©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Mutagenesis of the Ligand Binding Domain of the Human Retinoic Acid Receptor Identifies Critical Residues for 9-cis-Retinoic Acid Binding (*)

(Received for publication, March 7, 1995; and in revised form, June 13, 1995)

Bonnie F. Tate Joseph F. Grippo (§)

From the Department of Metabolic Diseases, Hoffmann-La Roche Inc., Nutley, New Jersey 07110

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

We have recently identified a small region (amino acids 405-419) within the ligand binding domain of a truncated human retinoic acid receptor alpha (Delta419) that is required for binding of 9-cis-retinoic acid (RA), but not all-trans-retinoic acid (t-RA). To probe the structural determinants of this high affinity 9-cis-RA binding site, a series of Delta419 mutants were prepared whereby an individual alanine residue was substituted for each amino acid within this region. These modified receptors were expressed in mammalian COS-1 cells and assayed for their ability to bind 9-cis-RA as well as t-RA. Only two of the mutants, M406A (mutation of methionine 406 to alanine), and I410A (mutation of isoleucine 410 to alanine) exhibit no detectable binding of 9-cis-RA when analyzed using saturation binding kinetics. Substitution of methionine 406 with the amino acids leucine, isoleucine, and valine yields mutant receptors that exhibit decreased binding for 9-cis-RA as the length or hydrophobicity of the R group decreases. Further substitution of methionine 406 with the small polar amino acid, threonine, results in a loss of detectable 9-cis-RA binding. Since amino acids 405-419 on a human RARalpha (hRARalpha) are predicted to form a short amphipathic alpha-helix, modeling of this structure into a helical wheel indicates that these two amino acids, methionine 406 and isoleucine 410, are actually positioned proximal to each other. Data presented here suggest that high affinity 9-cis-RA binding to a hRARalpha depends on an interaction with the two amino acids methionine 406 and isoleucine 410.


INTRODUCTION

The retinoic acid receptors (RARs) (^1)(1, 2, 3, 4) and the retinoid X receptors (RXRs, 5-8) have a prominent role in cellular differentiation and vertebrate development through their interaction with retinoic acids (RA), which are natural derivatives of vitamin A (retinol, (9) and (10) ). The RARs and the RXRs are members of a superfamily of ligand-dependent transcriptional regulators which also include the steroid hormone, thyroid hormone, and vitamin D receptors(11, 12) . Based on amino acid homologies and functional similarities, six regions of identity (A-F) have been ascribed to members of this superfamily, including the RARs and RXRs, through which modulation of transcription can occur(11, 12) . Regions A and B contain a ligand-independent transcription function (AF-1) while region C contains a highly conserved zinc finger DNA-binding domain. The functions of regions D and F are still relatively unknown, however, region E is known to contain a multiplicity of functions, namely, those of heterodimerization, ligand-dependent transactivation (AF-2) and ligand binding(13) .

Both the RARs and RXRs bind endogenous stereoisomers of RA within the ligand binding region and undergo a conformational change prior to their activation as transcriptional regulators(14, 15, 16, 17) . The RXRs show a binding preference for 9-cis-RA (18, 19, 20, 21) while the RARs bind 9-cis-RA as well as the stereoisomer, all-trans-RA (t-RA), with equal affinity(18) . An analysis of ligand binding to mouse RARs using saturation kinetics and Scatchard analyses gave dissociation constants (Kvalues) in the range of 0.2-0.7 nM for both 9-cis-RA and t-RA(22) . Competition of [^3H]t-RA or [^3H]9-cis-RA by 9-cis-RA and t-RA indicates that the two ligands can compete with one another for binding to the RARs(22) . These binding analyses led to the seemingly obvious conclusion that a common binding pocket on an RAR exists for both t-RA and its configurational isomer, 9-cis-RA.

Recently, however, we found that there are actual differences in the binding pocket for these two ligands and that a distinct region is required for the binding of 9-cis-RA, but not t-RA, to a hRARalpha(17) . Truncation of 43 amino acids to position 419 (removal of the F domain) on a hRARalpha produced a mutant which had a binding affinity for both t-RA and 9-cis-RA with normal K's of 0.2-0.3 nM for both ligands. Truncation of 58 amino acids to position 404, however, produced a mutant which had a normal binding affinity for t-RA (K = 0.3 nM), but which demonstrated no detectable binding of 9-cis-RA. We concluded that the binding sites for t-RA and 9-cis-RA on a hRARalpha were overlapping but distinct and that the region from amino acids 405 to 419 defined a high affinity binding determinant for 9-cis-RA. Interestingly, this region also corresponded with the highly conserved AF-2 domain found in the majority of receptors of the steroid hormone, thyroid hormone, and vitamin D superfamily(23) . Therefore, this same mutant which lost the ability to bind 9-cis-RA (Delta404) also failed to initiate transcription on a retinoic acid-responsive element after binding t-RA. Addition of these 15 amino acids (405-419) to a Delta404 resulted in a recovery of both 9-cis-RA binding and transactivation ability and yielded a fully functional receptor (Delta419, (17) ).

Since we had isolated a high affinity 9-cis-RA binding determinant to within 15 amino acids, we were then able to analyze those motif(s) responsible for this high affinity binding. Therefore, we have further characterized the region from amino acids 405 to 419 using alanine scanning mutagenesis on a truncated hRARalpha and have found that the integrity of two amino acids, methionine 406 and isoleucine 410, appear critical for high affinity 9-cis-RA binding. Based on a Chou and Fasman (24) algorithm which predicts that this region forms a short alpha-helical structure, these two amino acids actually exist in close proximity to each other. From our data we propose a model of 9-cis-RA binding to a truncated hRARalpha that may involve a contact with these two amino acids, methionine 406 and isoleucine 410.


EXPERIMENTAL PROCEDURES

Materials

All-trans-[11,12-^3H]retinoic acid was purchased from DuPont NEN. All-trans retinoic acid, 9-cis-retinoic acid, 9-cis-19-pentyl retinoic acid ((2E,4E,6Z,8E)-3-methyl-7-hexyl-9-(2,6,6-trimethyl-1-cyclohexen-1yl)-2,4,6,8-nonatetraenoic acid), 9-cis-20-butyl-retinoic acid ((2E,4E,6Z,8E)-3-penyl-7-methyl-9-(2,6,6-trimethyl-1-cyclohexen-1-yl)-2,4,6,8-nonatetraenoic acid), and [10-^3H]9-cis-retinoic acid were synthesized and purified by the Department of Medicinal Chemistry, Hoffmann-La Roche.

Construction of Receptor Mutants

Mutant RARalpha receptors were generated by modifying the full-length human RARalpha cDNA that was cloned into the EcoRI site of the expression vector pSG5 (25) . The plasmid pSG5-hRARalpha was linearized with BamHI and digested with SmaI to remove sequences at the 3` end of hRARalpha. Amino acid mutations were incorporated into the design of synthetic single-stranded oligonucleotides. The synthetic single-stranded oligonucleotides were phosphorylated at 37 °C for 60 min in the presence of T4 polynucleotide kinase (Life Technologies, Inc.), 70 mM Tris (pH 7.6), 100 mM KCl, 10 mM MgCl(2), 5 mM dithiothreitol, and 0.5 mM ATP and then annealed with their corresponding opposite strands by heating to 100 °C for 3 min and slowly cooling to room temperature. Each mutant receptor was then generated by insertion of the synthetic double-stranded oligonucleotides encoding the amino acids of interest and a stop codon into the SmaI-BamHI site. The integrity of the mutants and the presence of in-frame TGA codons was confirmed by sequencing.

Cell Culture and Transfections

COS-1 cells (African green monkey kidney cells derived from SV40-transformed CV-1 cells; ATCC) were maintained in Dulbecco's minimal essential medium (Life Technologies, Inc.) containing 100 units/ml penicillin, 100 µg/ml streptomycin, and 10% heat-inactivated fetal calf serum. COS-1 cells were transiently transfected by electroporation as described previously (20) . Typically, 5 10^5 cells were electroporated with a total of 25 µg of receptor expression vector. The cells were then seeded into 15-cm plates and, after 72 h at 37 °C, harvested for the preparation of nucleosol.

Nucleosol Preparations

COS-1 cells transfected with the expression vectors containing cDNAs encoding wild-type or mutant receptors were harvested and nucleosol fractions were prepared essentially as described previously for the use in retinoic acid binding assays(18, 20, 26) . Briefly, nuclei from transfected cells (4 plates) were disrupted in 4.0 ml of lysis buffer containing 10 mM Tris (pH 8.0), 1.5 mM EDTA, 2 mM dithiothreitol, 10% glycerol, 1 mM phenylmethylsulfonyl fluoride, 10 µg/ml leupeptin, 10 µg/ml aprotinin, and 800 mM KCl. Aliquots were stored at -80 °C until further use.

Retinoic Acid Binding Assays

Binding assays with [^3H]t-RA and [^3H]9-cis-RA were performed as detailed previously(20, 22) . Aliquots of nucleosol (15-25 µl) were incubated at 4 °C for 4 h after the addition of ligand contained in ethanolic solutions that did not exceed 2% of the total assay volume. Receptor bound radioactivity was then separated from free by chromatography over a desalting PD-10 column (Pharmacia LKB Biotechnology Inc.), radioactive eluate was collected and tritium counts were determined as described previously(22) . Binding of tritiated ligand in the presence of a 100-fold molar excess of unlabeled ligand was defined as nonspecific binding. Specific binding was defined as the total binding minus the nonspecific binding. For total binding assays, the concentration of [^3H]t-RA or [^3H]9-cis-RA was 5 nM, and %t-RA binding activity was defined as femtomoles (fmol) of 9-cis-RA binding/fmol of t-RA binding 100, where the amount of nucleosol used in the assays was normalized to yield between 300 and 500 fmol of t-RA binding for each expressed mutant receptor. For saturation kinetics and Scatchard analyses, [^3H]t-RA or [^3H]9-cis-RA was added at increasing concentrations from 4 10 to 5 10M. Affinity of wild-type or mutant receptors for t-RA and 9-cis-RA was determined by the method of Scatchard(27) . For competitive binding assays, incubations were performed with increasing concentrations of unlabeled competing ligand and a fixed nominal concentration of radioligand (5 nM [^3H]t-RA).

Analysis of Data

Data shown in Fig. 3-5 are representative of at least two independent experiments performed in duplicate. The data in Table 1and Table 2are the means of two to four independent experiments performed in duplicate.


Figure 3: Saturation binding analysis of [^3H]t-RA or [^3H]9-cis-RA binding to the Delta404, M406A, and E415,418A mutant receptors. Nucleosol from COS-1 cells transiently transfected with the indicated mutant receptors was incubated with the indicated concentrations of [^3H]t-RA (A, B, and D) or [^3H]9-cis-RA (E) in the absence or presence of a 100-fold molar excess of unlabeled t-RA (A, B, and D) or 9-cis-RA (E). Nonspecific binding activity (open circle) was subtracted from total binding activity (open square) to generate specific binding activity (triangle). Scatchard analysis (C) revealed that M406A (closed triangles, K = 4.2 nM) had a 14-fold lower binding affinity for [^3H]t-RA than Delta404 (open triangles, K = 0.3 nM). Neither mutant bound [^3H]9-cis-RA with any apparent affinity. Scatchard analysis (F) also revealed that the double mutant, E415,418A, bound both [^3H]t-RA (open triangles) and [^3H]9-cis-RA (closed triangles) with apparent equal affinity (K values = 0.8 and 0.9 nM, respectively).








RESULTS

Binding of [^3H]9-cis-RA to Mutant Receptors

To investigate the contribution of individual amino acids to 9-cis-RA binding within the region from serine 405 to glycine 419 on the truncated receptor, Delta419, each residue (except methionine 413 and leucine 414 which were deleted) was replaced with alanine as depicted in Fig. 1. Substitution of alanine allows an analysis of the importance of the amino acid R groups for 9-cis-RA binding.


Figure 1: Schematic of the truncated, mutant human retinoic acid receptor alpha sequences. A schematic representation of the hRARalpha receptor truncated to amino acid position 419 (Delta419) is indicated at the top. The dark region corresponds to the ligand binding domain and the black lines indicate the region containing amino acids that have been replaced by alanine (from amino acid 405 to 419). The amino acid sequences are shown below. Deletions are symbolized by Delta and the numbers refer to the positions in the amino acid sequence. The hRARalpha mutants were made as described under ``Experimental Procedures'' by ligation of synthetic, double-stranded oligonucleotides.



For each mutant receptor depicted in Fig. 1, nucleosol was isolated from mammalian COS-1 cells transiently transfected with the corresponding receptor expression vector. [^3H]t-RA or [^3H]9-cis-RA was then incubated with these nucleosol fractions for 4 h at 4 °C since little isomerization has been shown to occur under these conditions(22) . The results are shown in Fig. 2and 9-cis-RA binding is calculated as %t-RA binding activity, where total binding of t-RA has been normalized to yield between 300 and 500 fmol of transfected receptor binding sites for each assay (from 15 to 25 µl of nucleosol).


Figure 2: Percent t-RA binding activities of hRARalpha Delta419 alanine mutants. The mutant receptors were transiently expressed in COS-1 cells and nucleosol was prepared as described under ``Experimental Procedures.'' Nucleosol was incubated with [^3H]t-RA or [^3H]9-cis-RA and total binding was calculated in the presence of 100-fold molar excess cold ligand. %t-RA binding was then reported as the total binding with [^3H]9-cis-RA/total binding with [^3H]t-RA (normalized to 300-500 fmol) 100. The truncation mutants Delta404 and Delta419 are also indicated.



The alanine substitutions that most strikingly disrupt 9-cis-RA binding are those at methionine 406, isoleucine 410, and proline 407. These substitutions result in only 5.6, 4.0, and 14% of 9-cis-RA binding activity, respectively, relative to that of t-RA. The parent receptor, Delta419, exhibits 89% 9-cis-RA/t-RA binding activity (Fig. 2). Alanine substitution of other amino acids proximal to methionine 406 also reduce 9-cis-RA binding but not as severely. For example, P408A and L409A bind 9-cis-RA at 43 and 53% of t-RA binding capacity, respectively. Interestingly, even though serine 405 is adjacent to methionine 406, S405A has a normal capacity for 9-cis-RA. This may be due to the fact that serine 405 lies N-terminal to the start of the putative amphipathic alpha-helix.

Using saturation kinetics and Scatchard analyses (27) we also determined the dissociation constants (K values) of select mutant receptors for binding 9-cis-RA and t-RA. The results are summarized in Table 1. Mutant receptors with changes in the amino acids of interest from Fig. 2as well as mutants with changes in each part of the alpha-helical structure were analyzed. The K values for 9-cis-RA binding to the mutant receptors emulate those results seen in Fig. 2where substitutions between amino acids 406 and 410, the amino-terminal portion of the alpha-helix, most affect 9-cis-RA binding (Table 1). Both M406A and I410A exhibit no detectable binding (NB), while P407A, P408A, and L409A exhibit K values of 14, 7.9, and 2.7 nM, respectively (Table 1). Receptors with mutations in the carboxyl-terminal portion of this region exhibit K values more in the normal range, 0.9 nM for both the deletion mutant ML413,414Delta and the double mutant E415,418A.

Binding of [^3H]t-RA to Mutant Receptors

Mutations within the amino-terminal portion of the alpha-helix that affect 9-cis-RA binding also affect t-RA binding. Binding affinity decreases to 1.8 nM for P408A and to 4.2 nM for M406A in comparison to both Delta419 (0.3 nM) and Delta404 (0.3 nM, Table 1). The fact that these perturbations of t-RA binding are confined to the first half of this alpha-helical structure demonstrates the critical nature of this region for ligand binding.

Representative saturation curves and Scatchard plots shown in Fig. 3lend further support to these interpretations. Both Delta404 (Fig. 3A) and M406A (Fig. 3B) exhibit saturable binding for [^3H]t-RA yielding a K of 0.3 nM for Delta404, similar to wild-type hRARalpha(18) , and a K of 4.2 nM for M406A (Fig. 3C). Scatchard analysis of E415,418A, however, shows a K of 0.9 for [^3H]9-cis-RA and a similar K of 0.8 for [^3H]t-RA (Fig. 3, D-F).

Amino Acid Substitutions at Methionine 406

To further probe the interaction of 9-cis-RA with this region on Delta419, additional mutants were made in which methionine 406 was changed to leucine, isoleucine, valine, or threonine. The results are shown in Fig. 4, where the R groups are also depicted. When normalized to %t-RA binding with methionine at position 406 (represented by Delta419), substitution of leucine, isoleucine, and valine yields 9-cis-RA binding values of 89, 72, and 54%, respectively (Fig. 4). Substitution of methionine with either alanine or threonine yields much lower values of 6 and 3%, respectively. This data suggests that the length, and perhaps the hydrophobicity, of the R group at position 406 is important for high affinity binding of 9-cis-RA.


Figure 4: Percent t-RA binding capacities and R group structures of different amino acids at position 406. Percent t-RA activity was calculated for each mutant receptor containing the indicated amino acid at position 406 as described for Fig. 2. The amino acid R group is indicated to the right of each bar, and the percent t-RA binding activity for a wild-type methionine (Delta419) at position 406 has been normalized to 100% as described under ``Experimental Procedures.''



Alkyl Substitutions on 9-cis-RA

Since we lost 9-cis-RA binding by replacing the longer side chain of methionine at position 406 by a methyl group from alanine, we next asked whether 9-cis-RA analogs with alkyl substitutions at positions 9 and 13 could restore high affinity binding of this ligand. Interestingly, a 9-cis-RA derivative which has a methyl to hexyl extension at position 9 competes well with t-RA for binding to M406A with an IC of 25 nM (Table 2). However, this same derivative competes with t-RA for binding to Delta419 with an IC of only 432 nM. This 17-fold difference in IC value suggests that a hydrophobic interaction is occurring between the methyl group at position 9 on 9-cis-RA and the methionine R group at amino acid position 406 on Delta419. A 9-cis-RA derivative which has a pentyl group at position 13, however, competes poorly with t-RA for binding to either M406A or Delta419, further indicating the importance of position 9 on 9-cis-RA. Taken together, the results obtained using these two analogs of 9-cis-RA suggest that a 9-cis-RA analog with an alkyl substitution at position 9 may be capable of restoring a high affinity binding interaction on M406A.


DISCUSSION

A number of the functions that allow the RARs to act as hormone-dependent transcription factors are contained within the ligand binding domain, namely, heterodimerization, hormone-dependent transactivation, and ligand binding (reviewed in (13) ). All of these functions are dependent on the ability of the RARs to bind their cognate ligands. While it is well established that the RARs have a high affinity for the ligands, t-RA and 9-cis-RA(18) , little is actually known about critical amino acid interactions that may distinguish between the binding of these two stereoisomers.

Recently, we reported that a truncated hRARalpha contains unique binding determinants for 9-cis-RA within the carboxyl-terminal region of the ligand binding domain(17) . In this study, we have determined the specific amino acids within this region (amino acids 405-419) that constitute this high affinity binding of 9-cis-RA by using alanine scanning mutagenesis(28) . This region in RARalpha is predicted to form an amphipathic alpha-helix that is well conserved among nuclear receptors(23) . In fact, analyses of the majority of substituted mutants shown, the exceptions being P407A and P408A, using a Chou and Fasman algorithm (24) indicates that the predicted secondary structure of this alpha-helix is not perturbed (data not shown). Overall, alanine scanning of amino acids 405 to 419 on a truncated hRARalpha demonstrates that the amino-terminal end of this region appears most important for high affinity 9-cis-RA binding. Specifically, amino acids 406, 407, 410, and to a lesser extent, 408, are critical as their replacement with alanine significantly alters the ability of the resulting receptors to bind 9-cis-RA.

When we modeled this putative amphipathic alpha-helical region between amino acids 404 and 420 of the hRARalpha into a helical wheel(29, 30) , the importance of methionine 406 and isoleucine 410 becomes clear. Since one turn in an alpha-helix is approximately 3.6 amino acid residues long, these two amino acids actually lie in close proximity to one another (Fig. 5). In addition, with the ability of the proline residues to initiate and stabilize the formation of the alpha-helix(24) , a decrease in binding capacity for 9-cis-RA with the mutation of prolines at positions 407 and 408 is not surprising.


Figure 5: Helical wheel analysis of a potential amphipathic alpha-helix on a hRARalpha (amino acids 406-419). The amino acid sequence of the carboxyl-terminal end of the ligand binding domain of the hRARalpha is projected showing the alpha-helical structure. The helix, as modeled here using a Chou and Fasman algorithm (24) and software from IntelliGenetics, Inc., is amphipathic with charged residues to the lower right and hydrophobic residues to the upper left. Those residues found to be most critical for 9-cis-RA binding as well as for maintaining the integrity of the t-RA binding pocket are shown in boxes (methionine 406 and isoleucine 410).



Interestingly, both M406A and I410A bind t-RA with lower affinity than Delta404, K values of 4.2 and 1.6 nM, respectively. We previously showed that Delta404 bound t-RA as well as the wild-type receptor which led us to the conclusion that amino acids up to and including 404 of a hRARalpha formed the t-RA binding pocket. A 10-fold decrease in t-RA binding affinity with the removal of just one additional amino acid to position 403 indicated to us that this was also a sensitive region for t-RA binding(17) . An incorrect three-dimensional interaction of amino acids 405-419 with the rest of the receptor may occur with the substitution of alanine at amino acid positions 406 or 410. This perturbation in three-dimensional structure could then ``block'' the t-RA binding pocket and result in a decreased affinity for t-RA.

We also examined the ability of some of the mutant receptors to activate gene transcription on an RARbeta retinoic acid responsive element in the presence of t-RA using transient transactivation assays. We found that although mutations in the carboxyl-terminal portion of this 405-419 region appear to have little effect on either 9-cis-RA or t-RA binding, they do have a profound effect on transactivation when measured in the presence of up to 10 µMt-RA. Similar to Delta404(17) , the mutants L409A, I410A, and ML413,414Delta all exhibit a dominant negative phenotype when compared to wild-type hRARalpha. M406A, on the other hand, yields results similar to wild-type when measured with concentrations of t-RA greater than or equal to 100 nM. (^2)These type of results have been seen previously with the estrogen and glucocorticoid receptors (23) and further emphasize the importance of this AF-2 region for members of the superfamily of ligand-dependent transcriptional regulators.

Our observations suggesting that the interaction of a methionine at amino acid position 406 with 9-cis-RA may be due to the length or hydrophobicity of the R group, led us to do the converse experiment where we modified the ligand, 9-cis-RA. This type of experiment has been done before with the visual pigment, rhodopsin, a member of the large family of G protein-linked receptors. Rhodopsin binds its ligand, 11-cis-retinal, by means of a protonated Schiff base linkage with the -amino group of lysine 296 on the receptor. Zhukovsky et al.(31) showed that when they made a rhodopsin mutant which had alanine in place of lysine at position 296, the receptor would no longer bind 11-cis-retinal. However, this mutant K296A would readily bind 11-cis-retinal when it was presented as a Schiff base with n-alkylamine(31) . We also found that the loss of 9-cis-RA binding by a methionine to alanine substitution could be overcome by a ligand modification which substitutes a hexyl for a methyl group on the 9 position of 9-cis-RA. It appears that an extension of the hydrocarbon chain on the ligand at this particular position can compensate for the decrease in length or hydrophobicity of the R group at amino acid position 406. In fact, that the truncated hRARalpha receptor yields close to wild type activities when a functional alkyl group is swapped from the receptor to the ligand suggests that the methyl group at the 9 position of 9-cis-RA is likely, but not necessarily, involved in a hydrophobic interaction with the methionine side chain at position 406.

There are other reports of a methionine interacting with a steroidal ligand, for example, a methionine to arginine substitution in a naturally occurring androgen-insensitive mutant caused a decrease in ligand binding activity to 5% of wild type(32) . Although this androgen mutation was found in the middle of the ligand binding domain of a human androgen receptor (codon 807), it was still in a region that is well conserved among the glucocorticoid, progesterone, estrogen, and mineralocorticoid receptors. This region was also predicted to form an alpha-helix. Methionine residues have also been shown to be important in the ligand binding domains of the glucocorticoid and progesterone receptors. Carlstedt-Duke et al.(33) used affinity labeling to determine that methionine 622, cysteine 754, and cysteine 656 of the rat glucocorticoid receptor interact with steroid ligands(33) . Their data indicated that methionine 622 and cysteine 754 are in close proximity when the protein folds. These investigators also showed that methionine 759 in the progesterone receptor corresponded to methionine 622 in the rat glucocorticoid receptor and played a similar role in ligand binding(34) . Strömstedt et al.(34) found that methionine 909 in the human progesterone receptor was labeled after the receptor was covalently charged with [^3H]promegestone. This methionine 909 lies within the same region of the receptor as methionine 406 in the hRARalpha, i.e. the conserved alpha-helix.

A recent report by Tairis et al.(35) showed that two positively charged amino acids, arginine 269 and lysine 220, are crucial for the binding of t-RA to a mouse RARbeta. Tairis et al.(35) postulated that these two amino acids may interact with the negatively charged carboxyl group on RA. Considering that protease mapping experiments imply the involvement of the entire ligand binding domain in the binding of ligands to RARs(14, 15, 16, 17) , the involvement of lysine 220, arginine 269, methionine 406, and isoleucine 410 in the binding of 9-cis-RA is not difficult to envision. In fact, all four amino acids are conserved within the RAR family. A ligand-induced three-dimensional conformation may occur upon binding of RA to an RAR such that the ligand binding domain ``folds'' around the ligand leaving lysine 220 and arginine 269 to interact with the carboxylate tail and methionine 406 and, possibly, isoleucine 410, to interact with the methyl group located at the 9 position on 9-cis-RA. We suggest that this interaction of 9-cis-RA with methionine 406 and isoleucine 410 gives this ligand a distinct binding domain compared to t-RA. The beta-ionone ring of both 9-cis-RA and t-RA would then likely fit into an overlapping hydrophobic pocket composed of a number of hydrophobic residues. While the complete structural details of this region await x-ray crystallographic determinations, clearly the results we describe here can provide insight into the nature of the interaction between the ligand binding pocket on a truncated hRARalpha and two different ligands.


FOOTNOTES

*
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed: Dept. of Metabolic Diseases, Hoffmann-La Roche, Inc., 340 Kingsland St., Nutley, NJ 07110-1199. Tel.: 201-235-8674; Fax: 201-235-7636.

(^1)
The abbreviations used are: RAR, retinoic acid receptor; RA, retinoic acid; t-RA, all-trans-retinoic acid; RXR, retinoid X receptor; hRARalpha, human RARalpha.

(^2)
B. F. Tate, G. Allenby, J. R. Perez, A. A. Levin, and J. F. Grippo, manuscript in preparation.


ACKNOWLEDGEMENTS

We acknowledge and thank Dr. Gary Allenby, Dr. Arthur A. Levin, and Sonja Kazmer for their scientific and intellectual contributions. We also thank M. Rosenberger and A. Lovey for the synthesis of the 9-cis-RA analogs, the Department of Medicinal Chemistry for the synthesis of the [10-^3H]9-cis-RA, and E. J. Duker, L. Foppiani, J. D. Larigan, and J. F. Levine from the Department of Biotechnology for their gene sequencing and oligonucleotide services.


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