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Molecular Endocrinology 18 (12): 2839-2853
Copyright © 2004 by The Endocrine Society

Retinoic Acid Receptor-{alpha} Is Stabilized in a Repressive State by Its C-Terminal, Isotype-Specific F Domain

Behnom Farboud and Martin L. Privalsky

Section of Microbiology, Division of Biological Sciences, University of California at Davis, Davis, California 95616

Address all correspondence and requests for reprints to: Martin L. Privalsky, Section of Microbiology, Division of Biological Sciences, One Shields Avenue, University of California at Davis, Davis, California 95616. E-mail: mlprivalsky{at}ucdavis.edu.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Retinoic acid receptors (RARs) are hormone-regulated transcription factors that play multiple roles in vertebrate development and differentiation. Three isotypes of RARs, {alpha}, ß, and {gamma}, are encoded by distinct genetic loci and possess distinct transcriptional properties. Typically, RAR{alpha} represses target gene transcription in the absence of hormone, whereas RARß and {gamma} fail to repress under these conditions. This inability of RARß and RAR{gamma} to repress transcription is due to intramolecular interactions between helix 3 and helix 12 of the hormone binding domains of these isotypes that inhibit corepressor binding while favoring coactivator binding. We report here that the converse ability of RAR{alpha} to repress requires the integrity of the receptor F domain, a domain that maps C-terminal to helix 12, varies in sequence among different nuclear receptors, and is of poorly understood function. The F domain appears to help stabilize helix 12 of RAR{alpha} in a more open position that enhances corepressor binding and inhibits coactivator binding in the absence of hormone. Intriguingly, the RAR{alpha} F domain is isotype autonomous in its function. We speculate that the RAR{alpha} F domain may dock elsewhere on the receptor surface, and this intramolecular interaction may maintain RAR{alpha} helix 12 in an open, repression-competent conformation.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
NUCLEAR HORMONE RECEPTORS are a large class of ligand-regulated transcription factors that regulate many physiological processes, including reproduction, development, and homeostasis (1, 2, 3). Nuclear hormone receptors include those that bind and respond to steroids, such as the glucocorticoid and progesterone receptors, as well as receptors for nonsteroid hormones, such as thyroid, vitamin D3, and retinoic acid receptors (RARs) (4, 5). These receptors function primarily by binding to specific DNA recognition sequences, denoted response elements, in promoters of their target genes and regulating transcription in response to hormone ligand (1, 2, 3). Many nuclear hormone receptors, including certain RARs, display a dual mode of transcriptional regulation and are capable both of repressing transcription in the absence of hormone and of activating transcription in the presence of hormone (6, 7). This binary mode of transcriptional regulation is a reflection of the ability of these receptors to associate alternatively with coregulatory proteins denoted coactivators and corepressors (reviewed in Refs.7, 8, 9, 10). In the absence of hormone, nuclear hormone receptors typically bind to corepressors, such as silencing mediator of retinoid and thyroid hormone receptor (SMRT) and nuclear receptor corepressor (N-CoR); SMRT and N-CoR, in turn, function as bridging factors that can recruit additional components of a larger, corepressor complex, including histone deacetylases (such as histone deacetylase 3), transducin ß-like protein 1, transducin ß-like related protein 1, G protein pathway suppressor 2 protein, and SMRT/HDAC1-associated repressor protein (reviewed in Ref.7). Addition of hormone agonist results in release of the corepressor complex, and recruitment of a series of coactivator proteins, including the p160s (steroid receptor coactivator 1 (SRC-1), glucocorticoid receptor interacting protein 1 (GRIP1) and activator of thyroid and retinoic acid receptors (ACTR), p300/cAMP response element binding protein-binding protein, pCAF, and the vitamin D receptor-interacting protein/thyroid hormone receptor associated protein/mediator complex (7, 11). Corepressors and coactivators regulate gene expression by modifying the chromatin template and by interacting with the general transcriptional machinery so as to antagonize or facilitate the formation of a preinitiation transcriptional complex (12, 13, 14, 15, 16).

Nuclear hormone receptors recruit SMRT and N-CoR through an interaction of L/V-X-X-I/V-I amino acid motifs within the corepressor with a docking surface on the receptor’s hormone binding domain (17, 18, 19, 20). Intriguingly, portions of the same corepressor docking surface also form the principal binding site for the L-X-X-L-L motifs found in many coactivators (21, 22). The choice between corepressor vs. coactivator binding is controlled by the position of an adjacent receptor domain: helix 12. In the absence of hormone ligand, the helix 12 of most nuclear hormone receptors is thought to assume a relatively extended conformation that exposes the corepressor docking surface, permitting corepressor recruitment (Fig. 1AGo) (17, 20, 22, 23, 24, 25). Binding of hormone agonist by the receptor drives a conformational change in helix 12 that positions it against the body of the receptor; the reoriented helix 12 then occludes key portions of the corepressor docking surface, causing corepressor release, as well as forming a charge clamp that completes a high-affinity binding site for coactivator (Fig. 1AGo) (17, 19, 20, 21, 22, 26, 27). Different ligands can position helix 12 in yet additional conformations; antagonists, for example, can operate helix 12 so as to further enhance corepressor binding while preventing coactivator binding (22, 28).



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  		Fig. 1. A Structural Model for the Conformational Changes Conferred by Hormone Ligand on RAR{alpha} and -ß

A, A model of the relationship between helix 12 and the corepressor and coactivator docking surfaces on the RARs is presented. The locations of helix 3 and 12, detailed in the text, are indicated, as are the proposed docking surfaces for corepressor (black oval) and coactivator (oval with dense hatch marks). Oval with light hatch marks depicts an occluded corepressor binding site. B, A schematic representation of RAR subdomains. The "A/B" domain is the N-terminal hypervariable region; "C" is the DNA-binding domain; "D" is the hinge region; "E" is the hormone-binding domain; and "F" is the hypervariable C-terminal domain. The location of helix 3 and helix 12 are highlighted, and the sequence of the helix 12 and F domains are shown for RAR{alpha}, -ß, and -{gamma}.

 
Many nuclear hormone receptors are expressed as a series of interrelated isotypes or isoforms. There are three major isotypes of RAR in vertebrates, denoted {alpha}, ß, and {gamma}, that are encoded by three distinct genetic loci (29, 30). These three isotypes share significant sequence relatedness, yet mediate distinct biological functions (29, 30). We have previously reported that these three RAR isotypes differ in their interactions with corepressors in vitro and in their transcriptional properties in vivo (31, 32, 33). RAR{alpha} can efficiently recruit the SMRT corepressor in the absence of hormone and represses transcription in vivo, whereas RARß and -{gamma} interact only weakly with SMRT and activate, rather than repress, transcription in the absence of hormone. These differences in the transcriptional properties of RAR{alpha}, -ß, and -{gamma} map primarily to amino acid differences within helix 3 of the receptor hormone binding domain (31, 32). The helix 3 domain of RARß and -{gamma} appears to help stabilize helix 12 in a closed, agonist-like position even in the absence of hormone, thereby preventing repression and producing a constitutive level of activation (Fig. 1AGo). In contrast, the amino acid sequence in helix 3 of RAR{alpha} disrupts the helix 3/helix 12 interaction in the absence of hormone, causing helix 12 to assume an open conformation and permitting binding of corepressor to its docking surface (Fig. 1AGo). Addition of hormone agonist to RAR{alpha} appears to overcome the disfavorable helix 3 sequence sufficiently to gate helix 12 closed, thereby releasing corepressor and binding coactivator (Fig. 1AGo) (32). Notably, other members of the nuclear hormone receptor family also can assume an agonist-like conformation of helix 12 in the absence of a hormone ligand, although the detailed structural basis for this phenomenon varies from receptor to receptor (34, 35, 36). Therefore the position of helix 12 is programmed differently in different receptor classes and isotypes, resulting in distinct cofactor interactions and distinct transcriptional regulatory properties.

Despite the importance of the helix 3/helix 12 interaction in determining the transcriptional properties of the different RAR isotypes, we also noted in our prior experiments a possible, unanticipated contribution of the receptor F domain. The F domain is a receptor region that extends C-terminal to helix 12 itself and is highly variable in sequence and length among different receptors and receptor isotypes (Fig. 1BGo). The three-dimensional structure of the F domain of most nuclear hormone receptors is unknown, and its function(s) are poorly understood. We had earlier determined that a frame-shift mutation that scrambled the F domain sequence of RARß resulted in enhanced corepressor binding (32). This indication that mutations in the receptor F domain can influence cofactor recruitment led us to more closely examine the role of the wild-type F domains. We report here that the F domain in the wild-type RAR{alpha} (wtRAR{alpha}) functions to enhance corepressor binding and to destabilize coactivator binding, apparently by helping to stabilize the helix 12 domain in an extended position in the absence of hormone. The F domains of RARß and -{gamma} also contribute to the equilibrium between corepressor and coactivator binding, although playing a more minor role in determining the transcriptional properties of these two isotypes. We propose that the F domain of nuclear hormone receptors can function, at least in part, to determine the positioning of the adjacent helix 12, and thereby can help define the balance between corepressor binding and coactivator binding, and between transcriptional repression and activation.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
The F Domain of RAR{alpha} Is Required for Transcriptional Repression
We wished to better understand the contributions of the F domain and of receptor helix 12 to transcriptional regulation by RARs. We first examined the role of these C-terminal domains on RAR regulation of a retinoic acid-response element (RARE)-reporter gene in a transient transfection assay of CV-1 cells. As previously shown, wtRAR{alpha} repressed reporter gene expression in the absence of hormone, whereas both wtRARß and wtRAR{gamma} activated reporter gene expression under the same conditions (Fig. 2AGo) (31, 32). All-trans retinoic acid (ATRA) relieved repression by wtRAR{alpha} and converted it into a transcriptional activator (Fig. 2AGo) but only modestly further increased the constitutive activation properties of RARß and -{gamma} (Fig. 2AGo). Deletion of the entire C-terminal domain of RAR{alpha}, -ß, and -{gamma}, including helix 12 and the F domain sequence ({Delta}12/F), converted all three receptor isotypes into constitutive repressors (Fig. 2AGo); this result is consistent with an established requirement for helix 12 for the release of corepressors and the acquisition of coactivators (6, 7). Unexpectedly, however, deletion of only the F domain of RAR{alpha} (RAR{alpha}{Delta}F) resulted in a loss of repression in the absence of hormone, resulting in the RAR{alpha}{Delta}F mutant behaving as a constitutive activator in the absence of hormone, and as a stronger than wild-type activator in the presence of ATRA (Fig. 2AGo). Deletion of the F domain of RARß and -{gamma} (RARß{Delta}F and RAR{gamma}{Delta}F), in contrast, had relatively little effect on the inherent activation properties of these isotypes relative to their wild-type counterparts (Fig. 2AGo).



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  		Fig. 2. Deletion of the F Domain from RAR{alpha} Results in Transcriptional Activation in the Absence of Hormone

A, Deletion of only the F domain of RAR{alpha} (RAR{alpha}{Delta}F) yields a constitutive transcriptional activator. CV-1 cells were transfected with either a TK-luciferase reporter lacking an RARE (TK-luciferase) or a TK-luciferase reporter containing three tandem RAREs (RARE-TK-luciferase), together with the pSG5-receptor vector indicated and a pCH110 (lacZ) construct as an internal transfection control. Cells were incubated in the absence or presence of 500 nM ATRA hormone for 24 h before cell harvest and analysis. Relative luciferase activity (absolute luciferase activity/absolute ß-galactosidase activity) was calculated, and the average and SEM are provided for at least three independent experiments. The dashed horizontal line represents basal reporter transcription (i.e. the activity of the pTK-Luc reporter lacking RAREs). B, Varying the receptor expression plasmid DNA concentration does not alter the direction of the RAR{alpha}{Delta}F transcriptional response. Transient transfections of CV-1 cells were performed as described in panel A, but the concentrations of the pSG5-receptor vector were varied as indicated below the panel. For comparison, the relative luciferase is also presented for the TK-luciferase reporter lacking RAREs and for the RARE-TK-luciferase reporter in the absence of ectopic receptor. Average and SEM are provided for at least three independent experiments.

 
To determine whether the enhanced transcriptional response of RAR{alpha}{Delta}F was due to increased expression of the mutant receptor, receptor protein levels were examined by immunoblotting. Rather than an increase, deletion of the F domain actually caused a moderate reduction in the steady state level of the RAR{alpha}{Delta}F mutant compared with that of wild-type receptor (data not shown); this is consistent with reports that increased transcriptional activity can lead to increased nuclear hormone receptor turnover (37, 38, 39). We also tested a range of expression vector DNA inputs. Introduction of the unliganded RAR{alpha}{Delta}F mutant activated the reporter gene over a wide range of expression vector concentrations (from 0.04 to 40 ng; only endogenous RAR activity was detected at vector inputs of 0.004 ng or less) (Fig. 2BGo); conversely, introduction of the unliganded wtRAR{alpha} repressed the reporter gene under the same conditions (Fig. 2BGo). Analogous results were observed using a variety of reporter vector inputs (data not shown). The enhanced ability of the RAR{alpha}{Delta}F mutant to activate reporter expression in the presence of ATRA was also observed over a range of expression vector inputs (Fig. 2BGo). We conclude that the loss of repression and gain of activation function by RAR{alpha}{Delta}F are not due to enhanced RAR accumulation but, instead, reflect an inherent change in the transcriptional properties of the mutant receptor.

Vertebrate cells, including the CV-1s used here, express low levels of endogenous RARs that mediate a modest response to ATRA even in the absence of ectopic RAR expression (note the "no receptor" lane in Fig. 2BGo). To exclude interference from these endogenous RARs, and to avoid potential cross-talk from other nuclear receptors that might recognize the DR-5 element in our RARE-luciferase reporter, we repeated these experiments using Gal4-RAR fusions and a Gal4–17mer thymidine kinase (TK)-luciferase reporter system. As anticipated, the Gal4–17mer reporter did not respond to ATRA in the absence of an ectopically introduced receptor [Fig. 3Go; Gal4 DNA-binding domain (DBD) alone]. Introduction of a Gal4DBD-RAR{alpha} construct strongly repressed the Gal4–17mer reporter in the absence of hormone and activated this reporter in the presence of ATRA (Fig. 3Go), recapitulating the results seen with native receptors. Similarly, Gal4DBD-RARß and Gal4DBD-RAR{gamma} conferred a constitutive Gal17-mer reporter activation in the absence of ATRA that was further increased by addition of ATRA (Fig. 3Go). As with the native receptor constructs, the Gal4DBD-RAR constructs bearing the {Delta}12/F deletions mediated a potent repression of transcription in this system in either the presence or absence of hormone, whereas deletion of only the F domain from RAR{alpha} resulted in constitutive activation in the absence of hormone and a greater than wild-type enhancement of activation in the presence of ATRA (Fig. 3Go). Comparable {Delta}F deletion mutants of Gal4BDB-RARß or Gal4DBD-RAR{gamma} functioned equal to, or slightly weaker than, the corresponding wild-type constructs (Fig. 3Go). These data indicate that the integrity of the F domain of RAR{alpha} is important for efficient target gene repression in the absence of hormone, with elimination of the F domain resulting in enhanced target gene activation.



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  		Fig. 3. Gal4DBD-RAR{alpha} Represses, Whereas Gal4DBD-RAR{alpha}{Delta}F Activates Reporter Gene Expression

CV-1 cells were transiently transfected with a nonrecombinant Gal4-DBD expression vector (Gal4DBD alone) or with GAL4-DBD fusion proteins bearing the hormone-binding domains of the various RAR isotypes as indicated. A pGL3-GAL4–17mer luciferase reporter vector and pCH110 (lacZ) reporter were also included in each transfection. Cell were incubated in the absence or presence of 500 nM ATRA hormone for 24 h and harvested and analyzed as in Fig. 2Go. The dashed horizontal line represents basal level expression of the pGAL-17mer Luc reporter when introduced with the nonrecombinant Gal4DBD. Relative luciferase activity is presented; the average and SEM are provided for at least three independent experiments.

 
Deletion of the F Domain Does Not Detectably Alter the Ability of RAR{alpha} to Bind or Respond to Hormone Agonist
We next examined whether the RAR{alpha}{Delta}F mutation altered the affinity of the receptor for hormone ligand. Hormone binding by nuclear receptors results in a conformational change in the receptor that rearranges and compacts the hormone binding domain (6, 24, 25, 26, 27, 28). As a result, although the apo-receptor is extensively degraded by many proteases, the hormone-liganded receptor forms a condensed polypeptide core that is highly resistant to further proteolytic degradation (24, 40, 41, 42, 43). This acquisition of a protease-resistant conformation in response to ligand is a useful measure of hormone binding by the nuclear receptors (e.g. Ref.24), and we employed this method to compare the ability of wtRAR{alpha} and RAR{alpha}{Delta}F to bind ATRA in vitro. Both wtRAR{alpha} and RAR{alpha}{Delta}F were extensively degraded by elastase in the absence of hormone but acquired an elastase-resistant core at concentrations of ATRA greater than 5 nM, consistent with the proposal that F domain deletion does not alter receptor avidity for ATRA (Fig. 4AGo), and in agreement with prior studies of the effects of C-terminal RAR truncations on hormone binding (44, 45). We also tested the ability of the wild-type and mutant RAR{alpha} constructs to induce reporter gene activation over a range of hormone concentrations in vivo (Fig. 4BGo). Although, as noted, RAR{alpha}{Delta}F conferred basal activation in the absence of hormone, the ATRA concentration required to further stimulate RAR{alpha}{Delta}F to half-maximal activity was the same as for wtRAR{alpha}, with the two hormone efficacy curves paralleling one another (Fig. 4BGo). Similarly, RARß{Delta}F displayed a hormone efficacy response virtually identical to that of wtRARß (Fig. 4BGo). We conclude that the F domain does not contribute to the ability of RAR{alpha} to bind or respond to ATRA but alters the transcriptional properties of this receptor by some other mechanism.



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  		Fig. 4. Truncation of the F Domain of RAR{alpha} Does Not Alter the Ability of RAR{alpha} to Bind to or Respond to ATRA

A, RAR{alpha} and RAR{alpha}{Delta}F bind to hormone at comparable ATRA concentrations. Radiolabeled receptors, synthesized using an in vitro coupled TnT system, were incubated with elastase in the presence of different concentrations of ATRA, as indicated below the gel. The resulting proteolytic products were resolved by SDS-PAGE and were visualized and quantified by phosphor imager analysis. ATRA binding results in formation of a protease-resistant RAR core derived from the hormone-binding domain (solid arrow). Data are presented graphically as the fraction of protease-resistant receptor core (solid arrow) remaining at each time point relative to full-length receptor (noted by the open arrow). The average and SEM are provided for at least three independent experiments. B, RAR{alpha} and -ß (and their F domain truncations) respond to ATRA ligand in a comparable manner in transfected cells. Transient transfections of CV-1 cells were performed as described in Fig. 2Go; cells were treated with the different concentrations of ATRA as noted below the panel. Data are presented as relative luciferase activity; average and SEM are provided for at least three independent experiments (SEM values less than the diameter of the corresponding plot symbol are not depicted).

 
The Ability of the Unliganded RAR{alpha}{Delta}F Mutant to Activate, Rather Than Repress, Transcription Correlates with Reduced Corepressor and Enhanced Coactivator Binding in Vitro
We next used a glutathione-S-transferase (GST) protein interaction system to examine the effects of our helix 12 and F domain mutations on the interaction of RARs with corepressors and coactivators in vitro. GST-corepressor or GST-coactivator constructs representing the receptor-binding domains were synthesized in and purified from Escherichia coli. wtRAR{alpha}, or the RAR{alpha}{Delta}F mutant, were synthesized by in vitro translation, producing radiolabeled proteins of the correct molecular weights (e.g. Fig. 4AGo; note RAR{alpha}{Delta}F migrates slightly faster than wtRAR{alpha}). As expected, the GST-SMRT corepressor construct interacted strongly with wtRAR{alpha} in the absence of hormone, and this interaction was destabilized by ATRA (Fig. 5AGo). wtRARß and -{gamma}, in contrast, interacted only weakly with GST-SMRT in either the absence or presence of ATRA (Fig. 5AGo). Deletion of only the F domain of RAR{alpha} resulted in a modest, but reproducible, reduction in the interaction of this isotype with GST-SMRT, whereas the already low binding of the unliganded RARß and RAR{gamma} to GST-SMRT was not significantly altered by deletion of their F domains (Fig. 5AGo). Deletion of both helix 12 and the F domain permitted efficient GST-SMRT binding by all RAR isotypes tested (Fig. 5AGo).



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  		Fig. 5. Deletion of the F Domain from RAR{alpha} Reduces Interaction with SMRT, But Enhances Association with ACTR and SRC1 in the Absence of Hormone in a GST-Pull-Down Assay

A, Deletion of the F domain from RAR{alpha}, but not RARß or -{gamma}, reduces binding to SMRT. Each radiolabeled receptor (indicated below the panel) was synthesized in vitro by a coupled transcription-translation method and was incubated with GST-SMRT immobilized on glutathione-agarose. Binding reactions were conducted in the absence (open bars) or presence (solid bars) of 1 µM ATRA. The receptors remaining bound to the GST-SMRT after washing were eluted from the agarose beads, resolved by SDS-PAGE, and quantified by phosphor imager analysis; the percent of receptor bound to the GST-SMRT, relative to input (total radiolabeled receptor present in the assay), is presented. The average and SEM values from at least three independent experiments are shown. B and C, Deletion of the F domain from RAR{alpha}, -ß, and -{gamma} enhanced receptor interaction with ACTR and SRC1 in the absence of hormone. The protein-protein interaction assay was performed as described in panel A, but using GST-coactivator constructs. The percent of receptor binding to GST-ACTR (panel B) or to GST-SRC1 (panel C), relative to input (total radiolabeled receptor present in the assay), was calculated; the average and SEM values from at least three independent experiments are shown.

 
Next, the interaction of the various RAR isotypes and mutants with the p160 coactivators was examined using a similar GST-pull-down methodology. All three wtRAR isotypes interacted very strongly with GST-SRC1 or GST-ACTR in the presence of ATRA, but only relatively weakly in the absence of hormone (Fig. 5Go, B and C). Deletion of both helix 12 and the F domain (RAR{Delta}12/F) of RAR{alpha}, -ß, or -{gamma} resulted in a near complete loss of coactivator binding in either the absence or presence of ATRA, consistent with the known contribution of helix 12 to the coactivator binding surface (Fig. 5Go, B and C). Deletion of the F domain alone resulted in a small, but consistent increase in coactivator binding for all three isotypes compared with the corresponding wild-type constructs; this increase was generally observed both minus and plus hormone agonist (Fig. 5Go, B and C); only the interaction of RARß with GST-ACTR in the presence of ATRA was not increased by the {Delta}F mutation.

The interactions of nuclear receptors with corepressors and coactivators can be influenced by receptor dimerization and by DNA binding (46, 47). We therefore also examined the binding of the wtRAR{alpha} and the RAR{alpha}{Delta}F mutant to SMRT and to ACTR using an EMSA. RAR{alpha}/RXR{alpha} heterodimers were assembled on an RARE oligonucleotide, and the ability of increasing amounts of a SMRT or ACTR protein construct (encompassing the appropriate receptor-interaction domains) to bind to and form a supershifted complex was tested. Mirroring our GST interaction assays, deletion of the F domain from RAR{alpha} significantly reduced the ability of RAR{alpha}{Delta}F/RXR/DNA complexes to associate with the SMRT construct in the absence of hormone (Fig. 6AGo). Conversely, the ACTR coactivator construct interacted only weakly with the wtRAR{alpha}/RXR/DNA complex in the absence of ATRA and more strongly with the RAR{alpha}{Delta}F mutant/RXR/DNA complex under the same conditions (Fig. 6BGo). As anticipated, ATRA released corepressor and induced strong coactivator binding to both wt and mutant receptor/DNA complexes (data not shown). These results provide additional evidence indicating that the intact F domain in the wtRAR{alpha} favors corepressor binding and destabilizes coactivator binding.



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  		Fig. 6. Deletion of the F Domain from RAR{alpha} Reduced Interaction with SMRT and Enhanced Association with ACTR in an EMSA Protocol

A, RXR/RAR{alpha}{Delta}F heterodimers bound to DNA interact with SMRT less efficiently than do analogous RXR/wtRAR/DNA complexes. wtRAR{alpha} or the RAR{alpha}{Delta}F mutant was bound as an RXR heterodimer to a radiolabeled RARE probe, as indicated. Each receptor/DNA complex was then incubated with differing amounts (2-fold dilutions) of GST-SMRT (or GST alone) in the absence of hormone. The resulting products were resolved on a nondenaturing TBE gel and were visualized and analyzed by phosphor imager analysis. The amount of receptor/DNA complex supershifted by GST-SMRT was quantified and plotted. A representative electrophoretogram (bottom panel), and the average data from three independent experiments, ± SEM (top panel) are presented. B, RXR/RAR{alpha}{Delta}F heterodimers bound to DNA interact with ACTR more efficiently than do analogous RXR/wtRAR/DNA complexes. The receptors indicated were bound to a radiolabeled RARE probe as in panel A; each receptor/DNA complex was then incubated with differing amounts (2-fold dilutions) of GST-ACTR (or GST alone) in the absence of hormone. The resulting products were resolved on a nondenaturing TBE gel and were visualized and analyzed by phosphor imager analysis. The amount of receptor/DNA complex supershifted by GST-ACTR was quantified and plotted. A representative electrophoretogram (bottom panel), and the average data from three independent experiments ± SEM (top panel) are presented.

 
Deletion of the RAR{alpha} F Domain Shifts the Equilibrium from Corepressor Binding toward Coactivator Binding
The corepressor and coactivator docking surfaces on the nuclear receptors partially overlap, indicating that the binding of these auxiliary factors is mutually competitive (17, 19, 20, 32). Given that deletion of the F domain of RAR{alpha} decreased corepressor binding and augmented coactivator binding, we next determined whether the {Delta}F mutation would alter the equilibrium between coactivator and corepressor binding when both cofactors were present simultaneously. We titrated the protein concentration in the EMSA so that all of the RAR/RXR/DNA complexes were initially bound to SMRT corepressor; this receptor-corepressor complex was then incubated with increasing amounts of an ACTR coactivator construct. In the absence of hormone, ACTR was inefficient at displacing SMRT from the wtRAR{alpha}/RXR/DNA complex but was effective at displacing SMRT from the RAR{alpha}{Delta}F mutant/RXR/DNA complex, forming an ACTR/RAR{alpha}{Delta}F/RXR/DNA complex (Fig. 7Go). We conclude that the F domain of wtRAR{alpha} helps favor corepressor binding over coactivator binding, and that deletion of the F domain results in a change in the cofactor equilibrium toward corepressor release and coactivator binding.



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  		Fig. 7. In the Absence of Hormone, Coactivator Effectively Competes with Corepressor for Binding to RAR{alpha}{Delta}F, But Not to wtRAR{alpha}

Indicated receptor protein was bound to radiolabeled RARE probe such that each receptor heterodimer formed approximately the same amount of DNA-bound complex. The DNA-bound complex was then incubated with GST-SMRT protein such that all of the DNA-bound complex was supershifted by SMRT. Various amounts of GST-ACTR protein (beginning at an ~4-fold excess to the GST-SMRT construct and doubling in each subsequent lane) were then added, and the resulting receptor/cofactor/DNA complexes were resolved on a nondenaturing TBE gel and visualized and analyzed by phosphor imager analysis; the GST-SMRT and GST-ACTR supershifted complexes could be distinguished by their distinct mobilities. Data are presented as the percent of the receptor/DNA complex shifted from a SMRT to an ACTR complex (solid squares) or remaining bound by SMRT (open squares). Data from a representative experiment are provided; comparable results were obtained in three independent experiments.

 
The F Domain of wtRAR{alpha} May Help Stabilize Helix 12 in an Open, Corepressor-Permissive Conformation
Carboxypeptidase Y (CPY) is an exopeptidase that removes amino acids sequentially from the C terminus of protein substrates and is a useful probe of the solvent accessibility of the C terminus of nuclear receptors (24, 32, 48). The wtRAR{alpha} C terminus is known to assume an open, solvent-accessible position in the absence of hormone, and a more closed, sequestered conformation in the presence of hormone; consistent with this model, the C terminus of wtRAR{alpha} was rapidly degraded by CPY in the absence of hormone, but was relatively protected from CPY on addition of ATRA (Fig. 8Go). Notably, in the absence of hormone the C terminus of the RAR{alpha}{Delta}F mutant was less sensitive to degradation by CPY than was the C terminus of wtRAR{alpha}, consistent with the apo-RAR{alpha}{Delta}F assuming a more sequestered C-terminal conformation than the apo-wtRAR{alpha} (Fig. 8Go). Addition of ATRA further protected the RAR{alpha}{Delta}F C terminus, indicative of an additional conformational change in response to binding hormone agonist (Fig. 8Go) and consistent with the enhanced release of corepressors, binding of coactivators, and transcriptional activation by RAR{alpha}{Delta}F seen in vivo and in vitro in response to hormone agonist. These results suggest that the F domain of wtRAR{alpha} helps stabilize an open, corepressor-accessible conformation of this receptor; deletion of this domain results in a more closed conformation that, as shown with RARß, occludes the corepressor binding surface and favors coactivator binding instead.



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  		Fig. 8. Deletion of the F Domain from RAR{alpha} Confers Partial Resistance to CPY Degradation in the Absence of Hormone

Radiolabeled receptors, synthesized using an in vitro coupled TnT system, were incubated with CPY for the indicated times in the presence (solid circles and squares) or in the absence (open circles and squares) of 1 µM ATRA. The resulting proteolytic products were resolved by SDS-PAGE and were visualized and quantified by phosphor imager analysis. The fraction of full-length receptor remaining at each time point is presented for both wild-type (wt) and F domain truncated ({Delta}F) receptors. A representative experiment is provided; analogous results were obtained in three independent experiments.

 
The Function of the F Domain of RAR{alpha} Is Isotype Specific and Cannot Be Replaced by the F Domain of either RARß or -{gamma}
The F domains of the different RAR isotypes are highly divergent (Fig. 1BGo), and we wished to determine whether or not these different F domains can functionally substitute for one another. We constructed chimeric RARs in which the F domain of one isotype was exchanged with that of the other two and analyzed these chimeras in transient transfection experiments using the RARE-luciferase reporter (Fig. 9AGo). Replacement of the RAR{alpha} F domain with that of either RARß (denoted RAR{alpha}-ßF) or RAR{gamma} (denoted RAR{alpha}-{gamma}F) resulted in an RAR{alpha}{Delta}F-like phenotype: a loss of repression, moderate constitutive reporter gene activation in the absence or hormone, and increased reporter gene activation in the presence of ATRA (Fig. 9AGo). Conversely, appending the F domain of RAR{alpha} in place of that of either RARß or -{gamma} did not confer repression on these isotypes or otherwise significantly affect their transcriptional properties (Fig. 9AGo). Substitution of either the ß- or {gamma}-sequences in place of the wtRAR{alpha} F domain reduced SMRT corepressor binding in vitro in the absence of hormone; the binding of coactivator was not detectably altered in this context (Fig. 9Go, C and D). Reciprocally, replacing the RARß F domain with that of RAR{alpha} modestly increased corepressor binding and slightly reduced ACTR binding in the absence of hormone relative to wtRARß (Fig. 9Go, B and C). Substituting the RAR{gamma} sequences in place of the RARß F domain reduced both SMRT and ACTR binding in the absence of hormone (Fig. 9Go, B and C). We conclude that the different F domains are not autonomous in their properties and are not interchangeable, but rather each are context specific in their actions.



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  		Fig. 9. The Functions of the F Domains from RAR{alpha}, -ß, and -{gamma} Are Not Exchangeable

A, Chimeric RAR{alpha} receptors with the F domain from RARß (RAR{alpha}-ßF) or from RAR{gamma} (RAR{alpha}-{gamma}F) are constitutive activators in CV-1 cells. Transient transfections were performed as described in Fig. 2Go. Cells were incubated in the absence of presence of 500 nM ATRA hormone for 24 h before analysis. Data are presented as relative luciferase activity; averages and the SEM values for at least three independent experiments are provided. The dashed horizontal line represents the basal level of gene expression from the pTK-Luc reporter lacking RAREs. B, The F domains of RARß and -{gamma} do not enhance corepressor binding when exchanged for the native RAR{alpha} domain. The GST interaction assay was conducted as described in Fig. 5Go. Radiolabeled receptors were bound in the absence (open bars) or presence (solid bars) of 1 µM ATRA. The radiolabeled receptors remaining bound to the GST-SMRT after extensive washing were eluted, resolved by SDS-PAGE, and quantified by phosphor imager analysis. The percent of receptor bound to GST-SMRT relative to input was plotted. The average and SEM from at least three independent experiments are shown. C and D, Fusion of the F domain of RARß or -{gamma} in place of the F domain from RAR{alpha} does not alter the ability of RAR{alpha} to bind coactivators in vitro. GST pull-down experiments were performed as above, but using either GST-ACTR (panel C) or GST-SRC1 (panel D). Radiolabeled receptors were bound in the absence (open bars) or presence (solid bars) of 1 µM ATRA. The receptors remaining bound to the GST coactivators after extensive washing were eluted, resolved by SDS-PAGE, and quantified by phosphor imager analysis. The percent of receptor remaining bound to each GST coactivator, relative to input, was plotted. The average and SEM values from at least three independent experiments are shown.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
MATERIALS AND METHODS
 REFERENCES
 
The F Domain Contributes to the Distinctive Transcriptional Properties of the RAR{alpha} Isotype
The {alpha}-, ß-, and {gamma}-isotypes of RAR are believed to have arisen through a process of gene duplication and divergence that occurred early in the evolution of the vertebrate lineage (29, 30). Genetic disruption experiments have demonstrated that these three different isotypes perform unique, although partially overlapping, roles in physiology and development (29, 30). Consistent with this mix of redundant and unique functions, the sequences of the RAR DNA and hormone binding domains are well conserved among the three different isotypes, whereas the N-terminal A/B domain and C-terminal F domain are significantly more variable as to sequence and length. The A/B domains of many nuclear hormone receptors contain autonomous transcriptional regulatory properties and can make contacts with corepressors and coactivators in addition to those described for the hormone-binding domain (49, 50, 51, 52). The distinct biological actions of each isotype are therefore determined, in part, by the unique contributions of these differing A/B domains to transcriptional regulation. The function of the C-terminal F domain, however, is less understood. Mutations in the F domain do not typically exert as dramatic an effect on receptor function as do mutations in the DNA-binding or hormone-binding domains. Adding to the difficulty in assigning a function to the F domain, this region has not been visualized in most of the nuclear hormone receptor structures determined to date. The experiments reported here help clarify these questions by revealing that the F domain can serve as a modulator of the transcriptional properties of the RARs and appears to function by stabilizing RAR{alpha} in a repressive state in the absence of hormone and by attenuating RAR{alpha}-mediated activation in the presence of ATRA.

The F Domain Contributes to Transcriptional Regulation by RAR{alpha} by Stabilizing Corepressor Binding and by Destabilizing Coactivator Binding in the Absence of Hormone
The wtRAR{alpha} repressed target gene expression in CV-1 cells in the absence of hormone and activated in the presence of ATRA. An RAR{alpha} lacking the F domain (RAR{alpha}{Delta}F) lost the ability to repress and, instead, activated reporter gene expression even in the absence of hormone. The same RAR{alpha}{Delta}F mutant also activated reporter gene expression more strongly in the presence of ATRA than did wtRAR{alpha}. These alterations in the transcriptional properties of the RAR{alpha}{Delta}F mutant were observed over a wide range of reporter and receptor inputs, were recapitulated when assayed as a Gal4DBD-receptor fusion (indicating that the switch from repressor to activator is independent of contributions of the receptor A/B and DNA binding domains), and were not due to simple overexpression of the RAR{alpha}{Delta}F mutant protein in the transfected cells or to detectable changes in the affinity for hormone. Instead, the altered transcriptional properties of the RAR{alpha}{Delta}F mutant most closely correlated with a reduction in SMRT binding and an increase in p160 coactivator binding in GST-pull-down and EMSA interaction assays. In competition assays performed in the absence of hormone, ACTR efficiently competed with SMRT for binding to the RAR{alpha}{Delta}F mutant, but not to the wtRAR{alpha}. These results suggest that the F domain confers repression by the wtRAR{alpha} by increasing its affinity for corepressor and decreasing its affinity for coactivators in the absence of hormone. Deletion of the F domain also increased the ability of RAR{alpha} to bind to SRC-1 and ACTR in the presence of ATRA; increased coactivator binding may therefore also contribute to the enhanced transcriptional activity of the {Delta}F mutant in the presence, as well as in the absence, of hormone agonist.

Our observations suggest that differences in the levels or types of coactivators and corepressors that are expressed in different cells might be able to shift the equilibrium between repression and activation by RARs. Consistent with this idea, the ability of wtRAR{alpha} to repress or activate reporter gene expression can vary in different cell types (e.g. Refs.31 and 53); notably, deletion of the F domain generally reduced repression and/or further enhanced activation by RAR{alpha} when tested in a variety of cell lines (our unpublished results). In contrast, overexpression of ectopic SMRT or ACTR in CV-1 cells failed to have any significant effect on the repression or activation properties of the wtRAR{alpha} or {Delta}F mutant in the absence of hormone (our unpublished results); however, given that both SMRT and ACTR function in multicomponent complexes and appear to already be in excess in many cells, it is not possible to reach a simple conclusion from this type of overexpression experiment.

The F Domain of wtRAR{alpha} May Function by Maintaining Helix 12 in an Open Conformation
The helix 12 of the hormone-binding domain of the nuclear receptors is believed to be gated into at least two distinct positions (25). In the absence of hormone, helix 12 of many nuclear hormone receptors can form an open or extended conformation that allows access of corepressor to a nearby docking surface (6, 17, 20, 22, 23, 24, 25). Binding of hormone agonists, however, can realign helix 12 into a closed or sequestered position that occludes the corepressor-docking site and forms instead a coactivator-binding surface (17, 20, 22, 24, 25, 26, 40). We have shown that both thyroid hormone receptors and RAR{alpha} follow this prototypic model and assume an open conformation in the absence of hormone and a closed conformation in the presence of hormone (24, 32). RARß and -{gamma}, in contrast, appear to assume a constitutively closed conformation in both the absence and presence of hormone due to isotype-specific stabilizing contacts between helices 3 and 12. As a result RARß and -{gamma} fail to bind corepressor efficiently, fail to repress, and instead activate target gene transcription both minus and plus hormone (32). Although RAR{alpha} lacks this stabilizing interface between helices 3 and 12, thereby favoring an open helix 12 position, our current study suggests that additional sequences, mapping to the F domain, are also necessary to help maintain the RAR{alpha} helix 12 in an open, repressive conformation. Using CPY as a probe of the solvent accessibility of the RAR C terminus, we determined that the RAR{alpha}{Delta}F mutant is more resistant to CPY degradation in the absence of hormone than is the wtRAR{alpha}. This is consistent with the {Delta}F mutation inducing a more sequestered, more agonist-like C-terminal conformation compatible with the loss of corepressor and gain of coactivator binding observed for this mutant. One potential limitation to this simple interpretation is that deletion of the RAR{alpha} F domain might alter the rate of CPY degradation of the receptor C terminus independent of the conformation of this domain. Arguing against this as a potential artifact, however, is that although CPY degradation of the RAR{alpha}{Delta}F mutant is slower than wild type in the absence of hormone, the susceptibility to CPY of mutant and wtRAR{alpha} are near equal in the presence of ATRA.

How might the F domain contribute to the positioning of helix 12? A suggestion comes from the x-ray structure of the progesterone receptor (PR), one of the few nuclear receptors for which an electron density map of the F domain is available (54). In this structure, the PR F domain curves around the surface of the hormone-binding domain and forms a ß-strand that packs antiparallel to an ß-strand structure located between receptor helices 8 and 9. Interestingly, the PR F domain sequence is conserved in the mineralocorticoid, androgen, and glucocorticoid receptors, with the F domain required for agonist binding/transcriptional activation by the latter (40, 55, 56, 57, 58, 59). Although the amino acid sequences (and perhaps functions) of the F domains of the steroid receptors and of RAR{alpha} diverge, the RAR{alpha} F domain may also make intramolecular contacts with distal receptor surfaces that, in the case of RAR{alpha}, help stabilize an open, repression-competent helix 12 conformation. This hypothesis is consistent with the nonautonomous nature of the F domains when substituted in different RAR isotype backgrounds (see below). Alternatively, the preponderance of prolines and glycines in the wtRAR{alpha} F domain might favor an unstructured conformation that destabilizes the closed conformation (rather than stabilizing the open conformation), or the conformation of the F domain may act though influencing the folding of the adjacent helix 12.

Although the Receptor F Domains Also Influence Cofactor Recruitment by RARß and -{gamma}, the Effects Appear to Be Secondary to the Stronger Contributions of the Helix 3/Helix 12 Interaction and Are Not Transferable between Receptor Isotypes
Deletion of the F domain of either the RARß or RAR{gamma} isotype enhanced coactivator binding in vitro but had relatively little effect on the already weak corepressor binding displayed by these isotypes. Presumably due to the inherent lack of repression and strong constitutive activation properties of RARß and -{gamma} (a result of the helix 3/helix 12 interactions described above), the F domain deletions of the RARß and -{gamma} isotypes did not result in detectably increased reporter gene activation in transfected cells. In addition, the F domains of RAR{alpha}, -ß, and -{gamma} are not functionally interchangeable when exchanged among the different isotypes. For example, the F domains of RARß and -{gamma} did not substitute for that of {alpha}, nor did the F domain of RAR{alpha} detectably alter the transcriptional properties of either RARß or -{gamma} when substituted for the corresponding native domains. Notably, each of these F domains is highly divergent in sequence from one another. These data suggest that the F domains of RAR do not function in a autonomous manner, but are context dependent and are influenced in their actions by the remainder of the receptor molecule. More generally, our experiments also suggest that the in vivo ability of a given RAR isotype (or mutant) to activate target gene expression in the absence of hormone correlates more closely in vitro with an absence of corepressor binding rather than a gain of coactivator binding. This implies that SMRT binding is dominant to p160 coactivator binding in the absence of hormone, a proposal consistent with our own EMSA competition experiments, and with reports that corepressor release is necessary for transcriptional activation by RAR{alpha} (60, 61).

The F Domain Modulates the Transcriptional Activities of a Diverse Series of Nuclear Receptors
Our results with RAR{alpha} are not without precedent. The F domain of HNF-4{alpha}1, an orphan member of the nuclear receptor family, has also been shown to destabilize coactivator binding and to stabilize corepressor binding (62). The A/B domain of HNF-4{alpha}1 appears to participate in this phenomenon by antagonizing, directly or indirectly, the effects of the F domain on corepressor association (62); the actions of the RAR{alpha} F domain, in contrast, can be observed in both the presence and absence of the RAR A/B domain. In estrogen receptor, deletions of the F domain are able to alter the receptor’s response to selective receptor modulators, such as tamoxifen, enhancing the agonist properties of these ligands and diminishing their antagonist-like functions (63, 64, 65). The F domain is also a target for posttranslational modifications that may further modulate its functions; the F domain of RAR{alpha}, for example, can be phosphorylated at multiple positions that may be able to further modify the properties we report here (66); similarly, the F domain of the estrogen receptor is modified by an O-linked N-acetylglucosamine moiety (67). Taken as a whole, these studies indicate that the variable F domains serve, at least in part, to fine tune the transcriptional properties of the different nuclear receptors, permitting them to display distinct transcriptional responses when expressed as different isotypes in response to different ligands, or potentially, in response to different protein modification pathways operative in cells.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
REFERENCES
 
Molecular Clones
The mammalian expression vector pSG5.1 was constructed by enzymatic digestion of pSG5 (Stratagene, La Jolla, CA) with PvuII and BglII. The multiple cloning site from pSP72 (Promega Corp., Madison, WI) was excised by digestion with EcoRI and treated with Mung Bean Nuclease followed by digestion with BglII. The resulting fragments were resolved by gel electrophoresis, purified using a QIAquick Gel Extraction Kit (QIAGEN, Valencia, CA) and ligated to produce a pSG5-derived vector with an expanded multiple cloning site. The molecular clones of human RAR{alpha}, RARß, and RAR{gamma} correspond to cDNAs with accession numbers NM_000964, NM_000965, and NM_000966, respectively. The corresponding coding sequences of the receptors were amplified by PCR introducing HindIII (5') and BamH1 (3') sites for subsequent ligation into pSG5.1. RAR{alpha}{Delta}H12/F (containing a stop codon in place of codon 403), RAR{alpha}{Delta}F (containing a stop codon in place of codon 416), RARß{Delta}H12/F (containing a stop codon in place of codon 396), RARß{Delta}F (containing a stop codon in place of codon 409) RAR{gamma}{Delta}H12/F (containing a stop codon in place of codon 405), and RAR{gamma}{Delta}F (containing a stop codon in place of codon 418) were generated by a QuikChange site-directed mutagenesis protocol (Stratagene).

For expression in E. coli, pGEX-MPa was constructed by digestion of pGEX-KG (68) with BamH1 and Sma1. The annealed oligonucleotides 5'-GATCCGGTAC CGCATGCAGA TCTCCC-3' and 5'-GGGAGATCTG CATGCGGTAC CG-3'were then ligated into the cleaved pGEX-KG to expand the multiple cloning site. pGEX-MPc was constructed by digesting pGEX-KG with BamH1 and Sma1 and ligating the annealed oligonucleotides 5'-GATCCATGGT ACCGCATGCA GATCTCCC-3' and 5'-GGGAGATCTG CATGCGGTAC CATG-3'. A pGEX-MPa-SRC1 construct (representing codons 568–891, corresponding to the cDNA with accession no. NM_003743) was constructed by cleaving both SRC1 and pGEX-MPa with EcoR1 and Nco1 and ligating the resulting fragments. SMRT S1+S2 (silencing domains 1 and 2; corresponding to codons 1055–1495 of the cDNA with accession no. U37146) was amplified by PCR to introduce a BamH1 (5') and a stop codon plus an Xho1 site (3') for insertion into pGEX-MPc. pGEX-KG-ACTR (representing codons 621–821) was previously described (69).

The pGL3-GAL-17mer reporter plasmid and the pSG5-Gal4DBD-RAR{alpha}, -ß, and -{gamma} hormone-binding domain fusions were previously described (31). The pTK-Luc-RARE3 reporter, containing three copies of the RARE found within the human RARß promoter, was obtained from A. Dejean (Pasteur Institute, Paris, France). All subclones and mutations were confirmed by DNA sequence analysis.

Transient Transfection Assays
CV-1 cells were maintained in DMEM (Invitrogen, Carlsbad, CA) supplemented with 10% heat-inactivated fetal bovine serum (HyClone, Laboratories, Inc., Logan, UT) in a humidified atmosphere of 5% CO2 at 37 C. For transfections, cells were trypsinized and plated at a density of 14.5 x 103 cells per cm2 in 24-well plates and allowed to attach overnight. Immediately before transfection, the cells were rinsed with PBS and placed in DMEM supplemented with 10% heat-inactivated, hormone-depleted fetal bovine serum. Transfections were performed using the Effectene protocol as recommended by the manufacturer (QIAGEN, Valencia, CA) using 50 ng of an RARE-pTK-Luc reporter vector (or a pTK-Luc vector lacking RAREs as a negative control), 10 ng of the appropriate pSG5.1-RAR expression vector (or as otherwise noted), 50 ng of pCH110 (used as an internal transfection control), sufficient pUC18 to bring the total DNA to 250 ng, and 2 µl of Enhancer reagent and 2.5 µl of Effectene per well. The culture medium was replaced 24 h later with fresh medium containing either all-trans retinoic acid (ATRA) or an equivalent amount of ethanol carrier. After an additional 24 h at 37 C, the cells were harvested and lysed, and the luciferase and ß-galactosidase activities were determined as previously described (70). For the Gal4-DBD repression assay, the transfections were conducted as described above but using 100 ng of a pGL3-GAL17mer reporter plasmid, 5 ng of each pSG5-Gal4DBD-RAR plasmid, 50 ng pCH110, and 95 ng pUC18.

In Vitro Protein-Protein Interaction Assay
GST-corepressor and GST-coactivator protein fusions were produced in E. coli strain BL-21 cells (Stratagene) transformed by an appropriate pGEX vector (pGEX-MPc-SMRT, pGEX-KG-ACTR, or pGEX-MPa-SRC1) (68). The bacteria were lysed by sonication, and the GST fusion proteins were bound to a glutathione-agarose matrix as previously described) (68). 35S-radiolabeled RARs were synthesized in vitro using the TnT-coupled reticulocyte lysate system (Promega Corp., Madison, WI). Each radiolabeled receptor (typically 2–5 µl of TnT product per reaction) was then incubated with the immobilized GST fusion protein of interest (~50 ng of GST fusion protein immobilized to 10 µl of agarose matrix per reaction) in a total volume of 120 µl of HEMG buffer (4 mM HEPES, pH 7.8; 100 mM KCl; 0.2 mM EDTA; 5 mM MgCl2; 0.1% Nonidet P-40; 10% glycerol; 1.5 mM dithiothreitol) at 4 C. The binding reactions were performed in 96-well multiscreen filter plates (Millipore Corp., Bedford, MA) placed on a rotating platform to ensure thorough mixing. After a 3-h incubation, the filter wells were washed by centrifugation four times with 200 µl ice-cold HEMG buffer per well, and any radiolabeled RAR proteins remaining bound to the immobilized GST fusion proteins were eluted with 50 µl of 10 mM glutathione in 50 mM Tris-HCl, pH 7.8. The eluted proteins were resolved by SDS-PAGE and were visualized and quantified using a Storm phosphor imager (Amersham Biosciences, Piscataway, NJ).

Protease Resistance Assay
35S-radiolabeled RAR proteins were synthesized in vitro using the TnT reticulocyte lysate system. For each time point in the CPY assays, 1 µl of the TnT reaction products was diluted to 16 µl in 50 mM Tris-HCl (pH 6.8) containing either 1 µM ATRA or an equivalent amount of ethanol carrier and incubated 10 min on ice. Four microliters (0.5 U) of CPY (Sigma Chemical Co., St. Louis, MO) were then added to each sample, and the tubes were transferred to 10 C to initiate digestion. At each time indicated, proteolysis was terminated by the addition of 20 µl of 2x SDS-PAGE loading buffer, and the samples were rapidly frozen and stored on dry ice. The samples were subsequently quickly thawed and denatured by heating to 95 C for 10 min and resolved by SDS-PAGE, and the proteolytic degradation products were visualized and quantified by Storm phosphor imager analysis. For each time point for the elastase assays, 1 µl of the TnT reaction products was diluted to 16 µl in 50 mM Tris-HCl (pH 7.4) containing the indicated concentration of ATRA or an equivalent amount of ethanol carrier and incubated 10 min on ice. Four microliters (0.05 U) of elastase (Sigma) were then added to each sample, and the tubes were transferred to room temperature to initiate digestion. After 10 min, proteolysis was terminated by the addition of 20 µl of 2x SDS-PAGE loading buffer, and the samples were rapidly frozen and stored on dry ice. The samples were subsequently denatured, resolved, and visualized as performed above.

EMSA
The RARE oligonucleotide probe was prepared by annealing the plus strand (5'-TCGAAAGGGT TCACCGAAAG TTCACTCGCA-3') and minus stand (5'-TCGATGCGAG TGAACTTTCG GTGAACCCTT-3') and was radiolabeled by Klenow polymerase fill-in using 32P-{alpha}-dGTP (3000 Ci/mmol) (PerkinElmer, Boston, MA) and the remaining three unlabeled deoxynucleotide triphosphates. Human RXR{alpha} was expressed in Sf9 cells using recombinant baculovirus and protein isolated as previously described (71). RARs were expressed in COS-1 cells by transient transfection. The COS-1 cells were plated at a density of 14.5 x 103 cells per cm2 in 10-cm plate and allowed to attach overnight. Immediately before transfection, the cells were washed with PBS and placed in DMEM supplemented with 10% heat-inactivated, hormone-depleted fetal bovine serum. Transfections were performed by an Effectene (QIAGEN) protocol using 5 µg of the appropriate pSG5.1-RAR expression vector, 40 µl of Enhancer reagent, and 50 µl of Effectene. After 48 h, cells were harvested and nuclear isolates were prepared. Briefly, cells were extensively washed with PBS and collected in 0.5 ml of 10 mM HEPES, pH 7.9; 10 mM KCl; 1 mM EDTA; 0.05% Nonidet P-40; 0.5 mM dithiothreitol (DTT); and complete protease inhibitor cocktail (Roche Applied Sciences, Indianapolis, IN) solution. The cell lysate was then Dounce homogenized with 20 strokes, and nuclei were isolated by centrifugation at 5000 x g for 5 min at 4 C. Nuclei were then resuspended in 50 µl of 20 mM HEPES, pH 7.9; 420 mM KCl; 1 mM EDTA; 25% glycerol; 0.5 mM DTT; and complete protease inhibitor cocktail (Roche Applied Sciences) and placed on a rotator for 30 min at 4 C. Nuclear extract supernatant was isolated by centrifugation at 14,000 x g for 30 min at 4 C. To perform the EMSAs, proteins and probe (50,000 counts/pmol) were incubated as indicated at room temperature for 15 min in 20 µl binding buffer (7.5 mM Tris-HCl, pH 7.5; 150 mM KCl; 2.3% glycerol; 10 mg/ml BSA; 1.5 mM MgCl2; 2 µg poly-(dI)·(dC); and 2 mM DTT). The complexes were then resolved by electrophoresis in a 5% polyacrylamide gel using a 0.5x TBE buffer system (45 mM Tris-borate, 1 mM EDTA) at 180 V for 90 min. Bound probe was visualized and quantified by Storm phosphor imager analysis.


   ACKNOWLEDGMENTS
 
We are grateful to L. Liu for superb technical assistance. We also thank Michael Goodson for construction of the pGEX-MPa and pGEX-MPc vectors.


   FOOTNOTES
 
This work was supported by Public Health Service (PHS) Grant DK53528 from the National Institute of Diabetes, Digestive, and Kidney Diseases. B.F. was funded, in part, by the PHS predoctoral training award, T32GM07377, from the National Institute of General Medical Sciences.

Abbreviations: ACTR, Activator of thyroid and retinoic acid receptor; ATRA, all-trans retinoic acid; CPY, carboxypeptidase Y; DBD, DNA-binding domain; DTT, dithiothreitol; GST, glutathione-S-transferase; N-CoR, nuclear corepressor; PR, progesterone receptor; RAR, retinoic acid receptor; RARE, retinoic acid response element; SRC, steroid receptor coactivator; SMRT, silencing mediator of retinoid and thyroid hormone receptor; TK, thymidine kinase; wtRAR, wild-type RAR.

Received for publication June 8, 2004. Accepted for publication August 16, 2004.


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 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
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NURSA Molecule Pages Link:

Nuclear Receptors:   RARα  |  RARβ  |  RARγ  |  PR
Coregulators:   SRC-1  |  AIB1  |  SMRT
Ligands:   all-trans-Retinoic acid



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