites.

Based on the crystal structure of RARgamma , Renaud et al. (15) have reported that there are 24 amino acid residues distributed over eight structural elements in the ligand binding domain that are positioned within 4.5 Å of RA and therefore delineate the ligand binding pocket. These eight structural elements include H1, H3, H5, beta -turn, loop 6-7, H11, loop 11-12, and H12. Furthermore, Wurtz et al. (16) have suggested that the ligand binding pockets of all nuclear receptor holo-ligand binding domains have a similar architecture involving these structural elements. When the amino acid sequences of the three RAR subtypes are compared, only 3 of these 24 amino acid residues lining the ligand binding pocket are variable (Ser232, Ile270, and Val395 in RARalpha ). All three of these divergent residues in RARgamma are associated with alpha -helices, which form the hydrophobic portion of the ligand binding pocket and interact with the beta -ionone ring and/or the isoprenoid side chain of RA. These three divergent residues have been suggested to play a role in the determination of the ligand specificity of the three RAR subtypes (15, 16).

Our data demonstrate that Lys227 and Arg276 of RARalpha , when mutated to Ala and Gln, behave differently in both retinoid binding assays and retinoid-dependent transactivation assays when compared with the homologous mutations in RARbeta . These data suggest that there are other structural features of the ligand binding pocket besides the three divergent residues described above that are unique to each receptor subtype. Of the eight structural elements demonstrated to form the ligand binding pocket of RARgamma , four of these (H1, H3, C-terminal portion of H5 including Arg278, and beta  turn) contribute to defining that portion of the pocket involved in the interaction with the carboxylate group of RA. All of the amino acid residues of these four structural elements demonstrated to be within 4.5 Å of RA in RARgamma are conserved among the three RAR subtypes. However, when one considers the conservation of all amino acids that constitute these four structural elements of RARalpha and RARbeta , only H1 and H3 contain residues that are not conserved. Six of the 18 residues in H1 (Ala182, Val184, Gly185, Glu186, Ile188, and Val190 of RARalpha ) and 4 of the 23 residues in H3 (Ile222, Asp223, Ser232, and Thr237 of RARalpha ) differ between RARalpha and RARbeta . In addition, 3 of the 5 positively charged amino acids reported to be part of the positively charged electrostatic guidance field in RARgamma are located on H3, whereas the other 2 are located on H5. Therefore, it is likely that these divergent residues in H1 and H3 may play an important role in defining the orientation of and electronic environment associated with Arg269 in RARbeta compared with Arg276 in RARalpha . Thus, consideration of these divergent residues in H1 and H3 and their effect on the positioning of the positively charged amino acid residues in H3 and H5 may be useful in the construction of subtype-selective retinoids.


FOOTNOTES

*   This work was supported in part by National Institutes of Health Grant DK44517 (to D. R. S.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
§   Supported by a Temple University graduate student fellowship.
   Supported by the M.D./Ph.D. Program of Temple University School of Medicine.
Dagger Dagger    Recipient of National Institutes of Health Research Career Development Award HD01076. To whom correspondence should be addressed: Dept. of Biochemistry, Temple University School of Medicine, 3420 N. Broad St., Philadelphia, PA 19140. Tel.: 215-707-3266; Fax: 215-707-7536.
1   The abbreviations used are: RA, retinoic acid; RAR, retinoic acid receptors; RXR, retinoid X receptor; RARE, retinoic acid-responsive element; CAT, chloramphenicol acetyltransferase; EMSA, electrophoretic mobility shift assay; s, sense; as, antisense; TBE, Tris borate/EDTA.

ACKNOWLEDGEMENTS

We thank Erin Fitzgibbons, Sijie Zhang, Zeng-ping Zhang, Susan Horvath, and Gladys Yumet for their technical assistance. In addition, we thank Prof. Pierre Chambon for the pSG5-RAR constructs, Dr. Ronald Evans for the RARE-CAT reporter construct, and F. Hoffmann-LaRoche for the retinoids used in these studies.


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