Protein-Protein Interaction Domains and the Heterodimerization of Thyroid Hormone Receptor Variant {alpha}2 with Retinoid X Receptors

Yifei Wu, Ying-Zi Yang and Ronald J. Koenig

Division of Endocrinology and Metabolism University of Michigan Medical Center Ann Arbor, Michigan 48109-0678


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Heterodimerization between thyroid hormone receptors (TRs) and retinoid X receptors (RXRs) is mediated by a weak dimerization interface within the DNA- binding domains (DBDs) and a strong interface within the C-terminal ligand- binding domains of the receptors. Previous studies have shown that the conserved ninth heptad in the TR ligand-binding domain appears to play a critical role in heterodimerization with RXR. However, despite lacking the full ninth heptad, TR variant {alpha}2 (TRv{alpha}2) can heterodimerize with RXR on specific direct repeat response elements, but not on palindromic elements or in solution. Two possibilities may account for TRv{alpha}2-RXR heterodimerization on direct repeats. First, the DBD of TRv{alpha}2 may play a critical role in heterodimerization with RXR. Second, a specific sequence within the unique C terminus of TRv{alpha}2 may promote the formation of TRv{alpha}2-RXR heterodimers. In this study, we used receptor chimeras in which the DBD of RXR was replaced by either the TR DBD or an unrelated DBD from the metalloregulatory transcription factor AMT1 to address the role of the DBD dimerization interface in TRv{alpha}2-RXR heterodimerization. Gel mobility shift analyses showed that whereas TR{alpha}1 formed heterodimers with these chimeras, TRv{alpha}2 failed to do so. Deletion of the unique C terminus of TRv{alpha}2 had only a marginal effect on heterodimerization with RXR. Mutations within the DBD dimerization interface abolished heterodimerization of full-length TRv{alpha}2 with RXR but only marginally affected heterodimerization of full-length TR{alpha}1 with RXR. These data support the hypothesis that the TR-RXR DBD dimerization interface plays a critical role in TRv{alpha}2-RXR heterodimerization. Additional data show that the amino acid residues that make direct TR-RXR contacts within the DBDs also may play a role in receptor monomer binding to DNA, since mutations within these residues severely impair this interaction.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
The thyroid hormone receptors (TRs), as well as the receptors for retinoic acid and vitamin D, belong to the nuclear receptor superfamily of ligand-dependent transcription factors that control diverse aspects of development and cellular homeostasis (1). Transcriptional regulation by TRs occurs via binding to thyroid hormone response elements (TREs) in the promoter regions of target genes. TREs are usually composed of two or more half-sites related to the sequence AGGTCA, arranged as direct repeats with a 4-bp spacer (DR4) (2, 3), inverted repeats with no spacer (4, 5), or everted repeats with a 6-bp spacer (4, 6). Although TRs are capable of binding to TREs as monomers or homodimers, TRs preferentially bind to most TREs as heterodimers with retinoid X receptors (RXRs) (7, 8, 9).

Extensive structural and functional analyses have revealed that the nuclear receptors contain functionally separable domains for DNA binding and hormone binding. The DNA-binding domain (DBD) contains two zinc fingers and is highly conserved among members of the superfamily (1). The C-terminal region of the receptors is functionally complex, performing a variety of activities including ligand binding, receptor dimerization, and hormone-dependent transcriptional activation or repression (1). It is known that heterodimerization between TR and RXR is mediated by distinct dimerization interfaces within the DBDs and the C-terminal ligand-binding domains (LBDs) of the receptors (10, 11). The dimerization interface between the DBDs was elucidated through crystallographic analysis (10). In general, the TR-RXR interaction mediated through DBDs is believed to be relatively weak, since DBDs alone cannot form heterodimers in solution. However, the DBD interface specifies the requirement for the spacer of direct repeat response elements (12, 13). In contrast to this weak DBD interface, the dimerization domains within the C-terminal LBDs of TR and RXR have been shown to be the major heterodimerization interface, since these domains allow for the formation of dimers in solution before DNA targeting. Although the dimerization interface appears to involve multiple regions of the C-terminal LBD (11, 14, 15), a major role of the conserved ninth heptad repeat [TR{alpha}1 amino acids 367–374, part of helix 11 in the crystal structure of the TR LBD (16)] in heterodimerization with RXR has been documented by mutational analysis (11).

TR variant {alpha}2 (TRv{alpha}2) is an alternative splice product of the TR{alpha} gene and is widely expressed (17). TRv{alpha}2 lacks a full ninth heptad and possesses a unique 122-amino acid C-terminal sequence in place of TR{alpha}1 amino acids 371–410 (Fig. 1Go). As TRv{alpha}2 is missing the C-terminal 40 amino acids of the TR{alpha}1 LBD, it is not surprising that TRv{alpha}2 does not bind T3 and is not a functional thyroid hormone receptor. Although the physiological role of TRv{alpha}2 remains unknown, its weak dominant negative activity in transfection systems has been well demonstrated (18). Previous studies have shown that TRv{alpha}2 is capable of forming heterodimers with RXR on specific DR response elements in which a thymidine is present two nucleotides 5' to the downstream hexameric half-site (TNAGGTCA) (19, 20, 21). Although many natural TREs do not have a thymidine in this position, others do (such as TREs in the spot 14 and human 5'-deiodinase type I genes). In contrast, TRv{alpha}2-RXR heterodimers are not detected on inverted repeat or everted repeat elements and are only marginally detectable in solution. It is unclear why TRv{alpha}2 retains the ability to heterodimerize with RXR on DRs. Two possibilities may account for this. First, the DBD of TRv{alpha}2 may play a critical role in heterodimerization with RXR. Second, a specific sequence within the unique C terminus of TRv{alpha}2 may promote the formation of TRv{alpha}2-RXR heterodimers.



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Figure 1. Schematic Diagram of TR{alpha}1 and TRv{alpha}2

These two proteins are identical for their first 370 amino acids but then diverge completely. The sequences of the full ninth heptad (amino acids 368–374) in TR{alpha}1 as well as the truncated ninth heptad (amino acids 368–370) in TRv{alpha}2 are shown and underlined. Numbers indicate the amino acid positions in the receptors.

 
In this report, we present evidence in support of the former hypothesis. Moreover, we show that the amino acid residues within the DBD that make direct TR-RXR contacts also may play an important role in receptor monomer binding to DNA.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Mutations within the Dimer Interface of the TR or RXR DBD Severely Impair the DNA Binding of TR or RXR Monomers
Previous studies have revealed that TRv{alpha}2 can form heterodimers with RXR on a subset of DRs that contain a downstream octameric TR-binding half-site (TNAGGTCA) (19, 20, 21). It also has been shown that RXR in RXR-TR heterodimers occupies the 5'-half-site of a DR4 response element (10, 12, 13). Based on the three-dimensional crystal structure of the RXR-TR DBDs bound to a DR4, the amino acid residues that make the dimer interface within the DBDs of TR and RXR have been defined (10). Three arginine residues from the second zinc finger of RXR interact with three amino acids from around the TR first zinc finger as well as one amino acid in a more C-terminal region of the DBD known as the T box. These residues were mutated to alanines singly or in clusters (Fig. 2Go) to assess whether the DBD dimerization interface is required for the formation of TRv{alpha}2-RXR heterodimers on the octamer-containing response element 8DR4 (Table 1Go).



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Figure 2. Schematic Diagram of Alanine Substitutions within the TR{alpha} and RXR{alpha} DBDs

The amino acid sequences of the second zinc finger of RXR{alpha} as well as the first zinc finger and T box of TR{alpha} are shown. The amino acid residues that make the DBD dimer interfaces, as demonstrated by the crystal structure of TR-RXR DBDs bound to a DR4 (10 ), are underlined. The alanine replacements individually or in clusters are shown. Numbers indicate the amino acid positions in the receptors.

 

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Table 1. Oligonucleotide Probes Used in EMSAs

 
The wild-type and mutated DBDs were expressed either in rabbit reticulocyte lysate or in Escherichia coli. In vitro synthesized DBD proteins and a radiolabeled DNA probe (8DR4) were used to perform electrophoretic mobility shift assays (EMSAs). EMSAs revealed that the wild-type TR{alpha} DBD and RXR{alpha} DBD could bind to 8DR4 as monomers (Fig. 3Go, lanes 2 and 3) or as heterodimers (lane 7). Surprisingly, using reticulocyte lysate-translated receptors, all mutant RXR{alpha} DBD proteins showed virtually no binding to 8DR4 as monomers (Fig. 3Go, lanes 4–6) or as heterodimers with the wild-type TR{alpha} DBD (lanes 8–10). Similar results were observed with bacterially expressed proteins (data not shown). Furthermore, EMSAs revealed that the mutant TR{alpha} DBDs also have greatly diminished DNA binding as monomers (Fig. 4Go, lanes 2–4) or as heterodimers with the wild-type RXR{alpha} DBD (lanes 7–9). The observed loss of monomer binding to DNA complicates the use of full-length receptors bearing these DBD mutations to study the role of the DBD interaction in TRv{alpha}2-RXR heterodimerization, because loss of heterodimer-DNA complexes could simply reflect defective TR{alpha} or RXR{alpha} monomer-DNA binding. Furthermore, the loss of receptor monomer-DNA binding makes it difficult to prove that these mutations do indeed disrupt DBD heterodimerization on DNA.



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Figure 3. Impaired Binding of Mutant RXR{alpha} DBDs to the DNA Element 8DR4

A 32P-labeled oligonucleotide probe (8DR4) was incubated with reticulocyte lysate-translated wild-type or mutated RXR{alpha} DBDs in the presence or absence of the TR{alpha} DBD. Nondenaturing PAGE was performed to resolve the protein-DNA complexes. RXR{alpha} DBD peptides: wt, wild-type; 177, R177A; 187, R187A; 2, R2A. M, Monomer-DNA complexes; HTD, heterodimer-DNA complexes.

 


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Figure 4. Impaired Binding of Mutant TR{alpha} DBDs to the DNA Element 8DR4

In vitro translated wild-type or mutant TR{alpha} DBDs were incubated with a 32P-labeled DNA probe (8DR4) with or without the RXR{alpha} DBD. The protein-DNA complexes were resolved by nondenaturing PAGE. TR{alpha} DBD peptides: wt, wild-type; 123, D123A; 2, D2A; 3, 3A. M, Monomer-DNA complexes; HTD, heterodimer-DNA complexes.

 
At least two possibilities may account for the loss of the receptor monomer binding to DNA. First, the mutations within the DBD may disrupt the general conformation of the proteins. Second, the amino acid residues that are proposed to make direct TR-RXR interactions may also contribute to receptor monomer-DNA interactions. To begin to distinguish between these two possibilities, we carried out a proteolytic analysis by treating the wild-type and mutant DBDs of TR or RXR with various proteases. We hypothesized that if the mutations caused gross conformational changes in the DBDs, this would alter their susceptibility to protease digestion. We were particularly interested in three single DBD mutants (TR-D123A, RXR-R187A, and RXR-R177A), since these proteins have mutations in residues that, by crystallography, are not involved in DNA contacts, at least of the TR-RXR DBD heterodimer.

The wild-type and mutant TR{alpha} and RXR{alpha} DBDs were translated in reticulocyte lysate in the presence of both [35S]methionine and [35S]cysteine. The radioactively labeled DBDs were digested with increasing amounts of trypsin, chymotrypsin, or elastase. The digestion products were analyzed by Tricine-SDS-PAGE (22) and visualized by phosphorimager analysis. Figure 5AGo shows that the trypsin digestion patterns of the wild-type and mutant TR{alpha} DBDs are similar. Digestion with chymotrypsin or elastase also failed to reveal differences between the wild-type and mutant proteins (data not shown). Similar digestion patterns between the wild-type and mutant RXR{alpha} DBDs also were obtained after incubation with chymotrypsin (Fig. 5BGo), trypsin, or elastase (data not shown). Thus, this technique failed to provide evidence for gross conformational changes in the mutant DBDs. This suggests that the mutated residues of the DBD dimer interfaces may play a more direct role in protein-DNA interactions, at least for receptor monomers.



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Figure 5. Proteolytic Digestion Analysis of the Wild-Type or Mutated TR{alpha} and RXR{alpha} DBDs

A, In vitro translated [35S]methionine-[35S]cysteine-labeled TR{alpha} DBD was treated with different amounts of trypsin. The digested samples were analyzed by Tricine-SDS-PAGE. B, Similar to panel A, but for chymotrypsin-digested RXR{alpha} DBD. Molecular mass markers (in kilodaltons) are shown.

 
The DBD Interaction Is Necessary for TRv{alpha}2-RXR Heterodimerization on a Direct Repeat Response Element
Since mutations within the DBD dimer interface severely impaired TR{alpha} or RXR{alpha} DBD monomer binding to DNA, full-length receptors with these mutations were not initially used to study the role of the DBD interaction in TRv{alpha}2-RXR heterodimerization. Instead, we constructed receptor chimeras in which the RXR{alpha} DBD was replaced by either the TR{alpha} DBD or an unrelated DBD from the yeast metalloregulatory transcription factor AMT1 (23), referred to as RTR and RMR, respectively (Fig. 6Go). The AMT1 DBD was used because AMT1 binds DNA as a monomer (23). Also, AMT1 is not a zinc finger protein, and therefore it seemed unlikely that the AMT1 DBD would heterodimerize with the TR{alpha} DBD. Similarly, since TR{alpha} could not form homodimers on 8DR4 under our experimental conditions, it also appeared unlikely that the TR{alpha} DBD in the chimeric RTR would contribute to RTR-TRv{alpha}2 heterodimerization. Using RTR or RMR to evaluate the role of the DBD in TR{alpha}-RXR{alpha} heterodimerization would ensure that any observed failure of heterodimerization was not due to impaired DNA binding.



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Figure 6. Schematic Representations of RXR{alpha} and the Chimeric Constructs

The DBDs and LBD are indicated. The boundaries of each domain are labeled above the proteins with numbering referring to amino acid positions of the receptors. To facilitate the construction of RTR and RMR, SphI sites were first introduced into the RXR{alpha} cDNA immediately 5' and 3' to the DBD, creating RXR{alpha}-Sph. The numbers above RXR{alpha}-Sph indicate the positions of introduced SphI sites. The residues labeled with an asterisk reflect the changes from the wild-type RXR{alpha} (given in parentheses) to RXR{alpha}-Sph. TR{alpha} DBD (amino acids 48–150) is represented by a solid black background, and AMT1 DBD (amino acids 1–115) by a hatched box.

 
The full-length receptor proteins were produced in reticulocyte lysate in the presence of [3H]leucine, and the products were analyzed by SDS-PAGE. EMSAs using these in vitro synthesized full-length proteins showed that, as expected, TRv{alpha}2 and TR{alpha}1 are both capable of heterodimerizing with RXR{alpha} (Fig. 7Go, lanes 6 and 9) or RXR{alpha}-Sph (lanes 8 and 11). However, TRv{alpha}2 does not heterodimerize with RTR (lane 7), although TR{alpha}1 does (lane 10). Somewhat unexpectedly, an RXR homodimer band also was visible on this response element (e.g. lane 1).



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Figure 7. Heterodimerization between TRv{alpha}2 and RXR{alpha} Relies on the DBD Interaction

EMSA was performed using [3H]leucine-labeled TR{alpha}1, TRv{alpha}2, RXR{alpha}, RXR{alpha}-Sph, and chimeric RTR produced in reticulocyte lysate, and a 32P-labeled 8DR4 oligonucleotide probe. The amounts of proteins used in the binding reactions were equalized by normalization to [3H]leucine incorporation. M, TR monomer-DNA complexes; HMD, homodimer-DNA complexes; HTD, heterodimer-DNA complexes.

 
To verify these data, gel supershift experiments were performed using a specific anti-TRv{alpha}2 antibody (a generous gift from M. Lazar). Figure 8Go shows that the TRv{alpha}2-RXR{alpha} and TRv{alpha}2-RXR{alpha}-Sph heterodimer complexes were supershifted in the presence of the TRv{alpha}2-specific antibody (lanes 7 and 11), but that no supershifted complexes were observed when TRv{alpha}2 plus RTR was incubated with the antibody (lane 9). This confirms that TRv{alpha}2 and RXR{alpha} heterodimerize, but that TRv{alpha}2 and RTR do not.



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Figure 8. Gel Supershift Analysis Using a TRv{alpha}2-Specific Antibody

To confirm that TRv{alpha}2-RTR-DNA complexes do not form, EMSA incubations of TRv{alpha}2 with RXR, RXR{alpha}-Sph, or RTR were performed in the presence or absence of a specific TRv{alpha}2 antibody. SS, Antibody-supershifted complexes; HMD, homodimer-DNA complexes; HTD, heterodimer-DNA complexes.

 
As shown in Fig. 9Go, TRv{alpha}2 also was unable to heterodimerize with RMR on a DNA probe containing an upstream AMT1 site followed by an optimal TR- binding half-site spaced by 4 bp (lane 5), although RMR-TR{alpha}1 complexes did form on this probe (lane 6). Identical results were observed when the half-sites were spaced by 2 or 6 bp (data not shown).



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Figure 9. Heterodimerization between TRv{alpha}2 and RXR{alpha} Relies on the DBD Interaction

EMSA was performed using [3H]leucine-labeled TR{alpha}1, TRv{alpha}2, and chimeric RMR produced in reticulocyte lysate, and a 32P-labeled AMTTR4 oligonucleotide probe (see Table 1Go). The amounts of proteins used in EMSA were equalized by normalization to [3H]leucine incorporation. M, TR monomer-DNA complexes; HTD, heterodimer-DNA complexes; R, RMR-DNA complexes. Additional lanes not relevant to this experiment were deleted from the autoradiograph to produce this figure.

 
In addition, EMSAs were performed using deletion mutants of TR{alpha}1 and TRv{alpha}2 to assess the role of the unique C terminus of TRv{alpha}2 (amino acids 371–492) in heterodimerization with RXR. Deletion of TR{alpha}1 after amino acid 378, which leaves the full ninth heptad intact, had minimal impact on heterodimerization with RXR (Fig. 10Go, lane 12 vs. 4). Similarly, deletion of TRv{alpha}2 after amino acid 378 had no significant impact on heterodimerization (lane 10 vs. 6). Deletion after amino acid 370, which leaves intact only those residues common to TR{alpha}1 and TRv{alpha}2, also only marginally affected heterodimerization with RXR (lane 14).



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Figure 10. The Effect of C-Terminal Deletion of TR{alpha}1 or TRv{alpha}2 on Heterodimerization with RXR

Full-length or C-terminally deleted TR{alpha}1 and TRv{alpha}2 were incubated with a 32P-labeled DNA probe (8DR4) with or without RXR{alpha}. Protein-DNA complexes were resolved by nondenaturing PAGE. {alpha}1–9H is TR{alpha}1 in which amino acids 371–376 were replaced by TRv{alpha}2 amino acids 371–376. {alpha}2–378 is TRv{alpha}2 with a stop codon immediately after residue 378. {alpha}1–378 has a similarly placed stop codon in TR{alpha}1, and {alpha}-370 has a stop codon after amino acid 370. M, TR monomer-DNA complexes; HTD, heterodimer-DNA complexes. Additional lanes not relevant to this experiment were deleted from the autoradiograph to produce this figure.

 
Finally, EMSAs were performed using full-length TR{alpha}1, TRv{alpha}2, or RXR{alpha} bearing the alanine substitutions within the DBD dimerization interfaces (see Fig. 2Go). In the context of TR{alpha}1, these DBD mutations only minimally impaired TR-RXR-DNA complex formation (Fig. 11AGo, lanes 9–11 vs. 8), but the same mutations in the context of TRv{alpha}2 almost totally disrupted heterodimer-DNA binding (Fig. 11BGo, lanes 7–9 vs. 6). Similarly, mutations of the RXR DBD dimerization interface had no effect on TR{alpha}1-RXR-DNA complex formation (Fig. 11AGo, lanes 12–13 vs. 8), but abolished TRv{alpha}2-RXR-DNA complex formation (Fig. 11CGo, lanes 6–7 vs. 5).



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Figure 11. Effects of DBD Mutations on TR-RXR-DNA Interactions

A, Effect of DBD dimerization interface mutations on heterodimerization of full-length TR{alpha}1 with RXR. EMSA was performed using full-length TR{alpha}1 and RXR{alpha} (with or without DBD mutations) and a 32P-labeled 8DR4 oligonucleotide probe. The TR{alpha}1 and RXR{alpha} mutations are fully described in Fig. 2Go. TR{alpha}1 proteins: wt, wild-type; 123, D123A; 2, D2A; 3, 3A. RXR proteins: wt, wild-type; 177, R177A; 2, R2A. M, TR monomer-DNA complexes; HTD, heterodimer-DNA complexes. B, Effect of TRv{alpha}2 DBD dimerization interface mutations on heterodimerization of full-length TRv{alpha}2 with RXR. EMSA was performed using full-length TRv{alpha}2 and RXR{alpha} (with or without DBD mutations) and a 32P-labeled 8DR4 oligonucleotide probe. The TRv{alpha}2 mutations are fully described in Fig. 2Go. TRv{alpha}2 proteins: wt, wild-type; 123, D123A; 2, D2A; 3, 3A. HMD, homodimer-DNA complexes; HTD, heterodimer-DNA complexes. C, Effect of RXR DBD dimerization interface mutations on heterodimerization of full-length TRv{alpha}2 with RXR. EMSA was performed using full-length TRv{alpha}2 and DBD-mutated RXR{alpha} and a 32P-labeled 8DR4 oligonucleotide probe. The RXR{alpha} mutations are fully described in Fig. 2Go. RXR{alpha} proteins: wt, wild-type; 177, R177A; 187, R187A; 2, R2A. HTD, heterodimer-DNA complexes. Additional lanes not relevant to this experiment were deleted from the autoradiograph to produce this figure.

 
Taken together, these findings strongly indicate that the DBD interactions between TRv{alpha}2 and RXR{alpha} are essential for the formation of RXR-TRv{alpha}2 heterodimers on 8DR4. The C-terminal unique sequence of TRv{alpha}2 appears not to contribute to the dimerization interface. In contrast to TRv{alpha}2, since a strong LBD interface (full ninth heptad) is present in the TR{alpha}1, the TR{alpha}-RXR{alpha} DBD interaction is not necessary for formation of TR{alpha}1-RXR{alpha} heterodimers on these DNA probes.


    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
It is believed that heterodimerization with RXRs is an important mechanism by which TRs exert their biological activities. Mutational and crystallographic analyses have revealed that distinct dimerization interfaces within the DBD and the LBD of the receptors contribute to the formation of TR-RXR heterodimers (10, 11). Mutational analyses have shown that the conserved ninth heptad in the TR LBD is necessary for heterodimerization with RXR (11). In contrast, the DBD interaction is thought to be relatively weak and generally insufficient to support heterodimerization with RXR in the absence of DNA (21).

It has been shown that TR{alpha}1-RXR heterodimer complexes can be formed in solution, whereas TRv{alpha}2 is virtually incapable of heterodimerizing with RXR in solution before DNA targeting (20, 21). This difference reflects the incomplete ninth heptad of TRv{alpha}2 (20). Our data, as shown in Figs. 7Go, 9Go, and 11Go, indicate that TR{alpha}1 requires only the LBD dimerization interface to form heterodimers with RXR{alpha} on DNA; the DBD dimerization interface is dispensable. The fact that, despite lacking the full ninth heptad, TRv{alpha}2 can still form heterodimers with RXR on certain DRs raises the question as to which domain(s) within TRv{alpha}2 contribute to this heterodimerization. Two possibilities may be considered. First, a novel strong dimerization interface may be located in the unique 122-amino acid C-terminal sequence of TRv{alpha}2. Second, the DBD interface of TRv{alpha}2 may promote heterodimerization with RXR. In this study, we used receptor chimeras to examine the role of the DBD interaction in the binding of TRv{alpha}2-RXR heterodimers to 8DR4. The observations that TRv{alpha}2 failed to heterodimerize with the chimeras in which the RXR DBD was swapped with the TR DBD (RTR) or the AMT1 DBD (RMR), whereas TR{alpha}1 did heterodimerize, indicate that: 1) the DBD interaction between TRv{alpha}2 and RXR is necessary for this heterodimerization; and 2) a strong dimerization interface within the unique C-terminal region of TRv{alpha}2 (similar to that of TR{alpha}1) is not likely. To confirm these findings, a series of C-terminal truncation mutants of TR{alpha}1 and TRv{alpha}2 were studied. Previous studies showed that C-terminal deletion of TRv{alpha}2 to amino acid 387, which still contains the truncated ninth heptad, does not impair heterodimerization with RXR (21). However, further deletion to amino acid 347 abolished TRv{alpha}2-RXR heterodimerization. These data do not exclude the possibility that the unique TRv{alpha}2 sequence between amino acids 371–387 may have a critical role in the TRv{alpha}2-RXR heterodimerization, although, clearly, residues C terminal to 387 are not required. Our studies demonstrate that deletion of the entire unique C terminus of TRv{alpha}2 to create TR{alpha}-370 still does not prevent heterodimerization with RXR. This indicates that the region between amino acids 371 and 387 is not essential for the formation of TRv{alpha}2-RXR heterodimers and thus substantiates the critical role of the DBD interface in this process. Perhaps C-terminal deletion to residue 347 abolishes TRv{alpha}2-RXR heterodimerization because this removes the proximal half of the ninth heptad (see Fig. 1Go)

In this model, because a strong LBD interaction between TRv{alpha}2 and RXR is lacking, the binding of TRv{alpha}2 or RXR to the target DNA appears to become an obligatory first step to promote the heterodimerization. The DNA binding, in turn, allows two DBD interfaces to interact. It is interesting to note that the unique C-terminal region of TRv{alpha}2 is phosphorylated by casein kinase II, both when translated in reticulocyte lysate and when expressed within cells (24). This phosphorylation reduces the ability of TRv{alpha}2 to bind DNA as a monomer. The optimized octameric TR binding half-site may be necessary to compensate for this deficiency. Despite the low binding affinity of TRv{alpha}2 monomers to DNA, the cooperative binding of TRv{alpha}2 and RXR to the target DNA greatly promotes the protein-protein interaction and thereby enhances the DNA binding of TRv{alpha}2-RXR heterodimers. Furthermore, the inability of TRv{alpha}2 to heterodimerize with RXR on inverted repeats or everted repeats (19, 20) presumably reflects the inability of the DBD dimer interfaces to interact on these palindromic elements.

It has been demonstrated that the DBD interface plays an important role in discriminating differences in spacing between DR half-sites (12, 13), since the TR DBD can cooperatively and selectively bind to direct repeat elements spaced by 4 bp as a heterodimer with the RXR DBD. Other DBDs impose different spacing requirements. For example, the vitamin D receptor DBD specifies binding to a DR3 element, and the retinoic acid receptor DBD specifies binding to a DR5 (2, 3). Interestingly, we observed that TR{alpha}1 could heterodimerize with RMR on half-sites spaced by 2, 4, or 6 bp. This finding is consistent with the notion that the precise spacing requirements for DR half-sites is intrinsic to the DBD dimer interface, because the formation of TR{alpha}1-RMR heterodimers was solely promoted through the strong LBD dimerization interface, not the DBD interface.

An unexpected observation emerged when point mutations within the DBD dimer interface of TR{alpha} and RXR{alpha} were studied. Our data showed that the ability of DBD monomers to bind DNA was severely impaired or abolished by these substitutions. Based on the crystal structure of the RXR-TR heterodimer DBD bound to a DR4, these amino acids do not make direct contact with the DNA (10). Although some of the mutated residues are involved in water-mediated phosphate contact, others only contribute to the protein-protein interaction in the crystal structure. However, even the mutations that occurred within these latter residues still resulted in the loss of receptor DBD monomer binding to DNA. A trivial explanation for this finding would be global conformational disruption of the receptors by these alanine substitutions. However, proteolytic analysis showed that the digestion patterns of the wild-type DBDs by various proteases are similar to those obtained for the mutant DBDs, suggesting that the mutations may not disrupt the global structure of the receptors’ DBDs. Furthermore, an alignment of amino acid sequences among nuclear receptor superfamily proteins revealed that the alanine substitutions should be tolerable, since alanine and other small hydrophobic amino acids are used by several naturally occurring members of the superfamily. For example, the amino acid residue in the human glucocorticoid or androgen receptor corresponding to mouse RXR{alpha} amino acid R177 is alanine (25, 26). Therefore, these residues may not only participate in the intersubunit interactions, but also may play a role in stabilizing the DNA-protein complexes, at least for monomer-DNA interactions. Understanding receptor monomer-DNA interactions is relevant since several nuclear receptors can bind to DNA and activate transcription as monomers, including SF-1 (27), NGFI-B (27), and TR{alpha}1 (28, 29). However, we cannot exclude the possibility of conformational disruption caused by these alanine substitutions, as proteolytic analysis may not be sensitive enough to detect this. Efforts to study this possibility by nondenaturing PAGE were uninformative (data not shown). Further investigation will be required to better resolve this issue.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Expression of Proteins
Wild-type and mutated DBDs of mouse TR{alpha} (amino acids 40–159) and mouse RXR{alpha} (amino acids 133–228) were expressed in Escherichia coli as glutathione S-transferase (GST) fusion proteins using vector pGEX-KG (a gift from K. Guan) as described previously (30). The fusion proteins were purified by glutathione-Sepharose beads (Sigma Chemical Co., St. Louis, MO), and the receptor DBDs were cleaved from GST with thrombin. To produce the receptor DBDs in reticulocyte lysate, PCR-amplified DNAs encoding the wild-type and the mutated DBDs of TR{alpha} or RXR{alpha} were cloned into the BamHI-SalI sites of pCITE-4a(+) (Pharmacia, Piscataway, NJ). [3H]Leucine-labeled DBD proteins were generated using TNT T7/T3-coupled transcription translation (Promega, Madison, WI). For the generation of the full-length or truncated receptors, TR{alpha}1 (31), rat TRv{alpha}2 (17), RXR{alpha} (9), and chimeric cDNAs (see below), as well as point mutation or deletion mutant cDNAs, were transcribed from pBluescript plasmids and then translated in rabbit reticulocyte lysate (Promega) in the presence of [3H]leucine. Trichloroacetic acid-precipitable protein counts per minute were determined, and the products were analyzed by SDS-PAGE.

Oligonucleotide-Directed Mutagenesis and Constructions of the Chimeric Receptors and Deletion Mutants
The mutants with the alanine substitutions within the dimer interface of the TR{alpha} or RXR{alpha} DBDs (Fig. 2Go) were generated by PCR or the Stratagene QuikChange mutagenesis kit (Stratagene, La Jolla, CA). To construct the RTR and RMR chimeras, two SphI restriction sites flanking the DBD of mouse RXR{alpha} were first created using the Stratagene QuikChange kit. The resultant RXR{alpha}-Sph contained amino acid substitutions at position 134 (S to C) and 234 (D to G), when compared with wild-type RXR{alpha}. Next, PCR-generated DNAs encoding the DBDs of TR{alpha} (amino acids 48–150, with a residue substitution at amino acid 149 from E to R) or AMT1 (amino acids 1–115) (23) were exchanged with the corresponding region of RXR{alpha}-Sph to create RTR or RMR, respectively. The deletion mutants were constructed by insertion of stop codons at the appropriate positions using the Promega Altered Sites kit. To ensure the fidelity of the resulting constructs, all predicted mutations and PCR-based constructs were verified by DNA sequencing.

EMSAs
EMSAs were carried out essentially as previously described with minor modifications (20). Briefly, 3 x 104 cpm of 32P-labeled DNA were incubated with 1–1.5 x 104 cpm of [3H]leucine-labeled, reticulocyte lysate-translated full-length receptors or receptor DBDs; reticulocyte lysate-translated empty vector was used as a control. (Where indicated, 3–15 ng of E. coli-derived receptor DBDs were used.) Incubations were at room temperature for 40 min in a 35-µl reaction mixture containing 20 mM HEPES, pH 7.8, 50 mM KCl, 1 mM dithiothreitol, 0.1% Nonidet P-40, 20% glycerol, and 1.8 µg of poly(deoxyinosinic-deoxycytidylic)acid. After incubation, the samples were electrophoresed through 6% native polyacrylamide gels in 0.25x Tris-borate-EDTA buffer. The gels were dried and then subjected to autoradiography. For antibody supershift studies, 1 µl of antiserum was added to the EMSA reaction mixtures 30 min into the above incubations and then incubated for another 15 min before gel electrophoresis. The oligonucleotide probes used in the EMSA reactions are listed in Table 1Go.

Proteolytic Digestion Analysis
The limited proteolytic digestions were performed as described elsewhere with minor modifications (32). One microliter of 35S-labeled (methionine plus cysteine) DBD proteins (Promega TNT-coupled transcription translation system) was incubated with different amounts of chymotrypsin in 20 µl of reaction buffer (50 mM Tris, pH 7.8; 53 mM CaCl2) at room temperature for 10 min. After incubation, the proteolytic reactions were stopped by the addition of an equal volume of 2x SDS sample buffer and heated for 5 min at 95 C. The samples were analyzed on Tricine-SDS polyacrylamide gels (22). The gels were then incubated in 25% isopropyl-7% acetic acid overnight and vacuum dried. The radiolabeled proteolytic fragments were detected by PhosphorImager (Molecular Dynamics, Sunnyvale, CA) analysis. A similar procedure was used for trypsin or elastase digestion except that the digestion buffer for trypsin was 50 mM Tris, pH 7.5, and for elastase the digestion buffer was 100 mM Tris, pH 8.0. Preliminary experiments were performed with decreasing doses (in 0.5-fold decrements) of all these proteases until no digestion was observed. These experiments did not reveal any bands in addition to the ones seen in the data presented. Also, more prolonged electrophoresis failed to resolve additional bands.


    ACKNOWLEDGMENTS
 
We thank Mitchell Lazar for the TRv{alpha}2 antibody.


    FOOTNOTES
 
Address requests for reprints to: Ronald J. Koenig, M.D., Ph.D., Division of Endocrinology and Metabolism, University of Michigan Medical Center, 5560 MSRB-II, 1150 West Medical Center Drive, Ann Arbor, Michigan 48109-0678. E-mail: RKoenig{at}umich.edu

This work was supported by NIH Grant DK-44155.

Received for publication March 5, 1998. Revision received May 22, 1998. Accepted for publication June 18, 1998.


    REFERENCES
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
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