Mutational Analysis Reveals That All-trans-retinoic acid, 9-cis-Retinoic acid, and Antagonist Interact with Distinct Binding Determinants of RARalpha *

(Received for publication, December 4, 1996, and in revised form, April 11, 1997)

Siegfried Keidel Dagger §, François P. Y. Lamour Dagger and Christian M. Apfel par

From the Preclinical Research, Department of Infectious Diseases, F. Hoffmann-La Roche Ltd., CH-4070 Basel, Switzerland

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES


ABSTRACT

Retinoids exert their pleiotropic effects on cell differentiation and proliferation through specific nuclear receptors, the retinoic acid receptors (RARs) and retinoid X receptors (RXRs). Two biologically highly active natural retinoids have been identified, all-trans-retinoic acid (t-RA) and 9-cis-retinoic acid (9-cis-RA). The RXRs exclusively bind 9-cis-RA, whereas the RARs bind both isomers of RA with comparable affinity. Recently published results suggest that RARs have the same binding site for t-RA and 9-cis-RA but with different determinants (1-3). Antagonist binding on RARalpha has been suggested to induce distinct conformational changes in comparison with agonist binding. To elucidate the region minimally required for efficient binding of agonist (t-RA and 9-cis-RA) and antagonist Ro 41-5253 to the RARalpha , we generated N- and C-terminally truncated mutants of the receptor. Characterization of these deletion mutant proteins using protease mapping and ligand binding experiments revealed that different parts of the ligand-binding domain are necessary for t-RA, 9-cis-RA, and antagonist binding. Three distinct regions of the ligand-binding domain of the human retinoic acid receptor-alpha are required for binding of t-RA (RARalpha 187-402), 9-cis-RA (RARalpha 188-409), and the antagonist Ro 41-5253 (RARalpha 226-414).


INTRODUCTION

Retinoic acid (RA)1 has a broad spectrum of biological activities in vertebrate development and homeostasis (4-6). Due to their fundamental role in the control of cell differentiation and proliferation, RA and synthetic retinoids are clinically very useful, predominantly in the treatment of leukemia and nonmalignant hyperproliferative disorders of the skin. However, retinoids have undesirable side effects such as hypervitaminosis A syndrome and teratogenicity (4, 7-10). Two biologically active stereoisomers of RA have been identified, all-trans-retinoic acid (t-RA), and 9-cis-retinoic acid (9-cis-RA) (11-13). The direct biological effects of the retinoids are mediated by the activation of two subfamilies of nuclear receptors, the retinoic acid receptors (RARs) and the retinoid X receptors (RXRs) (13-16). Both subfamilies consist of three receptor subtypes referred to as RARalpha , -beta , and -gamma (14, 15, 17-20) and RXRalpha , -beta , and -gamma (13, 16, 21). RARs and RXRs belong to the nuclear hormone receptor superfamily, whose members act as ligand-inducible transcription enhancer factors (22-24). On the basis of homology to other nuclear hormone receptors, the sequences of RARs are divided into six distinct regions designated A through F. The C domain constitutes a highly conserved DNA-binding domain, while the E domain confers the ligand binding properties of each receptor (25). RARs seem to operate effectively only as heterodimeric RAR·RXR complexes (26-31), but RXR-independent transactivation by RARs has also been observed (32). The RXRs are able to activate genes via homodimers (33, 34), but act predominantly as coregulators, enhancing the binding of RA, vitamin D3, thyroid hormone, and peroxisome proliferator-activated receptors to their response elements via heterodimerization (26-31, 35). t-RA and 9-cis-RA are the natural ligands for RARs (14, 36), whereas 9-cis-RA and phytanic acid are the natural ligands for RXRs (11, 12, 37). Both RA isomers compete with each other for RAR binding (38). A synthetic retinoid Ro 41-5253 has been identified as a selective RARalpha antagonist (39), which, when bound to the receptor, induces a different conformational change as detected by limited proteolysis (40). Recently, the structure of the cocrystal hRARgamma -t-RA has been determined (1), and modeling of 9-cis-RA in the RARgamma binding pocket suggests that the two isomers also compete for the same binding site in this receptor. In addition, two residues, Met-406 and Ile-410, have been shown to be critical, specifically for the binding of 9-cis-RA to hRARalpha (2). Binding experiments using RA and different synthetic ligands have shown different requirements in the C-terminal part of the hRARalpha but no distinction between t-RA and 9-cis-RA binding specificity (41). It has also been demonstrated recently that Cys-235, Arg-217, and Arg-294 of hRARalpha play a role in antagonist binding, showing specific requirements for efficient antagonist binding to hRARalpha (3). As shown by competitive binding experiments, t-RA competes with the antagonist for RARalpha binding (40). This strongly suggests that the antagonist shares a common binding site with the RA isomers. To analyze the minimal structural requirements for an efficient binding of either t-RA, 9-cis-RA, or Ro 41-5253 to the hRARalpha , we generated N- and C-terminally truncated RARalpha mutants. Characterization of these deletion-mutant proteins using protease mapping and ligand binding experiments revealed that different parts of the ligand-binding domain of RARalpha are necessary for t-RA, 9-cis-RA, and antagonist binding.


EXPERIMENTAL PROCEDURES

Materials

[3H]t-RA (50 Ci/mmol) was obtained from NEN Life Science Products. 9-cis-[3H]RA (25 Ci/mmol) was obtained from Amersham Corp. [3H]Ro 41-5253 (28 Ci/mmol), unlabeled RA isomers and analogues were synthesized at F. Hoffmann-La Roche Ltd., Basel, Switzerland. All procedures using retinoids were carried out under dimmed light. The TNT® coupled reticulocyte lysate system was purchased from Promega. [35S]Methionine (>1000 Ci/mmol), 14C-methylated protein molecular weight markers, and AmplifyTM were obtained from Amersham. Restriction enzymes were obtained from Boehringer Mannheim. Trypsin (type I: from bovine pancreas) was purchased from Sigma.

Plasmids

The human RARalpha cDNA insert of pT7-RARalpha (42) was subcloned as a MscI-BamHI fragment into pSG5-MscI giving pSG5-RARalpha . pSG5-MscI was constructed by replacing the EcoRI-BamHI fragment of pSG5 (43) with a linker containing an MscI site. RARalpha deletion mutants were produced by PCR. The correctness of the sequence was confirmed by sequencing. For N-terminal deletions: removal of the EcoRI site of pET3a (44) was carried out by restriction with EcoRI, fill-in reaction and religation, yielding pET3a-Delta E. The linker (5'-TATGGAATTCTAGCGCTTA-3'), including a start codon and a subsequent EcoRI site, was ligated into the NdeI site of pET3a-Delta E giving pET3a-E. pSG5-M was constructed by ligation of the linker (5'-AATTGCTACCACCATGGAATT-3') in the EcoRI site of pSG5. The mutants were generated by PCR using pT7-RARalpha as template. The PCR products were restricted with EcoRI-BamHI (located in the 5' primer and 3' primer, respectively) and cloned into pET3a-E (expression in Escherichia coli for binding studies) or pSG5-M (in vitro translation for limited proteolytic digestion). For C-terminal deletions, the mutants were generated by PCR using pT7-RARalpha as template, 5' primers (positions 466-486), and 3' primer containing a BamHI site. The PCR products were restricted with SacI-BamHI and cloned into either the full-length pSG5-RARalpha restricted with SacI-BamHI (in vitro translation for limited proteolytic digestion) or the truncated pET3a-RARalpha -Delta 155 restricted with SacI-BamHI (expression in E. coli for binding studies).

Binding Assays, Kd and IC50 Determinations

RARalpha deletion mutants were expressed in E. coli BL21(DE3)pLys (44). Expression and solubility problems occurred with the N-terminal deletion mutants hRARalpha -Delta 188, -Delta 189, -Delta 225, -Delta 226, and -Delta 227 expressed in E. coli. These truncated proteins were synthesized with the TNT® coupled reticulocyte lysate system (Promega) for the determination of the IC50 values. For these last mutant receptors, a 40-60% specific binding was sufficient to allow the IC50 determination. Since Kd values, for either t-RA or 9-cis-RA, have been established for the C-terminal point mutants of hRARalpha (aa 405-419) (2), we determined the binding activity of the C-terminally truncated receptors by Scatchard analysis for the same ligands. From the N-terminally truncated hRARalpha (aa 187-227), no binding activity was determined previously, and we verified the binding activity by IC50 determination. The binding assays (Kd and IC50 determinations) were performed as described elsewhere (3).

In Vitro Transcription, Translation, and Limited Proteolytic Digestion

Human RARalpha (wt or truncated) in pSG5 was in vitro transcribed and translated in the presence of [35S]methionine by using rabbit reticulocyte lysates as specified by Promega. The limited proteolytic digestions were performed as described elsewhere (40).


RESULTS

Binding of t-RA to the N- and C-terminally Truncated RARalpha Mutants

An important region for the t-RA binding (aa 186-198) was defined previously in hRARalpha by a N-terminally truncated receptor (41). A C-terminal deletion to position 403 resulted in a moderate decrease in affinity for the same ligand (41). On the other hand, a C-terminal deletion to position 404 of hRARalpha exhibited a Kd of 0.3 nM for t-RA (2). To determine the precise region of the hRARalpha -LBD required for an efficient t-RA binding, we used binding assays and protease mapping to probe t-RA binding to N- and C-terminally truncated hRARalpha -LBD (Figs. 1 and 2).


Fig. 1. Effect of t-RA (lanes 14-20), 9-cis-RA (lanes 21-27), and Ro 41-5253 (lanes 28-32) on limited tryptic digestion of RARalpha wild type (wt) and C-terminal deletion mutants. Controls for wt and truncated receptor expression are in lanes 1-13. The presence of a proteolytic resistant fragment indicates that the indicated retinoid is able to bind the truncated receptors. In vitro synthesized [35S]methionine-labeled RARalpha deletion mutants were preincubated with Me2SO alone or the indicated concentrations of t-RA, 9-cis-RA, or Ro 41-5253. Trypsin solution was added giving a final concentration of 75 µg/ml. Alternatively, an equal volume of water was added. Incubations were for 10 min at room temperature. Samples were electrophoresed through a SDS-polyacrylamide gel, and the dried gel was autoradiographed. The sizes of molecular weight markers are indicated. The resistant protein fragment occurring in the presence of RA is marked by a diamond, and the resistant fragment characteristic of the antagonist is indicated by a star.
[View Larger Version of this Image (51K GIF file)]


Fig. 2. Effect of t-RA (lanes 11-16), 9-cis-RA (lanes 17-22), and Ro 41-5253 (lanes 23-27) on limited tryptic digestion of RARalpha wild type (wt) and N-terminal deletion mutants. Controls for wt and truncated receptor expression are in lanes 1-10. The experimental conditions were the same as described in the legend to Fig. 1.
[View Larger Version of this Image (61K GIF file)]

A Kd of 1.1 nM for t-RA was obtained with the hRARalpha -LBD (aa 155-462) (Table I). This value is in the range of the Kd published for the full-length hRARalpha expressed in E. coli or COS cells (0.67 nM (42), 1.7 nM (45)), and also for the hRARalpha -LBD expressed in E. coli (6.2 nM (46); 0.6 nM (3)).

Table I. t-RA, 9-cis-RA, and Ro 41-5253 binding properties of wt hRARalpha -LBD and C-terminally truncated mutants

The Kd values (nM) were obtained by Scatchard analysis. The Kd and IC50 values are the mean of two single experiments. For one analysis, each point was made in triplicate.

hRARalpha -LBD mutants (aa 155-462) t-RA (Kd) 9-cis-RA (Kd) Ro 41-5253 (IC50)

nM nM
  wt 1.1 1 16
 -Delta 414 NDa ND 59
 -Delta 413 ND ND 250
 -Delta 409 ND 36.5 >1000
 -Delta 408 ND 175 NB
 -Delta 402 13 NB
 -Delta 401 NBb

a ND, not determined.
b NB, no binding activity detectable.

Among the N-terminally truncated receptors, the hRARalpha -Delta 187 exhibited good protection against trypsin digestion when bound to t-RA. A strongly reduced signal was observed for the digestion of the hRARalpha -Delta 188 truncated receptor under the same conditions. At the C-terminal end of the receptor, the same tendency was observed with the two mutants hRARalpha -Delta 402 and hRARalpha -Delta 401. In regard to the binding activity of these mutant receptors, the N-terminally truncated hRARalpha -Delta 187 exhibited a Kd value 4.5-fold higher than the wild type hRARalpha -LBD, whereas a deletion of a single further amino acid abolished the binding activity in such a way that neither Kd nor IC50 could be determined (Table II).

Table II. Effect of truncation of the N-terminal portion of the hRARalpha on binding of t-RA, 9-cis-RA, and the antagonist Ro 41-5253

The IC50 values are the mean of two single experiments.



1 NB, no binding activity detectable.

Similar experiments were carried out using N-terminally truncated mutants of hRARgamma . We found that hRARgamma -Delta 189 (hRARalpha -Delta 187) was able to bind t-RA with an affinity comparable with the full-length hRARgamma , whereas hRARgamma -Delta 190 (hRARalpha -Delta 188) showed no detectable binding activity to this ligand (data not shown). Therefore, it appears that, while the C-terminal region of the D domain of RARalpha (aa 188-199) is required for efficient binding of the t-RA, the C-terminal part of the E domain (aa 403-419) and the entire F domain can be deleted without affecting the ability of the RARalpha to bind its natural t-RA ligand (see Fig. 3).


Fig. 3. Schematic presentation of the RARalpha fragments, which are minimally needed for high affinity binding of t-RA, 9-cis-RA, and Ro 41-5253. For comparison the RARalpha regions contained in the main proteolytic resistant fragments occurring after tryptic digestion of t-RA-, 9-cis-RA, or Ro 41-5253-liganded RARalpha (40) are shown.
[View Larger Version of this Image (15K GIF file)]

Binding of 9-cis-RA to the N- and C-terminally Truncated RARalpha Mutants

Two contradictory results have been published concerning the binding of 9-cis-RA to C-terminal deletion mutants of hRARalpha . Lefebvre et al. (41) observed that a deletion up to position 403 of the hRARalpha expressed in E. coli resulted in a moderate decrease in affinity for 9-cis-RA (41). On the other hand, a small region (aa 405-419) within the ligand binding domain of a truncated hRARalpha was demonstrated to be required for the 9-cis-RA binding (2). In our study, a Kd value of 1 nM was determined for the binding of 9-cis-RA to the hRARalpha -LBD. This value is in agreement with the range of the reported data for the truncated receptors expressed in E. coli (0.3 nM for the hRARalpha -Delta 419 (2) and 1.2 nM for the hRARalpha -LBD (3)).

After the limited trypsin digestion of the N-terminally truncated hRARalpha liganded to 9-cis-RA, the corresponding protection was almost completely abolished for the mutant hRARalpha -Delta 189, and a reduction of the signal was already observed for the mutant hRARalpha -Delta 188 (Fig. 2). At the C-terminal end of the receptor, the digestion of the hRARalpha -Delta 409 liganded to 9-cis-RA by trypsin yielded a band of the appropriate size, whereas the truncated hRARalpha at the next residue (hRARalpha -Delta 408) exhibited only a faint band (Fig. 1). IC50 and Kd determinations for the binding of the 9-cis-RA to the above mentioned truncated receptors confirmed the N- and C-terminal deletion borders of the hRARalpha concerning the RA isomer. No 9-cis-RA binding was detectable to the hRARalpha -Delta 189, whereas a 5-fold higher IC50 value was obtained for the binding of the t-RA isomer to the hRARalpha -Delta 188 in comparison with the hRARalpha -LBD (Table II). At the C-terminal end, a 35-fold higher Kd value, was obtained for the hRARalpha -Delta 409, while the hRARalpha -Delta 408 bound the 9-cis-RA more than 150-fold less efficiently than the hRARalpha -LBD (Table I). From these results, it is clear that the C-terminal region of the D domain of RARalpha (aa 189-199) is required for an efficient binding of 9-cis-RA. Furthermore, the C-terminal part of the E domain (up to aa 409) is also crucial for the binding efficiency of this ligand to the hRARalpha (see Fig. 3).

Binding of Ro 41-5253 to the N- and C-terminally Truncated RARalpha Mutants

The antagonist Ro 41-5253 has been reported to induce a different conformational change when bound to the hRARalpha (40). It has also been shown recently that Ro 41-5253, as well as other antagonists, exhibit distinctly different requirements for efficient binding to hRARalpha (3). In the present study, we determined an IC50 value of 16 nM for the binding activity of the antagonist Ro 41-5253 to the hRARalpha -LBD (Table I). Limited trypsin proteolysis of the N- and C-terminally truncated hRARalpha bound to Ro 41-5253 led to the definition of the N-terminal deletion border between the residue D226 and the residue Lys-227 and the C-terminal deletion border between the residue L414 and M413 of the E region of the hRARalpha -LBD. Concerning the binding activity of Ro 41-5253 to the truncated hRARalpha s, 24- and 15-fold higher IC50 values for hRARalpha -Delta 227 and hRARalpha -Delta 413 have been observed, respectively, in comparison with the hRARalpha -LBD (Tables I and II). Remarkably, the hRARalpha -Delta 226 and the hRARalpha -Delta 414 mutant exhibited only a 2- and 3.5-fold reduction in IC50, respectively, in comparison with the hRARalpha -LBD. These results evidence distinctly different requirements of the N- and C-terminal regions of the ligand-binding domain of the hRARalpha for binding Ro 41-5253 compared with the two other ligands t-RA and 9-cis-RA (see Fig. 3).


DISCUSSION

It has been shown that the binding of both t-RA and 9-cis-RA induces a different conformational change in hRARalpha to that observed with the binding of the antagonist Ro 41-5253 (40). At present, several arguments allow one to think that, depending on the chemical structure of the ligands, distinctly different determinants are required for the efficient binding to the RARs. Recently published results also showed distinct determinant requirements for the binding of different ligands to the estrogen receptor (47). The recently described crystal structure of the RARgamma ligand-binding domain bound to t-RA has clarified the ligand binding interactions. Modeling 9-cis-RA in the hRARgamma binding pocket revealed that the binding site of this receptor was likely to be very similar to that of t-RA (1). In addition, three residues have been shown to play a significant role in ligand binding (agonist and antagonist) to hRARalpha , whereas Cys-235, Arg-217, and Arg-294 have been shown to be important residues of the hRARalpha -LBD, specifically for antagonist interactions (3). Furthermore, Arg-269 of the hRARbeta (Arg-276 of hRARalpha and Arg-278 of hRARgamma ) has been shown to be a crucial residue for RA binding (48, 49). These findings and the highly conserved amino acid homology of the E domain of the RARs (92%) (23) suggest that the binding conditions of t-RA to the three subtypes of receptors RARalpha , -beta , and -gamma are very similar. It also indicates that these ligands may compete for a unique binding site in the RARs.

In the present study, the 30-fold higher Kd value for 9-cis-RA binding to the C-terminal deletion hRARalpha -Delta 409 reinforces the role of the residue Ile-410 in the 9-cis-RA binding as shown by Tate and Grippo (2). We have provided evidence by this study that the complete F domain can be removed without disturbing the ability of the hRARalpha to bind any ligand. The C-terminal part of the E domain of the hRARalpha (to aa 414) is sufficient for the efficient binding of the antagonist Ro 41-5253, whereas further deletion of the E domain of the hRARalpha to positions 409 or 402 for 9-cis-RA and t-RA, respectively, does not disturb the ability of the receptor to bind these two ligands. Interestingly, t-RA and 9-cis-RA require the C-terminal part of the D domain of the hRARalpha (from aa 187 and 188, respectively), while the D domain and the N-terminal part of the E domain (aa 155 to 226) are not required for the efficient binding of the antagonist Ro 41-5253.

From the crystal structure of the hRARgamma -LBD bound to t-RA and the modeled 9-cis-RA in the hRARgamma binding pocket, less distance was expected between the ligand 9-cis-RA and the C-terminal region of the hRARgamma (helix-11 and helix-12) in comparison with the t-RA spatial disposition (1). It was also clear that the C-terminal part of helix-1 overlaps with the C-terminal part of helix-3. The importance of residue Cys-235 of the hRARalpha in antagonist binding (3) indicates a role of the helix-3 in antagonist binding. Remarkably, all of helix-1 can be deleted without disturbing antagonist binding, whereas this completely abolishes agonist binding. An explanation for this could be that the C-terminal part of helix-1 may stabilize the spatial location of helix-3 required specifically for the binding of the agonists.

Taken together, these results indicate that three different fragments of hRARalpha are minimally required for the binding of t-RA, 9-cis-RA, and Ro 41-5253. The data shown here suggest that distinct determinants of hRARalpha are either directly involved in the binding of these three ligands or, alternatively, they are needed to maintain the binding pocket in an appropriate conformation as a prerequisite for the binding of each of the isomers of RA as well as Ro 41-5253.

Limited trypsin digestion of either wild type or truncated receptor yielded the same digested peptides corresponding to the potential cleavage sites (data not shown). The undigested mutant receptors showed positions in the gel that were in accordance with their calculated molecular weights (Fig. 1, lanes 2-13; Fig. 2, lanes 2-10). Also, the molecular weights of the fragments as determined from the SDS gel were in good accordance with the magnitude of the truncation of the mutants (Figs. 1 and 2). This indicates that the binding of either the agonists t-RA and 9-cis-RA or the antagonist Ro 41-5253 to the truncated receptors induces the same conformational change in all the mutants as that observed with the full-length RARalpha . In this study, we demonstrate that with trypsin digestion of 9-cis-RA-bound full-length hRARalpha , the resulting 30-kDa fragment is less protected against further proteolysis than the one derived from the analogous experiment with t-RA. This was evidenced by the time course of tryptic digestion of RARalpha bound to t-RA versus 9-cis RA-bound receptor (Fig. 4). These results were further confirmed by tryptic proteolysis with increasing protease concentrations (data not shown). These differences in the proteolytic resistance cannot be explained by distinct affinities of the RA isomers to the RARalpha , because both exhibit Kd values in the range of 1 nM (Tables I). The decreased stability of the 30-kDa fragment obtained from RARalpha bound to 9-cis-RA could reflect the higher off-rate of 9-cis-RA from RARalpha in comparison with the t-RA. Displacement assays have demonstrated that 9-cis-RA exhibits about 2-fold higher off-rates from murine RARalpha than t-RA (38). Time course experiment using Ro 41-5253 as ligand yielded also a decreased stability of the 25-KDa antagonist characteristic fragment compared with the stable 30-kDa fragment observed with t-RA (Fig. 4). Incubation of the receptor with Ro 41-5253 or 9-cis-RA instead of t-RA in the digestion assay could result in more unliganded receptors, or fragments, for a short period of time. During this time, they might change to a more relaxed conformation, more accessible to the protease.


Fig. 4. Time course of limited tryptic digestion of t-RA-, 9-cis-RA-, and Ro 41-5253-liganded RARalpha . In vitro synthesized [35S]methionine-labeled RARalpha was preincubated with Me2SO alone, with 100 nM of either t-RA or 9-cis-RA or with 10 µM of the antagonist Ro 41-5253. Trypsin solution was added, giving a final concentration of 100 µg/ml. Alternatively, an equal volume of water was added. Incubations were for the indicated time intervals at room temperature. Cleavage was immediately stopped by boiling with SDS sample buffer. Samples were electrophoresed through a SDS-polyacrylamide gel, and the dried gel was autoradiographed. The sizes of molecular weight markers are indicated. The resistant protein fragment occurring in the presence of RA isomers is marked by a diamond, and the resistant fragment characteristic of the antagonist is indicated by a star.
[View Larger Version of this Image (74K GIF file)]

In conclusion, we have evidenced that three distinct regions of the of the hRARalpha -LBD are required for the efficient binding of t-RA, 9-cis-RA, and the antagonist Ro 41-5253. t-RA binding requires the region of the hRARalpha -LBD from amino acid 187 to amino acid 402, 9-cis-RA requires the region of the hRARalpha -LBD from amino acid 188 to amino acid 409, while the antagonist requires the region of the hRARalpha -LBD from amino acid 226 to amino acid 414.


FOOTNOTES

*   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.
Dagger    Both authors contributed equally to this work.
§   Present Department at F. Hoffmann-La Roche Ltd.: Quality Control and Assurance.
   Present address: Biozentrum, Abt. Strukturbiologie, Rm. 379, Klingelbergstrasse 70, CH-4056 Basel, Switzerland.
par    To whom correspondence should be addressed: PRPI, 69/11a, F. Hoffmann-La Roche Ltd., CH-4070 Basel, Switzerland. Tel.: 41-61-688-5878; Fax: 41-61-688-2377.
1   The abbreviations used are: RA, retinoic acid; RAR, retinoic acid receptor; RXR, retinoid X receptor; t-RA, all-trans-retinoic acid; 9-cis-RA, 9-cis-retinoic acid; hRARalpha , human retinoic acid receptor alpha ; hRARalpha -LBD, human retinoic acid receptor alpha -ligand-binding domain; RARalpha , retinoic acid receptor alpha ; aa, amino acid; wt, wild type.

ACKNOWLEDGEMENTS

We thank M. Klaus (F. Hoffmann-La Roche Ltd., Basel, Switzerland) for synthesizing the retinoids, P. LeMotte, P. Lardelli and W. Keck for critically reading the manuscript. We acknowledge C. Lacoste, B. Rutten,and O. Partouche for their expert technical assistance.


REFERENCES

  1. Renaud, J. P., Rochel, N., Ruff, M., Vivat, V., Chambon, P., Gronemeyer, H., and Moras, D. (1995) Nature 378, 681-689 [CrossRef][Medline] [Order article via Infotrieve]
  2. Tate, B. F., and Grippo, J. G. (1995) J. Biol. Chem. 270, 20258-20263 [Abstract/Free Full Text]
  3. Lamour, F. P. Y., Lardelli, P., and Apfel, C. M. (1996) Mol. Cell. Biol. 16, 5386-5392 [Abstract]
  4. Lotan, R. (1980) Biochim. Biophys. Acta 605, 33-91 [CrossRef][Medline] [Order article via Infotrieve]
  5. Roberts, A. B., and Sporn, M. B. (1984) in The Retinoids (Sporn, M. B., Roberts, A. B., and Goodmann, D. S., eds), Vol. 2, pp. 210-286, Academic Press, Orlando, FL
  6. Eichele, G. (1989) Trends Genet. 5, 246-251 [CrossRef][Medline] [Order article via Infotrieve]
  7. Chytil, F. (1984) Pharmacol. Rev. 36, 93-100
  8. Orfanos, C. E., Ehlert, R., and Gollnick, H. (1987) Drugs 34, 459-503 [Medline] [Order article via Infotrieve]
  9. Smith, M. A., Parkinson, D. R., Cheson, B. D., and Friedman, M. (1992) J. Clin. Oncol. 10, 839-864 [Abstract]
  10. Vokes, E. E., Weichselbaum, R. R., Lipmann, S. M., and Hong, W. K. (1993) N. Engl. J. Med. 328, 184-194 [Free Full Text]
  11. Heyman, R. A., Mangelsdorf, D. J., Dyck, J. A., Stein, R. B., Eichele, G., Evans, R., and Thaller, C. (1992) Cell 68, 397-406 [Medline] [Order article via Infotrieve]
  12. Levin, A. A., Sturzenbecker, L. J., Kazmer, S., Bosakowski, T., Huselton, C., Allenby, G., Speck, J., Kratzeisen, C., Rosenberger, M., Lovey, A., and Grippo, J. F. (1992) Nature 355, 359-361 [CrossRef][Medline] [Order article via Infotrieve]
  13. Mangelsdorf, D. J., Borgmeyer, U., Heymann, R. A., Zhou, J. Y., Ong, E. S., Oro, A. E., Kakizuka, A., and Evans, R. M. (1992) Genes Dev. 6, 329-344 [Abstract]
  14. Giguère, V., Ong, E. S., Segui, P., and Evans, R. M. (1987) Nature 330, 624-629 [CrossRef][Medline] [Order article via Infotrieve]
  15. Petkovich, M., Brand, N. J., Krust, A., and Chambon, P. (1987) Nature 330, 444-450 [CrossRef][Medline] [Order article via Infotrieve]
  16. Mangelsdorf, D. J., Ong, E. S., Dyck, J. A., and Evans, R. M. (1990) Nature 345, 224-229 [CrossRef][Medline] [Order article via Infotrieve]
  17. Benbrook, D., Lernhardt, E., and Pfahl, M. (1988) Nature 333, 669-672 [CrossRef][Medline] [Order article via Infotrieve]
  18. Brand, N. J., Petkowich, M., Krust, A., Chambon, P., de Thé, H., Marchio, A., Fiollais, P., and Dejean, A. (1988) Nature 332, 850-853 [CrossRef][Medline] [Order article via Infotrieve]
  19. Krust, A., Kastner, P., Petkovich, M., Zelent, A., and Chambon, P. (1989) Proc. Natl. Acad. Sci. U. S. A. 86, 5310-5314 [Abstract]
  20. Zelent, A., Krust, A., Petkovich, M., Kastner, P., and Chambon, P. (1989) Nature 339, 714-717 [CrossRef][Medline] [Order article via Infotrieve]
  21. Cell 68, 377-395Leid, M., Kastner, P., Lyons, R., Nakshrati, H., Saunders, M., Zacharewski, T., Chen, J.-Y, Staub, A., Garnier, J.-M., Mader, S., and Chambon, P. (1992-II) Cell 68, 377-395
  22. Evans, R. M. (1988) Science 240, 889-895 [Medline] [Order article via Infotrieve]
  23. Lotan, R., and Clifford, J. L. (1991) Biomed. Phamacother. 45, 145-156 [CrossRef][Medline] [Order article via Infotrieve]
  24. Laudet, V., and Stehelin, D. (1992) Curr. Biol. 2, 293-295
  25. Trends Biochem. Sci. 17, 427-433Leid, M., Kastner, P., and Chambon, P. (1992-I) Trends Biochem. Sci. 17, 427-433
  26. Yu, V. C., Delset, C., Andersen, B., Holloway, J. M., Devary, O. V., Näär, A. M., Kim, S. Y., Boutin, J.-M., Glass, C. K., and Rosenfeld, M. G. (1991) Cell 67, 1251-1266 [Medline] [Order article via Infotrieve]
  27. Bugge, T. H., Pohl, J., Lonnoy, O., and Stunnenberg, H. G. (1992) EMBO J. 11, 1409-1418 [Abstract]
  28. Kliewer, S. A., Umesono, K., Mangelsdorf, D. J., and Evans, R. M. (1992) Nature 355, 446-449 [CrossRef][Medline] [Order article via Infotrieve]
  29. Marks, M. S., Hallenbeck, P. L., Nagata, T., Segars, J. H., Apella, E., Nikodem, V. M., and Ozato, K. (1992) EMBO J. 11, 1419-1435 [Abstract]
  30. Nature 355, 441-446Zhang, X., Hoffmann, P. B.-V, Graupner, G., and Pfahl, M. (1992-I) Nature 355, 441-446
  31. Apfel, C. M., Kamber, M., Klaus, M., Mohr, P., Keidel, S., and LeMotte, P. (1995) J. Biol. Chem. 270, 30765-30772 [Abstract/Free Full Text]
  32. Schräder, M., Wyss, A., Sturzenbecker, L. J., Grippo, J. F., LeMotte, P., and Carlberg, C. (1993) Nucleic Acids Res. 21, 1231-1237 [Abstract]
  33. Mangelsdorf, D. J., Umesono, K., Kliewer, S. A., Borgmeyer, U., Ong, E. S., and Evans, R. M. (1991) Cell 66, 555-561 [Medline] [Order article via Infotrieve]
  34. Zhang, X., Lehmann, J., Hoffmann, B., Dawson, M. I., Cameron, J., Graupner, G., Hermann, G., Tran, P., and Pfahl, M. (1992) Nature 358, 587-591 [CrossRef][Medline] [Order article via Infotrieve]
  35. Keller, H. J., Dreyer, C., Medin, J., Mahfoudi, A., Ozato, K., and Wahli, W. (1993) Proc. Natl. Acad. Sci. U. S. A. 90, 2160-2164 [Abstract]
  36. Allenby, G., Bocquel, M. T., Sauders, M., Kazmer, M., Speck, J., Rosenberger, M., Lovey, A., Kastner, P., Grippo, J. F., Chambon, P., and Levin, A. A. (1993) Proc. Natl. Acad. Sci. U. S. A. 90, 30-34 [Abstract]
  37. LeMotte, P. K., Keidel, S., and Apfel, C. M. (1996) Eur. J. Biochem. 236, 328-333 [Abstract]
  38. Allenby, G., Janocha, R., Kazmer, S., Speck, J., Grippo, J. F., and Levin, A. A. (1994) J. Biol. Chem. 269, 16689-16695 [Abstract/Free Full Text]
  39. Apfel, C. M., Bauer, F., Crettaz, M., Forni, L., Kamber, M., Kaufmann, F., LeMotte, P., Pirson, W., and Klaus, M. (1992) Proc. Natl. Acad. Sci. U. S. A. 89, 7129-7133 [Abstract]
  40. Keidel, S., LeMotte, P., and Apfel, C. M. (1994) Mol. Cell. Biol. 14, 287-298 [Abstract]
  41. Lefebvre, B., Rachez, C., Formstecher, P., and Lefebvre, Ph (1995) Biochemistry 34, 5477-5485 [Medline] [Order article via Infotrieve]
  42. Keidel, S., Rupp, E., and Szardenings, M. (1992) Eur. J. Biochem. 204, 1141-1148 [Abstract]
  43. Green, S., Issemann, I., and Sheer, I. (1988) Nucleic Acids Res. 16, 369 [Medline] [Order article via Infotrieve]
  44. Studier, F. W., and Moffat, B. A. (1986) J. Mol. Biol. 189, 113-130 [Medline] [Order article via Infotrieve]
  45. Ostrowski, J., Hammer, L., Roalsvig, T., Pokornowski, K., and Reczek, P. R. (1995) Proc. Natl. Acad. Sci. U. S. A. 92, 1812-1816 [Abstract]
  46. Crettaz, M., Baron, A., Siegenthaler, G., and Hunziker, W. (1990) Biochem. J. 272, 391-397 [Medline] [Order article via Infotrieve]
  47. Ekena, K., Weis, K. E., Katzenellenbogen, J. A., and Katzenellenbogen, B. S. (1997) J. Biol. Chem. 272, 5069-5075 [Abstract/Free Full Text]
  48. Tairis, N., Gabriel, J. L., Gyda, I. I. I M., Soprano, K. J., and Soprano, D. R. (1994) J. Biol. Chem. 269, 19516-19522 [Abstract/Free Full Text]
  49. Tairis, N., Gabriel, J. L., Soprano, K. J., and Soprano, D. R. (1995) J. Biol. Chem. 270, 18380-18387 [Abstract/Free Full Text]

©1997 by The American Society for Biochemistry and Molecular Biology, Inc.