©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Alteration in the Retinoid Specificity of Retinoic Acid Receptor- by Site-directed Mutagenesis of Arg and Lys(*)

(Received for publication, September 28, 1994; and in revised form, April 18, 1995)

Nikolaos Tairis (1) Jerome L. Gabriel (1) (2) Kenneth J. Soprano (2) (3) Dianne Robert Soprano (1) (2)(§)

From the  (1)Department of Biochemistry, the (2)Fels Institute for Cancer Research and Molecular Biology, and the (3)Department of Microbiology and Immunology, Temple University School of Medicine, Philadelphia, Pennsylvania 19140

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

Retinoic acid receptor-beta (RAR-beta) specifically binds retinoic acid (RA) and functions as a RA-inducible transcriptional regulatory factor. Simultaneous mutation of Arg and Lys of RAR-beta 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-betas, 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-beta 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.


INTRODUCTION

Retinoic acid (RA), (^1)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) .

Three RARs, termed RAR-alpha, RAR-beta, and RAR-, have been described (2, 5, 6, 7, 8, 9, 10, 11) . In addition, seven isoforms of RAR-alpha and RAR- and four isoforms of RAR-beta 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) .

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 and Lys of RAR-beta 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-beta 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.

In this report, we have further examined the role of Arg and Lys of RAR-beta in the binding of ligand by preparing and assaying mutants of RAR-beta, 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-beta and that these two amino acid residues play an important role in determining retinoid specificity of RAR-beta most likely by directly interacting with the carboxylate group of RA.


MATERIALS AND METHODS

Plasmid Constructs and Site-directed Mutagenesis

The entire coding sequences of mouse RAR-beta2 cDNA cloned into the mammalian expression vector, pSG5 (pSG5-RARbeta), was a generous gift from Prof. Pierre Chambon (Strasbourg, France) (11) . The wild type RAR-beta and the mutants K220A, R269A, and K220A/R269A were previously described(27) . Three additional mutants of wild type RAR-beta 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.

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-RARbeta was used as the template for the single mutants. For K220Q, two PCR fragments were synthesized using the primer pairs RARbeta-5s (5`-GGGAGGGATCCATCGAGGGTAGATTTGACTGTATGGAT-3`) plus K220Q-as (5`-CTCACTGAACTGGTCCCAGAG-3`) and RARbeta-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 RARbeta-5s and RARbeta-3as. The SacI-BstXI restriction fragment that contained the mutation was exchanged with that of the wild type pSG5-RARbeta to create the pSG5-RARbeta-K220Q mutant expression vector. The pSG5-RARbeta-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-RARbeta. Finally the double mutant pSG5-RARbeta-K220Q/R269Q expression vector was prepared exactly the same as the K220Q mutant, except that the template DNA was XbaI-linearized pSG5-RARbeta-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-beta2 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-RARbeta 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-beta-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 10M 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 beta-galactosidase (beta-gal) activity(32) . CAT activity was normalized with respect to beta-gal activity to control for transfection efficiency and expressed as a percentage of relative CAT activity. The normalized CAT activity of wild type RAR-beta at 10M 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-beta determined by extrapolation from the plotted points.

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-RARbeta expression construct, 4 µg of RARE-CAT reporter construct, 4 µg of pRSV-beta-gal, and 9 µg of the RAR-beta mutant construct or pSG5 carrier DNA). This resulted in a 1:3 molar ratio of wild type RAR-beta DNA to mutant RAR-beta DNA. In some experiments, the cells were treated with either 10 or 10M all-trans-RA. In other experiments, the cells were treated with a combination of 10M all-trans-RA and either all-trans-retinol (10 or 10M) or all-trans-retinal (10 or 10M). 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-RARbeta 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 [^3H]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 [^3H]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-beta 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.

To determine the K 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 [^3H]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 those mutants in which we could not determine the K 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 [^3H]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 Kdeterminations. 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.


RESULTS

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-beta singly (K220Q and R269Q) and simultaneously to Gln (K220Q/R269Q) on RA-dependent transactivation activity and RA binding. All three of these mutant RAR-betas 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-beta and our previously described complementary Ala mutant RAR-betas (27) .

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 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-beta demonstrates a similar relationship between the fold reduction in functional activity and all-trans-RA binding.


Figure 1: The effect of mutation of Lys and Arg of RAR-beta to Gln on transactivation by all-trans-RA. CV-1 cells were cotransfected with one of the RAR-beta expression vectors (wild type (WT), K220Q, R269Q, or K220Q/R269Q), RARE-CAT reporter construct, and pRSV-beta-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 beta-gal assays. CAT activity was normalized for efficiency of transfection using beta-gal activity and expressed as relative CAT activity. The percent relative CAT activity was calculated using the CAT activity of the wild type RAR-beta at 10M all-trans-RA as 100%. Each point represents the mean of four to six independent transfection assays ± S.E.






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 [^3H]RA and [^3H]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 [^3H]RA or [^3H]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 [^3H]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 [^3H]RA. K and IC values are presented in Tables I-III as the mean ± S.E. for at least three independent determinations.



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 and 10M) 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-beta. At a concentration of all-trans-RA below the EC value for all the mutants (10M), only 40, 25, and 15% of the wild type activity was detectable when cells were transfected with K220Q, R269Q, and K220Q/R269Q, respectively. At 10M 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-beta2 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.


Figure 3: The dominant negative effect of RAR-beta 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-RARbeta, 4 µg of RARE-CAT reporter construct, 4 µg of pRSV-beta-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 10M all-trans-RA. Cells were harvested 24 h later for CAT and beta-gal assays. Normalized CAT activity of the wild type RAR-beta 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.

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-beta 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-beta 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-beta also displayed low affinity for all-trans-retinol with no detectable specific binding of [^3H]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-beta. 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 10M 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.


Figure 4: The effect of mutation of Lys and Arg of RAR-beta to either Ala or Gln on transactivation by all-trans-retinol. CV-1 cells were cotransfected with one of the RAR-beta expression vectors (wild type (WT), K220A, K220Q, R269A, R269Q, K220A/R269A, and K220Q/R269Q), RARE-CAT reporter construct, and pRSV-beta-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-beta expression construct were treated with 10M all-trans-RA (ATRA). 24 h after addition of the retinoid, the cells were harvested for CAT and beta-gal assays. CAT activity was normalized for the efficiency of transfection using beta-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 10M all-trans-RA, which was set at 100%. Each point represents the mean of four to ten independent transfection assays ± S.E.





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-beta 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-beta.


Figure 5: The effect of mutation of Lys and Arg of RAR-beta 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-beta Mutants by Retinol and/or Retinal

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-beta 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 10M 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 10M (approximately the EC value of K220Q and R269Q) or 10M 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 10M 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 10M 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.


Figure 6: The dominant negative effect of RAR-beta 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-RARbeta, 4 µg of RARE-CAT reporter construct, 4 µg of pSV-beta-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 10M all-trans-RA along with either 10 or 10M all-trans-retinol. Normalized CAT activity of the wild type RAR-beta plus pSG5 at each retinol concentration was set to 100% relative activity. Values are mean ± S.E. of three to five independent experiments.



In similar experiments, cells were transfected with the wild type RAR-beta along with one of each of the mutant receptors and treated with 10M all-trans-RA along with either 10 or 10M 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 10M. 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 10M, there was a relief of the dominant negative effect to a level proportional to the functional activity of each receptor for all-trans-retinal.


Figure 7: The dominant negative effect of RAR-beta 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 10M instead of all-trans-retinol.




DISCUSSION

This work confirms and extends our previous report in which Arg and Lys of RAR-beta 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-betas 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-beta 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-beta may interact with the carboxylate group of RA and that the nature of this interaction determines specific, high affinity binding of RA.

Each of the single and the double Gln mutants of Arg and Lys displays a dramatic reduction (20- to geq 1000-fold) in RA-dependent transactivation activity and RA binding affinity when compared with that of the wild type RAR-beta 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.

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-beta 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.

Mutation of either Arg or Lys to either Ala or Gln results in the creation of a family of mutant RAR-beta 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-beta 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.

The exact reason for these changes in retinoid specificity and the understanding of how Arg 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-beta 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^6 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-beta 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.

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 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-beta 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.

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 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.

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 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 Kvalue 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-beta receptor. In other words, R269Q functions as a nuclear retinol receptor.

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 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-beta.


FOOTNOTES

*
This work was supported by National Institutes of Health Grants DK44517 (to D. R. S.) and CA64945 (to K. J. S.). 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.

§
A recipient of a Research Career Development Award from the National Institutes of Health (HD01076). To whom correspondence should be addressed: Dept. of Biochemistry, Temple University School of Medicine, 3420 North Broad St., Philadelphia, PA 19140. Tel.: 215-707-3266; Fax: 215-707-7536.

^1
The abbreviations used are: RA, retinoic acid; RAR, retinoic acid receptor; RXR, retinoid X receptor; RARE, retinoic acid response element; PCR, polymerase chain reaction; beta-gal, beta-galactosidase; CAT, chloramphenicol acetyltransferase.


ACKNOWLEDGEMENTS

We thank Prof. Pierre Chambon for the wild type RAR-beta 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.


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