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Molecular Endocrinology, doi:10.1210/me.2002-0124
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Molecular Endocrinology 16 (11): 2528-2537
Copyright © 2002 by The Endocrine Society

Real-Time Analysis of Molecular Interaction of Retinoid Receptors and Receptor-Interacting Protein 140 (RIP140)

Yixin Chen, Ann Kerimo, Shaukat Khan and Li-Na Wei

Department of Pharmacology, University of Minnesota Medical School, Minneapolis, Minnesota 55455

Address all correspondence and requests for reprints to: Li-Na Wei, Department of Pharmacology, University of Minnesota Medical School, 6-120 Jackson Hall, 321 Church Street Southeast, Minneapolis, Minnesota 55455. E-mail: weixx009{at}tc.umn.edu.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Receptor interacting protein 140 (RIP140) is a coregulator for a large number of transcription factors. RIP140 interacts with retinoic acid receptor (RAR) and retinoid X receptor (RXR) with or without ligands. The C-terminal domain of RIP140 (RIP-C') contains a novel sequence (1063–1076, LTKTNPILYYMLQK) and has been shown to interact with RAR and RXR ligand dependently in two-hybrid interaction and pull-down assays. To examine the kinetic characteristics of molecular interaction of RIP-C' with RAR and RXR, a surface plasmon resonance technology (BIAcore) was applied for real-time analyses of this molecular interaction with highly purified proteins. A modified pull-down assay using purified proteins was also conducted to obtain supporting data. The effect of retinoid ligands on this type of interaction was addressed. By using receptor mutants, it was demonstrated that the activation function-2 domain and the ability to form dimers of the receptors are required for an efficient interaction of receptor with RIP140. Finally, with a mutagenesis approach, we determined the effects of specific point mutations on the kinetics of RIP-C' interaction with RAR/RXR.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
SINCE THE INTRODUCTION of BIAcore in 1991 (1, 2), the use of surface plasmon resonance technology has facilitated the kinetic analysis of molecular interactions. Provided that one of the interaction partners can be covalently immobilized or captured to the sensor surface in the active form, this technology allows a real-time analysis of molecular interactions involving analytes with a wide range of molecular weights. Analysis of molecular interaction using surface plasmon resonance has been successfully applied for various types of molecular interactions, including protein-protein and protein-DNA interactions. Recently, surface plasmon resonance analysis has been used to determine the interactions between nuclear receptors, as well as the effects of ligands on receptor dimerization (3, 4, 5).

Receptor interacting protein 140 (RIP140, also named NRIP1) (6) is a coregulator for a large number of transcription factors (7, 8, 9, 10, 11, 12). Among these, nuclear receptors represent the largest group of transcription factors that interact with RIP140. The human RIP140 was first demonstrated as a ligand-dependent coactivator in a chimeric estrogen receptor system (7). The mouse RIP140 was cloned in this lab with the ligand binding domain (LBD) of orphan nuclear receptor TR2 as the bait and was shown to be a potent corepressor for TR2 in the absence of putative ligands (13). Later, many other labs, including ours, demonstrated a suppressive activity of RIP140 in nuclear hormone receptor- and other transcription factor-mediated gene expression (14, 15, 16, 17, 18). Recently, we have demonstrated a direct association of RIP140 with histone deacetylase 3 through its N-terminal domain (19).

While there are nine LXXLL motifs in RIP140, which were originally proposed as the interacting domain that mediate its ligand-dependent interaction (7), we have identified the carboxyl terminal region of this molecule (amino acid residues 977-1161, named RIP-C'), which lacks LXXLL but contains an interesting sequence (1063-1076, LTKTNPILYYMLQK). This region was later demonstrated as a novel retinoic acid (RA)-dependent retinoic acid receptor (RAR)- and retinoid X receptor (RXR)-interacting motif in two-hybrid interaction and glutathione-S-transferase (GST)-pull-down assays in our recent studies (20). Therefore, RIP140 is a unique coregulator featured by: 1) its ability to interact with nuclear receptors in both apo- and holo-forms; 2) the presence of nine copies of LXXLL motif in this molecule; 3) the presence of a novel RA-dependent, RAR- and RXR-interacting motif in its carboxyl terminus; 4) its direct interaction with histone deacetylases; and 5) its primarily suppressive activity on gene expression regulated by nuclear receptors in the presence or absence of receptor ligands.

The RARs and RXRs are the representative hormone nuclear receptors that are involved in a wide variety of biological processes (21, 22). RAR binds to all-trans RA (atRA) and 9-cis RA (9cRA), whereas RXR binds to 9cRA. Additionally, many synthetic compounds have been produced as RAR- or RXR-specific ligands. RIP140 interacts with RAR and/or RXR through their LBDs (23). The dissected RIP-C', which interacts with the LBDs of RAR and RXR ligand dependently in two-hybrid interaction and pull-down assays (23), contains LYYML, which diverts from the LXXLL box found in coactivators and the CoRNR box (L/IXXI/VI) found in corepressors. It is of significant interest to examine the nature of molecular interaction between RIP-C' with nuclear receptors and to address the effects of ligands on this interaction.

In this study, our primary aim was to examine the kinetic characteristics of molecular interaction of RIP-C' with RAR and RXR receptors using highly purified proteins. This was done in a BIAcore machine for real-time analyses of molecular interactions. Secondly, a modified pull-down assay using highly purified proteins was also conducted to obtain supporting data. Thirdly, the requirement of activation function 2 (AF-2) domains and the ability to form dimers of the receptors for an efficient interaction with RIP140 were demonstrated by using receptor mutants. Fourthly, the effect of retinoid ligands on this type of interaction was addressed. Finally, with a mutagenesis approach, we determined the amino acid residues of the peptide LTKTNPILYYMLQK, which are important for the interaction between RIP-C' and the RAR and the RXR.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Expression and Purification of RIP-C' and the LBDs of RAR and RXR
An EcoRI fragment containing the C-terminal 185 amino acid residues (977-1161) of RIP140, that was shown to interact with RAR and RXR, was fused to a His-Trx epitope (160 amino acid residues), designated as RIP-C', and expressed in Escherichia coli as described in Materials and Methods. The LBDs of RAR and RXR were fused to either an His or a GST epitope for expression in E. coli. From a 500-ml culture, approximately 3.5 mg pure RIP140-C' were obtained after the final purification step, which was estimated to be of greater than 95% purity according to the results in Coomassie blue-stained gels. The molecular mass of RIP-C' (a total of 345 amino acids) was estimated to be 42,100 Da and 42,700 Da by SDS-PAGE and gel filtration, respectively.

His-RAR and His-RXR were each purified over a TALON column affinity chromatography. His-RAR and GST-RXR were copurified as heterodimers in a two-step (Co pool, GF pool) procedure (24). The SDS-PAGE gel showed that the final monomer or heterodimer preparation was greater than 95% purity. The heterodimer of RAR and RXR contained an equal molar ratio of RAR/RXR. For quantitative analysis with a BIAcore where the His-RIP-C' was immobilized on the chip, His-RAR and His-RXR were further digested with thrombin protease to remove the His tag. The results of the purification of these proteins are shown in Fig. 1Go. Figure 1AGo shows the results of RIP-C' purification from an induced culture (lane 3), crude extract (lane 4), ammonium sulfate precipitation (lane 5), gel filtration (lane 6), and TALON column (lane 7). Lane 2 shows an uninduced control culture. Figure 1BGo shows the purified LBDs of His-RAR (lane 1), His-RXR (lane 3), and His-RAR/GST-RXR heterodimer (lane 5). The thrombin cleaved products are shown in lanes 2, 4, and 6. It is clear that each His-tag was very efficiently cleaved off by thrombin (lanes 2, 4, and 6).



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  		Figure 1. Purification of RIP-C', RAR, RXR and RAR/RXR Heterodimer

The protein was separated on a 10% SDS polyacrylamide gel and stained with Coomassie blue. A, Analysis the RIP-C' preparation at various stages of purification. A representative SDS-polyacrylamide gel of control (lane 2) and isopropyl-ß-D-thiogalactopyranoside-induced bacteria (lane 3), crude extracts (lane 4), precipitate of (NH4)2SO4 (lane 5), gel filtration (lane 6), and pool of the RIP-C' eluted from the TALON column (lane 7). B, Analysis of purified RAR (lanes 1 and 2), RXR (lanes 3 and 4), and RAR/RXR (lanes 5 and 6), before (lanes 1, 3, and 5) and after (lanes 2, 4, and 6) thrombin digestion.

 
Quantitative Analysis of RAR/RXR Interaction with RIP-C'
Our previous studies using two-hybrid interaction and pull-down assays demonstrated that the LXXLL-less C-terminal domain of RIP140 was responsible for its ligand-dependent interaction with RAR/RXR. Although our results were highly consistent, it was still possible that interaction mediated by the novel interaction motif of RIP140 could be complicated by other factors present in the cells or the reaction mixtures. To address this problem, it was desirable to examine this type of interaction with detecting systems that utilize proteins of high purity such as BIAcore. The His-RIP-C' was immobilized onto the NTA sensor chip, and the purified RAR, RXR, or RAR/RXR heterodimer (the analyte solution) was applied over the chip. The typical kinetic runs for these receptor combinations are shown in Fig. 2Go, and the deduced kinetic parameters are reported in Table 1Go.



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  		Figure 2. Kinetic Analysis of Immobilized His-RIP-C' Interaction with RXR (A and C) and RAR/RXR Heterodimer (B and D)

The overlaid sensograms are shown. A, Sensograms of apo-RXR at the concentration of 15 nM and 50 nM. B, Sensograms of RAR/RXR heterodimer at the concentration of 10 nM, 25 nM, and 50 nM. C, Sensograms of RXR (15 nM) in the presence and absence of ligands. D, Sensograms of RAR/RXR heterodimer (30 nM) in the presence and absence of ligands. atRA, 9cRA, and AGN194204 were each added at the final concentration of 1 x 10-5 M. Am80 was at the concentration of 8 x 10-8 M.

 

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  		Table 1. Kinetic Parameters for the Association of RIP-C' with RAR/RXR

 
It is interesting that the purified RAR alone was not able to interact with the immobilized RIP-C' in the presence or absence of retinoid ligands by using this detection method (data not shown). However, both the RXR alone and the RAR/RXR heterodimer were able to form stable complexes in the presence or absence of ligands. The presence of ligands indeed reduced the equilibrium dissociation constant (Kd) of the association of RIP-C' with RXR and RAR/RXR heterodimer. The association of apo-RXR alone with RIP-C' could be described in a simple equilibrium of the A + B {leftrightharpoons} AB type (Fig. 2AGo). The Kd of this interaction is 6.4 nM. Apo-RAR/RXR heterodimer also formed a stable complex with the RIP-C' with a Kd of 3.6 nM. This could also be described in a simple equilibrium of the A + B {leftrightharpoons} AB type where A represented the dimeric form of RAR/RXR and B represented RIP-C' (Fig. 2BGo).

To examine the effects of retinoid ligands on the interaction of RAR, RXR, or RAR/RXR with RIP-C' using this detection method, a number of retinoid ligands were used including atRA, Am80 (RAR agonists), 9cRA (agonist for both RAR and RXR), and AGN194204 (an RXR agonist). The highly purified receptor proteins were incubated with ligands at 4 C for 30 min before the injection into the flow cells. It is apparent that the addition of ligands increased the affinity of RXR and that of RAR/RXR to RIP140-C' as shown in the sensograms (Fig. 2Go, C and D). The calculated kinetic parameters are shown in Table 1Go. By comparing the Kd values of different receptor combinations and the nature of ligands used, it can be concluded that atRA is the best ligand for RAR/RXR heterodimer interaction with RIP-C', causing an approximately 5-fold decrease in the Kd of the heterodimer. Secondly, the Kd value of RXR interaction with RIP-C' was reduced for approximately 1.5-fold in the presence of agonist (6.4 vs. 4.1 or 4.7). Thirdly, the RAR/RXR heterodimer appeared to interact with RIP-C' much stronger than RXR alone, with or without ligands. Finally, the decreases of the Kd values in the presence of ligands was primarily attributed to the increases of the association rates.

The result that purified RAR was not able to interact with RIP-C' in the BIAcore assay prompted us to examine whether these highly purified proteins were in an active conformation. This was conducted by using a known RAR-interacting coactivator glucocorticoid receptor-interacting protein 1 (GRIP-1) (25) in a modified His-tag pull-down assay with the same highly purified proteins. In this experiment, all the proteins, including RAR, RXR, RAR/RXR dimer, and a negative control Nap-1 (26) were each tagged with a His-epitope and purified. The coactivator GRIP-1 was labeled in an in vitro transcription/translation reaction. Protein complexes were bound to TALON beads and washed extensively, followed by resolution in SDS-PAGE. As shown in Fig. 3Go, the purified RAR was able to interact with GRIP-1 in an RA-dependent manner (lanes 8 and 9). As expected, purified RXR, as well as RAR/RXR heterodimer, also interacted with GRIP-1 in a ligand-dependent manner (lanes 5–7, 10, and 11). As a negative control, the purified His-Nap-1 protein failed to interact with GRIP-1 in the same assay (lanes 2–4). The Coomassie blue-stained gel shown at the bottom of this figure confirmed an equal amount of protein in put in each lane. Therefore, it is confirmed that all the purified receptors are in their active conformations for interacting with a typical coactivator in a ligand-dependent manner.



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  		Figure 3. Modified Pull-Down Assay Using Purified Receptor Proteins

His-RAR, His-RXR, His-RAR/RXR, and His-Nap1 were each purified as described in the text. 35S-labeled GRIP-1 was incubated with each purified protein with or without ligands, and bound to TALON beads, followed by extensive washing and resolution by SDA-PAGE. A Commassie blue gel revealed the equal amount of protein input in each lane.

 
Peptide Competition in RIP-C' Interaction with RAR/RXR
Previously, we have dissected a 14-amino-acid peptide (residues 1063-1076, LTKTNPILYYMLQK) of the RIP-C', which was essential for interaction of RIP140 with RAR/RXR demonstrated in two-hybrid interaction tests (20). To provide further evidence for the necessity of this peptide in mediating this interaction, peptides of the wild-type sequence and specific mutations were produced and tested in RAR/RXR interaction with RIP-C' in BIAcore analysis. The effect of adding the wild-type peptide to the interaction mixtures, as presented in the RAR/RXR interaction sensograms, is shown in Fig. 4AGo. The wild-type peptide was added at a concentration range of 100-1000 nM, and the concentration of RAR/RXR was maintained at 30 nM. It appeared that the wild-type peptide was able to compete with RIP-C' interaction for about 50%, 75%, and 90% at the concentration of 100 nM, 500 nM, and 1000 nM, respectively (Fig. 4BGo). The RIP-C' sequence contains a motif, LYYML, similar to the LXXLL sequence seen in numerous coactivators. A computer model, based upon coactivator SRC-1 bond to holo-PPAR{gamma} (27) was created to predict essential amino acids that were important for the interaction between RIP-C' and receptors. This model predicted three amino acids, 1067Asn, 1068Pro, and 1073Met as important residues. The Pro could potentially be located at the amino-terminal end of the putative amphipathic helix formed by this sequence. The Met residue could offer a larger buried surface area that is more hydrophobic in character than Leu, as seen in a typical LXXLL motif. The Asn residue could be important due to its polar characteristic and a prediction of hydrogen bonds formed with the receptors. Based on the modeling, five mutant peptides were made as shown at the top of Fig. 4Go. M1 contains the Pro to Ala mutation, M2 contains the Met to Lys mutation, M3 contains the Met to Leu mutation (into wild-type LXXLL motif), M4 contains the Met to Ile mutation, and M5 contains the Asn to Gly mutation.



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  		Figure 4. Peptide Competition Experiment to Demonstrate Specific Interaction of the Dissected RIP-C' with RAR/RXR Heterodimer

A, Four overlaid sensograms of immobilized RIP-C' interaction with RAR/RXR (30 nM) in the presence of the wild-type peptide at the concentration of 0 nM, 100 nM, 500 nM, and 1000 nM, respectively. B, The competition of RIP-C' interaction by wild-type peptide at different concentrations, represented as the decrease in the resonance units ({Delta}RU).

 
These five mutant peptides at the concentration of 1000 nM were tested in the competition experiments as shown in the sensograms (Fig. 5AGo). Figure 5BGo shows the relative retention of the analytes ({Delta}RU) in each peptide competition analysis. It is clear that the wild-type peptide was most efficient (90%) in terms of competition with RIP-C'. The five mutant peptides were all competitive, but to different degrees in the range of 30–70%. The M1, M3, and M4 peptides, which had the 1068Pro to Ala, 1073Met to Leu, and 1073Met to Ile, respectively, competed for approximately 70%. These could be due to the fact that the M3 peptide has a typical coactivator LXXLL motif and the M4 peptide was mutated to a corepressor CoRNR box (L/IXXI/VI), both were expected to be competitive in receptor interaction. The M1 mutation remained reasonably efficient in competition, suggesting that the Pro mutation did not significantly affect the interaction of this peptide with receptors under this condition. On the contrary, the M2 and M5 peptides, mutated at the Met residue and the Asn, respectively, were much less efficient in competition, supporting the prediction that Met is critical for this interaction and the flanking Asn residue is also important.



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  		Figure 5. Effect of Mutant Peptides on RIP-C' Interaction with RAR/RXR

A, Overlaid sensograms of RIP-C' interaction with RAR/RXR (30 nM) in the presence of different peptides (100 nM). B, The competition of RIP-C' interaction with RAR/RXR by each peptide, represented as the difference in the resonance units ({Delta}RU).

 
Based on these results, it is concluded that the dissected peptide sequence LTKTNPILYYMLQK of the RIP-C' domain indeed mediates the interaction of RIP-C' with RAR/RXR. Within this peptide, the 1073Met and 1067Asn residues are essential for the interaction, whereas the 1068Pro residue is not as critical for this interaction.

In Vitro Association of RAR/RXR with RIP-C'
To further confirm receptor interaction mediated by this novel RIP-C' domain, a modified pull-down assay was performed, where purified protein, instead of crude protein expressed from in vitro transcription-translation, was used. The GST-RIP-C' was partially purified over a glutathione column and used as the bait by binding to glutathione beads. Purified his-RAR, -RXR, or -RAR/RXR heterodimer were then added to the GST-RIP-C'-saturated glutathione beads. The whole mixture was washed extensively and the proteins, the His-tagged RAR, RXR, or RAR/RXR, interacting with RIP-C' were recovered and analyzed by Western blotting using an anti-His antibody. The effect of ligands on receptor binding to RIP-C', including Am80, AGN194204, atRA, 9cRA was monitored in parallel reactions. Figure 6Go, A–C, shows the results of interaction with RAR, RXR, and RAR/RXR, respectively. It is interesting that the purified apo-RAR was able to interact with RIP-C' under this condition (Fig. 6AGo), and ligands, atRA, Am80, or 9cRA, were able to enhance this interaction. As predicted, both apo-RXR, and apo-RAR/RXR in the purified forms, also interacted with RAR/RXR. Consistent with the BIAcore data, ligands (9cRA and AGN194204 for RXR, and at RA, 9cRA, Am80, and AGN194204 for RAR/RXR heterodimer) enhanced this interaction. Therefore, these data further support the conclusion that, in the highly purified systems, the RIP-C' domain directly interacts with RAR and RXR and agonists can enhance this interaction.



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  		Figure 6. Interaction of RIP-C' with RAR (A), RXR (B), and RAR/RXR Heterodimer (C), Detected by the Modified Pull-Down Assay

Purified GST and GST-RIP-C' were each bound to glutathione-sepharose beads and incubated with purified His-tagged proteins. AtRA, 9cRA, and AGN194204 were each added at the concentration of 1 x 10-6 M and Am80 was at the concentration of 8 x 10-9 M. The reaction products were resolved by SDS-PAGE and detected with anti-His antibody.

 
Requirement of AF-2 Domain and Dimerization for an Efficient Interaction with RIP140
The enhancing effects of retinoids on the interaction of RAR and RXR with RIP-C' prompted three more questions regarding the nature of this interaction: 1) whether the dissected RIP-C' behaves similarly as the full-length RIP140 in terms of interaction with RAR and RXR; 2) if this interaction is AF-2 dependent; and 3) if the ability to form dimers is required for receptors to interact with RIP140. Due to technical difficulty in purifying the full-length RIP140, these questions were addressed in two hybrid interaction tests as shown in Fig. 7Go.



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  		Figure 7. Mammalian Two-Hybrid Interaction Tests for RIP140 Interaction with Wild-Type and Mutant Receptors

A, Interaction of the full-length RIP-140 (RIP-f) with RAR, RXR, and RAR/RXR in the absence (empty bars) or presence (filled bars) of ligands as indicated. B, Interaction of the RIP-C' domain with RAR (columns 4 and 5), RXR (columns 6 and 7), RAR/RXR (columns 8 and 9), AF2-deleted RAR (RAR-AF2, columns 18 and 19), AF2-deleted RXR (RAR-AF2, columns 20 and 21), the dimerization RXR mutant (RXRA/K, columns 16 and 17), or dimers of one wild-type receptor and one mutant receptor (columns 10–15), in the absence (empty bars) or presence (filled bars) of ligands as indicated.

 
The interaction of the full-length RIP140, as well as the dissected RIP-C', with RAR and RXR was examined in parallel as shown in Fig. 7Go, A and B. It appeared that RIP-C' (Fig. 7BGo, columns 4–7) behaved as the full-length RIP140 (RIP-f; Fig. 7AGo, columns 5–8) in terms of their ability to interact with RAR and RXR in a ligand-dependent manner. Both RIP-f and RIP-C' interacted with RXR more strongly than with RAR. Furthermore, both RIP-f and RIP-C' interacted with heterodimer much stronger than with either homodimer alone (Fig. 7AGo, lanes 9 and 10; Fig. 7BGo, lanes 8 and 9). The requirement for AF-2 domain of the receptors was demonstrated in the reactions using AF-2 deleted receptors (RAR-AF2 and RXR-AF2, Fig. 7BGo, comparing columns 4–7 and columns 18–21). Furthermore, an RXR deficient in dimerization with RAR (28) was also defected in interacting with RIP-C' (Fig. 7BGo comparing columns 6 and 7 with columns 16 and 17). More importantly, interaction of RIP140 with heterodimers consisting of one of these mutant receptors was dramatically affected (Fig. 7BGo, columns 10–15), supporting an essential role of receptor dimerization in their efficient interaction with RIP140.

Taken together, it is concluded that RIP-C' behaves very similarly as the full-length RIP140 for interacting with RAR, RXR, or RAR/RXR dimer. RIP140 interacts with heterodimers of RAR/RXR much stronger than with either receptor alone. Finally, for an efficient interaction with RIP140, the AF-2 domain, as well as the ability of the receptors to form dimers is crucial.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
MATERIALS AND METHODS
 REFERENCES
 
Previously, we have shown that RAR, RXR, and RAR/RXR heterodimer interact with RIP140 in a ligand-enhanced manner (20, 23). The LXXLL-containing regions of RIP140 are primarily responsible for ligand-independent interaction whereas a LYYML-containing carboxyl terminal domain mediates ligand-dependent interaction with RAR and RXR, as detected with two-hybrid interaction and GST-pull-down assays in our previous studies (20). We have also mapped the RIP140-interaction domain of RAR and RXR to their LBDs in our previous studies (23). The difference among the reported studies in terms of the interaction of RIP140 with hormone nuclear receptors is intriguing. Moreover, its interaction with RAR and RXR mediated by the novel RIP-C' domain that contains an LYYML sequence is of particular interest to us. Therefore, we aim to unambiguously demonstrate this molecular interaction using a highly purified system in this study. By using highly purified RIP-C' and the LBDs of RAR and RXR in the surface plasmon resonance measurements, we have now precisely determined the kinetic parameters of the association of RIP-C' with RAR or RXR, as well as the effect of retinoid ligands on the formation of these complexes. With competition and mutagenesis approaches, we have provided evidence for an essential role of the LTKTNPILYYMLQK motif in mediating this molecular interaction and identified important amino acid residues for this interaction. The association of RAR monomer was too weak to be detected, whereas RXR and RAR/RXR gave clear signals and exhibited dissociation constants of 6.4 and 3.6 nM, respectively, under physiological salt conditions. The agonists indeed enhanced the interaction as evidenced by the reduced Kd values, which was attributed to an increase in the association constants.

The LBD of the RXR was reported to be either a monomer using equilibrium sedimentation analysis or a dimer using gel filtration and laser light scattering, and was crystallized as a dimer. In contrast, the LBD of RAR was crystallized as a monomer (29). It is widely suggested that most nuclear receptors bind DNA as dimers (30). In our study reported here, although both RAR and RXR, as well as their heterodimer interact with RIP-C' in the modified pull-down tests (Fig. 6Go), only RXR and RAR/RXR interact with RIP-C' in BIAcore analyses (Fig. 2Go and Table 1Go). In consistence with these results, by using two hybrid interaction tests (Fig. 7Go) where the relative strength of the interactions can be compared, it is confirmed that RIP140 interacts with RXR consistently much stronger than with RAR, and heterodimeric RAR/RXR interaction with RIP140 is always the strongest among all the receptor combinations. It was first speculated that the highly purified RAR might be defected in its interaction with RIP-C' in the BIAcore analysis due to a defect in its conformation. Because all the purified receptors remain active in interacting with coactivator GRIP-1 in the modified pull-down assay (Fig. 3Go), the failure of RAR to interact with RIP-C' in BIAcore analyses could not be attributed to problems in protein folding of these highly purified protein preparations. Alternatively, it may be a result of the difference in the dynamic and kinetic features of molecular interactions detected by these two methods. This is supported by the positive ligand-dependent interaction of RAR with RIP140 in the mammalian two-hybrid interaction test (Fig. 7Go). However, interaction of RIP-f or RIP-C' with RAR is consistently much weaker than their interaction with RXR. Therefore, it can be concluded that interaction of RIP140 with RXR differs from its interaction with RAR. This is more clearly demonstrated in a detecting system using highly purified proteins, like BIAcore.

The effects of ligands on this interaction suggested a role of AF-2 domain in this type of interaction. This was addressed in mammalian two-hybrid interaction tests in which AF-2 deleted mutants, from both RAR and RXR, are significantly defected in interacting with RIP140 (Fig. 7Go). These RAR and RXR mutants remained defective provided alone or in combination with a wild-type partner. Another interesting property of receptors, i.e. the ability to form dimer, in interacting with RIP140 was also addressed in two-hybrid interaction tests by using a dimerization-deficient RXR (28) as shown in Fig. 7Go. It is apparent that the ability to interact with RIP140 was dramatically reduced in this dimerization-deficient mutant RXR, either provided alone or in conjunction with a wild-type RAR. The fact that receptor dimers, in particular heterodimers, are able to interact with RIP-C', which contains only one receptor-interacting motif, would suggest that one partner of the receptor dimer indirectly interacts with RIP140 through receptor dimerization. Alternatively, RIP140 may oligomerize and simultaneously interact with both partners of the receptor dimers. These questions remain to be addressed experimentally, particularly in the context of the full-length RIP140. Nevertheless, it can be concluded that ligand-dependent interaction between RIP140 and RAR/RXR requires the AF-2 domain of the receptors and their ability to form dimers. Dimerization of receptors and ligand binding may help to stabilize these complexes.

The nature of retinoids affects the interaction kinetics of receptor with RIP-C' (Table 1Go). Among all agonists tested, atRA appears to induce the highest binding affinity of RIP-C' with the RAR/RXR heterodimers. This seems to correlate with the affinity of ligands to receptors (comparing Am80 and atRA for RAR). Previously, it has been shown that the RAR-specific agonist Am80 exhibits approximately 2-fold lower binding affinity than atRA with respect to RAR binding (31). However, because the interaction kinetics conducted in this study were for the dissected RIP-C', it remains to be determined, once the full-length RIP140 can be purified, whether this correlation remains valid in the context of nature RIP140/RXR/RXR complexes. Furthermore, it has become obvious that different coregulator can form complexes with the same nuclear receptors; therefore, the kinetics of these molecular interaction probably play a crucial role in the formation of particular coregulator complexes. As such, the effects of different ligands on these molecular interaction deserve further attention. In particular, it is desirable that details in the kinetics of these molecular interactions can be examined more systemically. Our preliminary studies have shown atRA to be the most effective, among all the retinoids tested, in inducing reporter activation (data not shown). However, it remains to be examined whether and how the various ligands, which appear to induce various binding kinetics of RIP-C' with RAR/RXR, exert different effects on the suppressive activity of RIP140 on RA-targeted gene activation.

The effects of ligands in nuclear receptor conformation have been examined by biochemical and structural studies (32). Limited protease digestion has revealed that ligand confers a conformational change in receptor structure (33). Cheskis et al. (4) have demonstrated, also by surface plasmon resonance measurements, that 1,25-(OH)2D3 modulates the dimerization state of VDR. It is apparent from their sensograms that the retinoid ligand (9cRA) binding increased the affinity of RXR homodimerization both in solution and on DNA (3). The kinetic data obtained in our study indicate that, in the highly purified system, RIP-C' can interact with RAR/RXR or RXR homodimer in the absence of retinoid ligands with a relatively high affinity (Kd in the range of nM), and the addition of ligand enhances the association rate of receptors with RIP-C' (Table 1Go). The constitutive interaction of purified RIP-C' with purified RAR/RXR and RXR is consistent with the result of the modified pull-down assay, which also utilizes highly purified RAR and RXR (Fig. 6Go) rather than crude proteins made from in vitro transcription-translation as used in most typical GST-pull-down assays. One implication in this discrepancy is a possibility of some unknown factors participating in the interaction of RIP-C' with RAR and RXR, which were not present in the highly purified protein preparations. With regards to the effects of retinoid ligands on the increased on-rates, it can be speculated that retinoid ligand increases the concentration of dimer in solution and alters the conformation of LBD (29), thereby enhancing the formation of ternary complex composed of dimeric receptor and RIP-C'.

In our peptide competition experiments, the cold wild-type peptide of LTKTNPILYYMLQK sequence competed efficiently with RIP-C' for interacting with RAR/RXR. Mutation at either Met, Asn, or Pro residues all reduced the ability to compete, suggesting reduced affinities of these mutant peptides toward RAR/RXR. This result supports our hypothesis that Asn, Pro, and Me are important residues for interacting with RAR/RXR.

The suppressive effects of RIP140 in many hormone receptor-mediated gene expression and the enhanced interaction of RIP140 with holo-nuclear receptors suggest an interesting and testable hypothesis that a direct gene suppression can be elicited by hormones in the presence of RIP140. It would be important to compare the kinetic parameters of molecular interactions of RAR/RXR with RIP140 and that of RAR/RXR with other coactivators.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
REFERENCES
 
Construction of Bacterial Expression Vectors
The RIP-C', encoded in an EcoRI fragment (amino acid 977-1161) was fused to a His-tag E. coli vector pET32a (Novagen, Madison, WI) to generate a His-tagged RIP-C' fusion protein of 345 amino acids. For the modified GST-pull-down assay, a GST-RIP-C' generated previously (23) was used for the expression of GST-RIP-C'. The mouse RAR{alpha} LBD (amino acid residues 146-462) was fused to a His-tag vector pET-15b as a fusion protein of 351 amino acids. Two tagged fusions of RXRß-LBD were made, one with the LBD of the mouse RXRß (amino acids 148-411) fused to pET-28a vector as a His-tagged protein of 399 amino acids for purification of RXR, and the second with the same RXRß fragment fused to a GST- domain (pGEX-2T) (34) as a GST-fusion protein of approximately 540 amino acids for the purification of RAR/RXR heterodimer.

Protein Expression
To express RIP-C', E. coli strain AD494(DE3)(Lys) was transformed with the his-tagged RIP-C' fusion vector. Ammonia sulfate precipitation was used, followed by a Ultrogel AcA54 (Sigma, St. Louis, MO) column (100 x 1.5 cm) and a TALON metal affinity column (CLONTECH Laboratories, Inc., Palo Alto, CA). Fractions containing the purified protein were dialyzed against 20 mM HEPES (pH 7.4) containing 150 mM NaCl and frozen in -80 C. Purification of GST-RIP-C' was as described previously (23).

The LBDs of RAR and RXR were each expressed in E. coli as His-tagged fusions and purified over a TALON metal affinity chromatography. For purification of RAR/RXR heterodimer, the His-RAR and GST-RXR were expressed in separate E. coli cultures, and cultures were pooled for copurification. Briefly, it was first purified over a TALON metal affinity chromatography, followed by a sephacryl S-200-HR chromatography (Sigma) (100 x 1.5 cm) (24). The pooled protein was digested with bovine thrombin (Amersham Pharmacia Biotech, Piscataway, NJ) (2 U/µg) for 2 h at 30 C to remove the His-tag.

Molecular Weight Determination
The molecular weight (Mr) of each purified protein was estimated by gel filtration on a AcA54 column (100 x 1.5 cm) equilibrated with buffer A. The protein markers for the gel filtration were bovine albumin (Mr 67,000), egg albumin (Mr 45,000), {alpha}-chymotrypsinogen A (Mr 25,000), and equine myoglobin (Mr 17,800).

Surface Plasmon Resonance Measurements
Surface plasmon resonance measurements were carried out using a Biacore 1000 instrument (Biacore, Piscataway, NJ). All experiments were conducted at 25 C. Proteins were immobilized on sensor chips NTA as follows. The flow cells were washed in an eluent buffer containing 20 mM HEPES (pH 7.4), 50 µM EDTA, 0.005% Surfactant P-20, and 150 mM NaCl. The His-RIP-C' was injected at a flow rate of 5 µl/min to produce an increase of the baseline level of approximately 250 resonance units. For each concentration of the analyte, a control experiment was carried out without prior binding of the His-tagged protein. Experimental curves were analyzed with the BIAcore evaluation 3.1 software package. The formation of a surface-bound complex was analyzed according to the interaction type of A + B {leftrightharpoons} AB.

Modified GST- and His-Pull-Down Protein Interaction Tests with Purified Proteins
A modified GST-pull-down assay was conducted by using GST-RIP-C' to pull-down highly purified RAR and RXR, which were then detected by specific antibodies. Briefly, the GST-RIP140-C' was partially purified over a glutathione-agarose column as described previously (23). The partially purified GST and GST-RIP-C' fusion proteins were each bound to 20 µl glutathione-agarose beads (Sigma) in 1x PBS for 2 h with rotation. Beads were washed two times for 10 min each in 1x PBS containing 0.1% Triton X-100, and one time in a binding buffer (20 mM HEPES, 100 mM NaCl, 0.5 mM EDTA, 0.1% Triton X-100, 10% glycerol, protease inhibitor cocktail). The washed beads were incubated, in 300 µl binding buffer, with 1 µg of either highly purified His-RAR, His-RXR individually, or a combination of 0.5 µg each of RAR and RXR, in the absence or presence of various ligands. Complexes were centrifuged and washed three times with the binding buffer to remove nonspecific binding. Bound proteins were eluted in 30 µl of Laemmli sample buffer, boiled for 10 min, and analyzed by Western blotting with an anti-His antibody (Sigma). The modified His-pull-down assay was conducted similarly, except that the His-tag was used and the TALON beads were used for purification of the pull-down complexes. A GRIP-1 expression vector was kindly provided by Dr. M. R. Stallcup (UCLA, Los Angeles, CA) (25), and a Nap-1 (26) expression vector was kindly provided by Dr. C. M. Chiang (Case Western Reserve University, Cleveland, OH). The interacting protein was detected with a PhosphorImager (Molecular Dynamics, Sunnyvale, CA).

Mammalian Two-Hybrid Interaction Test
The mammalian two hybrid interaction tests, as well as vectors used in the assays such as the DR5-containing reporter, pM-RIP-f, Vp-RAR, Vp-RXR, and AF-2 deleted mutants Vp-RAR-AF2, and Vp-RXR-AF2 were as described previously (23). The RIP-C' was fused to the EcoRI site of the pM vector containing the DNA binding domain of GAL4. An RXR dimerization mutant with an A416K mutation (28) was kindly provided by Dr. J. W. Lee.


   ACKNOWLEDGMENTS
 
We thank Dr. Jim Thompson (University of Minnesota) for help in computer modeling and Dr. X. Hu (University of Minnesota) for help in plasmid construction. We thank the help of Dr. C. Pennell (Cancer Center, University of Minnesota) for help in BIAcore analysis. We thank Drs. H. Kagechika (University of Tokyo, Japan) and R. A. S. Chandraratna (Allergan, Irvine, CA) for the retinoid compounds. We also thank Dr. M. R. Stallcup and C. M. Chiang for expression vectors of GRIP1 and Nap1, respectively. This work was supported by Grants DK-54733, DK-60521, DA-11190, and DA-11806 from the NIH, and RPG-99-237-010CNE from the American Cancer Society (to L.N.W.).


   FOOTNOTES
 
This work was supported by Grants DK-54733, DK-60521, DA-11190, and DA-11806 from the NIH, and RPG-99-237-010CNE from the American Cancer Society (to L.N.W.).

Abbreviations: AF, Activation function; atRA, all-trans RA; 9cRA, 9-cis RA; GRIP, glucocorticoid receptor-interacting protein; GST, glutathione-S-transferase; Kd, dissociation constant; LBD, ligand binding domain; RA, retinoic acid; RAR, retinoic acid receptor; RXR, retinoid X receptor; RIP-C', C-terminal domain of RIP140; RIP140, receptor-interacting protein 140.

Received for publication April 2, 2002. Accepted for publication July 23, 2002.


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NURSA Molecule Pages Link:

Nuclear Receptors:   RARα  |  RXRβ
Coregulators:   RIP140  |  GRIP1
Ligands:   all-trans-Retinoic acid  |  9-cis-Retinoic acid



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