Identification of Critical Residues for Heterodimerization within the Ligand-Binding Domain of Retinoid X Receptor

Soo-Kyung Lee, Soon-Young Na, Han-Jong Kim, Jaemog Soh, Hueng-Sik Choi and Jae Woon Lee

College of Pharmacy (S.-K. L., H.-J.K., J.W.L.) Department of Biology (S.-Y.N., J.S.) Hormone Research Center (J.S., H.-S.C., J.W.L) Chonnam National University Kwangju 500–757, Korea


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Nuclear receptors regulate transcription by binding to specific DNA response elements as homodimers or heterodimers with the retinoid X receptors (RXRs). The identity box (I-box), a 40-amino acid region within the ligand-binding domains of RXRs and other nuclear receptors, was recently shown to determine identity in the heterodimeric interactions. Here, we dissected this region in the yeast two-hybrid system by analyzing a series of chimeric receptors between human RXR{alpha} and rat hepatocyte nuclear factor 4 (HNF4), a distinct member of the nuclear receptor superfamily that prefers homodimerization. We found that the C-terminal 11-amino acid region of the RXR I-box was sufficient to direct chimeric receptors based on the HNF4 ligand-binding domain to heterodimerize with retinoic acid receptors or thyroid hormone receptors. Furthermore, we identified the hRXR{alpha} amino acids A416 and R421 of the 11-amino acid subregion as most critical determinants of heterodimeric interactions; i.e. mutant HNF4s incorporating only the hRXR{alpha} A416 or R421 heterodimerized with retinoic acid receptor.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
The nuclear receptor superfamily is a group of transcriptional regulatory proteins linked by conserved structure and function (1). The superfamily includes receptors for a variety of small hydrophobic ligands such as steroids, T3, and retinoids, as well as a large number of related proteins that do not have known ligands, referred to as orphan nuclear receptors (2). The receptor proteins are direct regulators of transcription that function by binding to specific DNA sequences named hormone response elements (HREs) in promoters of target genes. Nearly all the superfamily members bind as dimers to DNA elements. While some apparently bind only as homodimers, thyroid hormone receptors (TRs), vitamin D receptor (VDR), retinoic acid receptors (RARs), the peroxisome proliferator-activated receptor, and several orphan nuclear receptors bind their specific response elements with high affinity as heterodimers with the retinoid X receptors (RXRs) (3, 4, 5, 6, 7, 8). Based on this high-affinity binding, such heterodimers have been considered to be the functionally active forms of these receptors in vivo. These heterodimers display distinct HRE specificities to mediate the hormonal responsiveness of target gene transcription, in that distinct HREs are comprised of direct repeats (DRs) of a common half-site with variable spacing between repeats playing a critical role in mediating specificity (2, 9). Accordingly, RARs activate preferentially through DRs spaced by two or five nucleotides, whereas VDR and TR activate through DRs spaced by three and four nucleotides, respectively. RXR-peroxisome proliferator-activated receptor heterodimers as well as RXR homodimers activate preferentially through DRs spaced by one nucleotide (referred to as DR1). In addition to DRs, response elements composed of palindromes as well as inverted palindromes referred to as everted repeats (ER) have been shown to mediate transcriptional activation by RXR-RAR and RXR-TR heterodimeric complexes (9). Such DNA-binding flexibility stands in contrast to the steroid hormone receptors, which bind exclusively as homodimers to inverted repeats (IR) spaced by three nucleotides (10).

Some orphan nuclear receptors bind DNA as homodimers. In contrast with the steroid receptor homodimers, orphan receptor homodimers can bind to both palindromic and DR-response elements. In particular, the hepatocyte nuclear factor 4 (HNF4) binds as a homodimer to DR1 and is a strong constitutive transcriptional activator (11). In contrast with HNF4, homodimers of the chicken ovalbumin upstream promoter transcription factors (COUP-TFs) such as EAR2, EAR3, and ARP1 are potent dominant repressors of both basal transcription and transactivation by several receptors, including RXR, RAR, VDR, and TR (12). Repression of these pathways by COUP-TF is believed to be accomplished in part by direct competitive binding and by the presence of a strong carboxy-terminal repressor domain (13). Interestingly, COUP-TF has an ability to heterodimerize with RXR, thereby titrating RXR into a transcriptionally inactive complex (14).

A dimerization interface has previously been identified within the DNA binding domains (DBDs) of RXRs, RARs, VDR, and TRs that selectively promotes DNA binding to cognate direct repeat HREs (15, 16, 17, 18, 19, 20). An additional dimerization interface that mediates cooperative binding to DNA, referred to as the I-box, has recently been mapped to a 40-amino acid region within the carboxy-terminal ligand binding domains (LBDs) of RAR, TR, COUP-TF, and RXR (21). In contrast to the interface within the DBDs, this dimerization motif promotes cooperative binding with similar efficiency to all three classes of repeats, DR, IR, and ER. The two dimerization domains appear to work in sequence and led to a two-step hypothesis for binding of heterodimers to DNA (2, 21). Accordingly, the LBD dimerization interface initiates the formation of solution heterodimers that, in turn, acquire the capacity to bind to a number of differently organized repeats. However, formation of a second dimer interface within the DBD restricts receptors to bind to DRs.

The I-box sequences are fairly well conserved among a subset of nuclear receptors including HNF4, a member of the homodimer subclass (11). This led us to test whether the I-box region of the HNF4 plays a similar role in the homodimeric interactions and, more importantly, to determine whether differences between the I-box sequences contribute to the preferences of HNF4 for homodimerization and RXR for heterodimerization. To answer these questions without complications associated with HRE binding and the dimerization interface within the DBDs, we exploited the yeast two-hybrid system in which LBDs of nuclear receptors are fused to either the DNA binding domain of the bacterial repressor LexA or the B42 transcriptional activation domain, as previously described (22, 23, 24). In this report, the HNF4 I-box was found to be sufficient for the homodimeric interactions. In addition, an 11-amino acid subregion within the RXR I-box was found to be essential for the heterodimeric interactions. From mutational analyses, the RXR amino acids A416 and R421 of the 11-amino acid subregion were also identified as particularly critical determinants of the heterodimeric interactions.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
The HNF4 I-Box Is Sufficient to Mediate the Homodimeric Interactions
To characterize dimerization properties of nuclear receptors, we exploited the yeast two-hybrid system that has been previously described (22, 23, 24). In the host strain used in this system, expression of ß-galactosidase (ß-Gal) is controlled by upstream LexA DNA-binding sites (operators). Thus, this yeast strain depends on transcriptional activation by a LexA protein for expression of ß-Gal. A series of nuclear receptors were fused to the full-length LexA repressor or the B42 transactivation domain. As previously described (23, 24), LexA or its fusions to most nuclear receptors are transcriptionally inactive in yeast. Protein-protein interactions between nuclear receptors, however, can bring the B42 transactivation domain to the LexA sites and activate the expression of the ß-Gal construct. The LexA portion of such chimeras contains its own dimerization domain and directs high-affinity binding to the LexA operators as a dimer, regardless of the status of dimerization between nuclear receptors. Therefore, an important merit of this system is the fact that dimerization properties of nuclear receptors can be directly assessed without complications associated with HRE binding. However, it should be noted that the exact nature of these interactions, for instance as to dimer vs. multimer, is not really known.

A dimerization interface referred to as the I-box has recently been mapped to a 40-amino acid region within the carboxy-terminal LBDs of RAR, TR, COUP, and RXR (21). The I-box sequences are moderately conserved among a subset of nuclear receptors including the homodimerizing HNF4, leading us to test whether the HNF4 I-box region plays a similar role in the homodimeric interactions. To this end, we constructed a series of chimeras between HNF4 and RXR. PCR was used to construct B42/HNF, a chimeric protein consisting of the B42 transactivation domain fused to the entire hinge and LBDs (D, E and F, amino acids 106–455) of the rat HNF4 (Fig. 1AGo). Chimeric receptors B42/H/X-298/389, B42/H/X-338/429, B42/X-HHH, B42/H-XXX, and B42/X/H-428/339 were similarly constructed (Fig. 1AGo). Western blot analyses were executed to confirm that expression levels for all the mutants constructed were comparable (data not shown). LexA/HNF4 showed some constitutive transcriptional activity in yeast, which was approximately 3.5-fold higher than that of LexA alone (data not shown). As shown in Fig. 1BGo, coexpression of B42/HNF led to approximately 5.5-fold enhancement of this constitutive activity, faithfully reflecting the HNF4-HNF4 homodimeric interactions in this yeast system. Furthermore, coexpression of B42/H/X-338/429 or B42/X-HHH led to approximately 4- and 7-fold enhancements of the transcriptional activity of LexA/HNF4, respectively. In contrast, the RXR I-box could not substitute the HNF4 I-box for the homodimeric interactions, as shown by inabilities of B42/H/X-298/389, B42/H-XXX, and B42/X/H-428/339 to enhance the transcriptional activity of LexA/HNF4. These results demonstrate that the HNF4 I-box is indeed a sufficient and transferable interaction interface for the homodimeric interactions.



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Figure 1. The I-box Directs Specificity in Dimerization

A, Schematic diagrams for chimeric nuclear receptors. Diagram shows chimeras containing the indicated amino acids derived from RXR (open) and HNF4 (stippled), where the I-box domains are highlighted (dotted in RXR and hatched in HNF4). Numbers refer to the amino acid boundaries of the receptor fragments consisting of the hinge and ligand binding D/E domains, the I-box domain, and the C-terminal F domain. B, Dimerization probed by the yeast two-hybrid system. Host cells in which ß-galactosidase expression is dependent on the presence of a transcriptional activator with a LexA DBD were transformed with plasmids expressing the indicated LexA or B42 chimeras and grown in liquid culture containing galactose, since expression of the B42 chimeras is under the control of the galactose-inducible GAL1 promoter (22). ß-Galactosidase readings were determined and corrected for cell density and for time of development (A415 nm/A600 nm) x 1000/min. Fold-activations by each B42 chimera are calculated by defining the reporter activity in the presence of B42/- as 1. The result is the average of at least six different experiments, and the SDs are less than 5%.

 
As expected, B42/RXR was able to interact with LexA/RAR and LexA/TR (Fig. 1BGo), and similar results were observed with chimeric receptors containing the RXR I-box sequences such as B42/H/X-298/389, B42/H-XXX, and B42/X/H-428/339 (Fig. 1BGo). In these interactions, LexA/RAR, which includes a full-length RAR in contrast to LexA/TR and other LexA chimeras consisting of only LBDs, shows higher ß-Gal activities than LexA/TR, probably due to the AF1 activation domain in the RAR A/B domains (25). Since LexA/TR and LexA/RAR show similar basal activities in yeast, this difference results in higher fold-activations with LexA/RAR than LexA/TR. For instance, coexpression of B42/RXR conferred approximately 44-fold activation to LexA/RAR and approximately 11-fold activation to LexA/TR (Fig. 1BGo). It is noteworthy that incorporation of only the RXR I-box sequences conferred to the resulting chimera the ability to heterodimerize with RAR and TR (Fig. 1BGo, compare interactions with B42/HNF and B42/H-XXX). Thus, we conclude that the RXR I-box was indeed a transferable interaction interface for heterodimerization, as reported previously (21).

Localization of a Critical Region of the RXR I-Box for Heterodimerization
As shown in Fig. 2Go, there is a high degree of sequence homology between the RXR I-box and the HNF4 I-box sequences. Nevertheless, the HNF4 I-box directs homodimerization, while the RXR I-box directs heterodimerization (21). Thus, we set out to dissect the RXR I-box region to identify sequences critical for heterodimerization by sequentially transferring part of the RXR I-box sequences into corresponding regions of the HNF4, or vice versa.



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Figure 2. Amino Acid Sequences of the I-Box Region

The I-box regions of USP (amino acids 429–468), human RXR{alpha} (amino acids 389–428), and rat HNF4 (amino acids 299–338) are shown. Helix 9 (H9) and helix 10 (H10), which were recently shown to constitute an interaction interface in the crystal structure of the RXR LBD (27), as well as the three most C-terminal (7, 8, 9) of the nine heptad repeats previously shown to be involved in dimerization (30–32), are indicated. Sequence homology determined with the NIH BLAST program is indicated between sequences of two receptors, in which + indicates conservative changes. Amino acids that are conserved between USP and RXR but not in HNF4 are shaded. Three artificial blocks designated to facilitate subsequent chimera constructions are boxed.

 
Examination of the I-box sequences reveals a high level of conservation between RXR and its Drosophila counterpart ultraspiracle (USP) (26), which heterodimerizes with many different members of the superfamily. Therefore, amino acids of the I-box critical for heterodimerization should be conserved between RXR and USP (Fig. 2Go). To facilitate analyses, we artificially divided the I-box region into three separate subregions each containing two to four residues that are conserved betwen RXR and USP but not in HNF4 (shaded in Fig. 2Go). These subregions were sequentially exchanged between B42/HNF and B42/X/H-428/339, resulting in a series of eight B42-fusion constructs shown in Fig. 3AGo. Western blot analyses were executed to confirm that expression levels for all the mutants constructed were comparable to each other (data not shown).



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Figure 3. An 11-Amino Acid Subregion of the RXR I-Box Directs the Heterodimeric Interactions

A, Schematic diagrams for chimeric nuclear receptors. Diagram shows chimeras containing the indicated amino acids derived from RXR (dotted) and HNF4 (hatched). Three smaller areas (stippled) around the three blocks subjected to exchanges are identical between RXR and HNF4, as shown in Fig. 2Go. B, Dimerization properties of each chimera with LexA/HNF4, LexA/TR, and LexA/RAR were probed by the yeast two-hybrid system, as described in Fig. 1BGo. Fold-activations by each B42 chimera are calculated by defining the reporter activity in the presence of B42/- as 1. The result is the average of at least six different experiments, and the SDs are less than 5%.

 
As shown in Fig. 3BGo, two things were evident from analyses of these chimeric receptors in the yeast two-hybrid system (22, 23, 24). First, none of the chimeric receptors was able to enhance the constitutive transcriptional activities of the LexA/HNF4, demonstrating that substitution of any of these HNF4 subregions with that of the RXR I-box blocked homodimerization. This suggests that important residues for the homodimeric interactions are present throughout the entire HNF4 I-box. Second, the C-terminal 11-amino acid subregion of the RXR I-box was sufficient to direct chimeric receptors to heterodimerize with RARs or TRs; i.e. B42/H-HHX, B42/X-HHX, B42/H-XHX, and B42/X-XHX efficiently interacted with LexA/RAR and LexA/TR (Fig. 3BGo).

Identification of Critical Amino Acids Responsible for Heterodimerization
As shown in Fig. 4Go, the 11-amino acid subregion of RXR is identical to that of HNF4 with the exception of five residues (RXR A416, K417, R421, A424, and R426). Among these, RXR K417, A424, and R426 (indicated by arrowheads in Fig. 4Go) are conserved changes from HNF4: i.e. HNF4 has glutamic acid, threonine, and glutamine (HNF4 E327, T334, and Q336) at these positions. However, RXR A416 and R421 (shaded in Fig. 4Go) that are conserved in USP are nonconserved changes from HNF4: i.e. HNF4 has glycine and leucine (HNF4 G326 and L331) at these positions. In contrast, COUP-TFs have glycine and arginine, and RARs and TRs have proline and lysine at these positions (Fig. 4Go). Accordingly, these two residues were identified as primary candidates for determining the heterodimerizing properties of the RXR I-box and subjected to mutational analyses.



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Figure 4. Amino Acid Sequences of the 11-Amino Acid Subregion of Various Receptors

Rat HNF4 (amino acids 326–336), human RXR{alpha} (amino acids 416–426), USP (amino acids 456–466), human COUP-TF (amino acids 363–373), human RAR{alpha} (amino acids 375–385), and rat TRß (amino acids 419–429) are shown. Two amino acids of this subregion that are conserved between USP and RXR but not in HNF4 are shaded. Three amino acids that are conserved changes between RXR and HNF4 are indicated as open arrows.

 
As shown in Fig. 5AGo, we introduced a series of point-mutations into the 11-amino acid subregion in the context of HNF4 resulting in three quadruple mutants (AR-AR, AR-KR, and AR-KA), three triple mutants (AR-K, AR-A, and AR-R), two double mutants (AR and PR), and three single mutants (A326, P326, and R331). Interactions of these point-mutants with LexA/HNF4 were largely unaffected among single, double, and triple mutants with an exception of AR-K, as shown in Fig. 5BGo. All the quadruple mutants were not able to interact with LexA/HNF4 (Fig. 5BGo). In contrast, quadruple mutants AR-KA and AR-KR showed relatively strong interactions with LexA/RAR (Fig. 5BGo). Quaduple mutant AR-AR, all the triple mutants, double mutant AR, and, most surprisingly, two single mutants (A326 and R331) were also able to interact with LexA/RAR. These interactions were relatively weak, but highly specific, as these mutants did not show any interactions with LexA/GR either in the presence or absence of its ligand, deoxycortisol (data not shown). Addition of the RAR ligands, all-trans-retinoic acid or 9-cis-retinoic acid, did not affect the interactions (data not shown). The inherently weaker interactions of LexA/TR with the heterodimerizing chimeras, however, were not evident with these point-mutants. In contrast, as expected from their RAR-based mutations, P326 and PR that incorporate both P326 and R331 were not able to stimulate ß-galactosidase activities above that of B42/HNF when coexpressed with LexA/RAR (Fig. 5BGo). Western blot analyses were executed to confirm that the expression levels for all the mutants tested were indeed comparable (Fig. 5CGo and data not shown).



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Figure 5. Point Mutants of the 11-Amino Acid Subregion of the HNF4

A, The HNF4 G326, E327, L331, T334, and Q336 subjected to point mutagenesis are as indicated (conserved changes by open arrows and nonconserved changes by closed arrows, determined with the NIH BLAST program). All the point mutants are identical to B42/HNF except the indicated amino acid changes. Dashed positions are identical to HNF4 residues. B, Dimerization properties of point mutants with LexA/HNF and LexA/RAR were probed by the yeast two-hybrid system, as described in Fig. 1BGo. All the point mutants are identical to B42/HNF except the indicated amino acid changes. Fold-activations by each B42 chimera are calculated by defining the reporter activity in the presence of B42/- as 1. The result is the average of at least six different experiments, and the SDs are less than 5%. C, Western blot analysis of the yeast strain EGY48 transformed with plasmids encoding indicated B42 chimeras was executed as described (23). All the point mutants are identical to B42/HNF except the indicated amino acid changes. Equivalent amounts (10 µg) of crude extracts for these samples as well as null extracts were examined using an antibody directed against Flu-tag, as described previously (22).

 
To confirm the interactions observed in yeasts, we incubated a glutathione S-transferase (GST) alone, GST-HNF, GST-RXR, GST-H-HHX, GST-AR, GST-A326, or GST-R331 with TR-LBD labeled with [35S]methionine by in vitro translation. All the point-mutants were identical to GST-HNF except the indicated amino acid changes. As shown in Fig. 6Go, TR-LBD bound specifically to GST-RXR and GST-H-HHX, but not to GST-HNF or GST alone, independently confirming the yeast results. In addition, TR-LBD was able to show relatively week but specific binding to GST-AR, GST-A326, and GST-R331. In yeasts, these weak interactions were only evident with LexA/RAR (Fig. 5Go), probably due to the AF1 activation domain in the A/B domains (25). As expected, luciferase labeled with [35S]methionine interacted with none of the GST proteins. Similar results were also obtained with RAR-LBD labeled with [35S]methionine (data not shown). Overall, these results, along with the yeast results, indicate that the RXR amino acids A416 and R421 are most critical to determine identity in the heterodimeric interactions.



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Figure 6. Pull-Down Assays

Luciferase and TR-LBD labeled with [35S]methionine by in vitro translation were incubated with glutathione beads containing GST alone, GST fusions to HNF4-LBD (GST/HNF), RXR-LBD (GST/RXR), H-HHX (GST/H-HHX), A326 (GST-A326), R331 (GST/R331), and AR (GST/AR). All the point mutants are identical to HNF4-LBD except the indicated amino acid changes. Beads were washed, and specifically bound material was eluted with reduced glutathione and resolved by SDS-PAGE.

 

    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
A dimerization interface has been identified within the DNA binding domains (DBDs) of RXRs, RARs, VDR, and TRs that selectively promote DNA binding to cognate direct repeat HREs (15, 16, 17, 18, 19, 20). Another dimerization interface referred to as the I-box has recently been mapped to a transferable 40-amino acid region within the carboxy-terminal LBDs of RAR, TR, COUP-TF, and RXR, which mediates cooperative binding to DNA (21). In contrast to the interface within the DBDs, this dimerization motif promotes cooperative binding with similar efficiency to all three classes of repeats, DR, IR, and ER. In the recently described crystal structure of the RXR LBD (27), a rotationally symmetrical dimerization interface was formed mainly by helix 10 (H10) and, to a lesser extent, helix 9 (H9) and the loop between helix 7 (H7) and helix 8 (H8). The I-box nicely overlaps with H9 and H10 (Fig. 2Go), whose sequences are well conserved among a subset of nuclear receptors including HNF4 (11) and COUP-TF (12) that prefer to homodimerize. In addition, the crystal structures of RAR and TR LBDs (28, 29) demonstrate that the I-box regions adopt a structure similar to that of the RXR (27) in an overall common fold that can be summarized as an {alpha}-helical antiparallel sandwich composed of 12 {alpha}-helices (H1-H12). As expected from this common fold and the conservation of the I-box sequences, the HNF4 I-box was expected to play an important role in the HNF4-HNF4 interactions. As shown in Fig. 1Go, the HNF4 I-box region is indeed sufficient for the homodimeric interactions (see the results with B42/H/X-338/429 and B42/X-HHH in Fig. 1Go). However, residues critical for homodimerization are clearly different from those for heterodimerization, since the HNF4-HNF4 interactions were not affected by any of the HNF4 point-mutations replacing the two residues found to be critical for the heterodimeric interactions (Fig. 5Go). One potential explanation of the inability of B42/H-HHX and similar mutants (shown in Fig. 3Go) to interact with LexA/HNF4 is that the overall structure of the chimeras is disrupted. However, this is ruled out by the ability of the chimeras to interact with LexA/RAR (Fig. 3Go).

The results described here define an 11-amino acid subregion of the I-box as a major determinant of dimerization specificity. This is consistent with the fact that this subregion is contained within H10 (Fig. 2Go), a major interface in the recently described crystal structure of the RXR LBD (27) and overlaps with heptad 9 previously shown to be critical for both homo- and heterodimerization of RXR (30, 31, 32). We also found that a chimeric RAR construct containing the RXR 11-amino acid subregion readily interacted with RAR, lending further support for the importance of this subregion for the heterodimeric interactions (our unpublished results). Within this subregion, RXR amino acids A416 and R421 are particularly critical, consistent with the fact that these two residues are conserved between RXR and USP, but not in the homodimerizing HNF4. As shown in Fig. 4Go, HNF4 has glycine and leucine, while COUP-TF has glycine and arginine at these positions, respectively. In contrast, RAR and TR, heterodimeric partners of RXR, have proline and lysine at these positions. As expected, mutant HNF4s incorporating only the RXR A416 or R421 heterodimerized with RAR. However, these and other HNF4 point-mutants we constructed showed distinct interactions with different receptors. R331, which resembles COUP-TF at the two target positions, weakly interacted with LexA/RXR (data not shown), consistent with a recent finding in which COUP-TF was shown to have an ability to heterodimerize with RXR (14). In contrast, PR, which resembles RAR and TR at these positions, did not interact with LexA/RXR (data not shown). Similarly, AR with the RXR sequences at these two positions interacted less efficiently with LexA/RAR than single mutants A326 or R331 (Fig. 5BGo). Overall, these results suggest that the two residues (RXR A416 and R421) are clearly important for identity in heterodimerization. Finally, it should be noted that changes of these two residues alone were not sufficient to switch specificity in the dimeric interactions (i.e. from homodimerization to heterodimerization). The change of specificity in dimerization was achieved with at least three mutations incorporated, as shown by AR-K, which weakly interacts with LexA/RAR, but not with LexA/HNF (Fig. 5Go). Similarly, all the quadruple mutants changed specificity in dimerization (Fig. 5BGo; compare results with LexA/HNF4 and LexA/RAR). It is also noteworthy that the effects of substitutions with the corresponding RXR sequences on the heterodimerizing potential of the resulting HNF4 chimeras are largely accumulative: i.e. A326, R331, AR, AR-K, AR-A, AR-R, and AR-AR show relatively weak fold-activations with LexA/RAR (3- to 7-fold, as shown in Fig. 5BGo), while the quadruple mutants AR-KR and AR-KA, as well as the quintuple mutant B42/H-HHX (Fig. 3BGo), which incorporates all the five RXR amino acids of the 11-amino acid subregion, showed much stronger fold activations with LexA/RAR (23-, 21-, and 43-fold activations, respectively). Accordingly, identity seems to be determined in the context of the overall structures in relation to neighboring residues, rather than solely by nature of the two residues. In addition, it will be interesting to test effects of the identified mutations in the context of full-length receptors.

In conclusion, we have identified an 11-amino acid subregion of the RXR I-box as a critical domain for the heterodimeric interactions and specifically identified two residues in this subregion as determinants of heterodimerization. Recently, we introduced random mutations into these two residues of RXR in an effort to make mutant RXR more selective in the process of heterodimerization. Consistent with the importance of these residues in heterodimerization, we were indeed able to find a series of mutant RXRs with significantly altered specificity (S.-K. Lee and J. W. Lee, unpublished). To understand in detail how these two residues fit into the overall structure of the I-box in each nuclear receptor and how they direct specificity in dimerization, more mutational studies as well as more structural information will be required.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Hormones
Deoxycortisol, 9-cis-retinoic acid, and all-trans retinoic acid were obtained from Sigma Chemical Co (St. Louis, MO).

Yeast Cells, Plasmids, and Expressions
EGY48 cells [MAT{alpha}leu2-his3-trp1-ura3-LEU2::pLexA-op6 LEU2({Delta}UASLEU2)], the lexA-ß-galactosidase reporter construct, and the LexA- and B42-parental vectors were as reported (22). LexA or B42 fusions to the full-length RAR and the LBDs of GR, RXR, HNF4, and TR were as previously described (23, 24). Two subsequent PCR steps according to a strategy that has been described (33) were employed for all the following plasmid constructions, by using Vent Polymerase (New England Biolabs, Beverly, MA) capable of proofreading to minimize unwanted mutations. A forward vector primer (B42 primer; 5'-GCC TCC TAC CCT TAT GAT-3') plus a reverse mutagenic primer (5'-CTT GAT CTT CCC GGG GTC ACT CAG-3') and a forward mutagenic primer (5'-CTG AGT GAC CCC GGG AAG ATC AAG-3') plus a reverse vector primer (ADH primer; 5'-GAC AAG CCG ACA ACC-3') were used to amplify B42/HNF-wild type as a template DNA at annealing temperature of 55 C. Both PCR products were mixed in an equal molar ratio and amplified using the B42 and ADH vector primers. Accordingly, B42/HNF includes a SmaI site just upstream of the HNF4 I-box, in which codons for amino acids Pro 296 (CCA) and Gly 297 (GGC) were silently changed to CCC (Pro) and GGG (Gly) to facilitate further subclonings. B42/H/X-298/389 encodes rat HNF4 amino acids 106–298 followed by human RXR{alpha} amino acids 389 to 462 to the C terminus. Similarly, B42/H/X-338/429 encodes HNF4 amino acids 106–338 followed by RXR amino acids 429–462 to the C-terminus. B42/X-HHH is identical to B42/RXR except that RXR amino acids 389–428 were replaced by HNF4 amino acids 299–338. B42/H-XXX is identical to B42/HNF except that HNF4 amino acids 299–338 were replaced by RXR amino acids 389 to 428. B42/X/H-428/339 encodes RXR amino acids 198–428 followed by HNF4 amino acids 339–455 to the C terminus. B42/H-XHH is identical to B42/HNF except that HNF4 amino acids 299- 308 were replaced by RXR amino acids 389–398. Similarly, B42/H-HXH is identical to B42/HNF except that HNF4 amino acids 312–322 were replaced by RXR amino acids 402–412, while B42/H-HHX is identical to B42/HNF except that HNF4 amino acids 326–336 were replaced by RXR amino acids 416–426. B42/H-XHX is identical to B42/H-HHX except that HNF4 amino acids 299–308 were further replaced by RXR amino acids 389–398. B42/X-XHH, B42/X-HXH, B42/X-HHX, and B42/X-XHX are identical to B42/H-XHH, B42/H-HXH, B42/H-HHX, and B42/H-XHX, respectively, except that HNF4 amino acids 106 and 299 were replaced by RXR amino acids 198–389. A326 and P326 are identical to B42/HNF except that amino acid Gly 326 was replaced by Ala and Pro, respectively. R331 is identical to B42/HNF except that amino acid Leu 331 was replaced by Arg. AR and PR are identical to R331 except that amino acid Gly 326 was further replaced by Ala and Pro, respectively. AR-R, AR-A, and AR-K are identical to AR except that amino acids Gln 336, Thr 334, and Glu 327 were replaced by Arg, Ala, and Lys, respectively. AR-AR is identical to AR-A except that amino acid Gln 336 was replaced by Arg. Similarly, AR-KR and AR-KA are identical to AR-K except that amino acid Gln 336 and Thr 334 were further replaced by Arg and Ala, respectively. All the constructs described here were sequenced to prevent any unwanted PCR mutations. In addition, expression levels of all the B42 chimeras were determined using a monoclonal antibody directed against Flu-tagging (gift of Dr. Kyung-Lim Lee) that resides just upstream of the B42 transactivation domain, as described (22). HNF4 sequences from B42/HNF, B42/RXR, B42/H-HHX, A326, R331, and AR were transferred to pGEX4T1 (Pharmacia, Piscataway, NJ) to express GST fusions. Vector for in vitro translation of the TR-LBD was as previously described (24).

Yeast ß-Galactosidase Assays
The cotransformation and transactivation assays in yeast were performed as described previously (23). Quantitative liquid ß-galactosidase assays were performed with the following changes as described (23). The yeast culture was initially diluted to an A600 nm of 0.05 and plated into 96-well culture dishes with the various concentrations of hormone. The cultures were then incubated in the dark at 30 C for 16 h. The A600 nm was determined, and then cells were lysed and substrate was added and A415 nm was read after 10–30 min. The normalized galactosidase values were determined as follows: (A415 nm/A600 nm) x 1000/min developed. For each experiment, at least six independently derived colonies expressing chimeric receptors were tested.

Pull-Down Assays
GST fusion proteins were produced in Escherichia coli and purified using glutathione-Sepharose affinity chromatography essentially as described (24). GST proteins were bound to glutathione-Sepharose 4B beads (Pharmacia) in binding buffer (50 mM KPO4, pH 6.0, 100 mM KCl, 10 mM MgCl2, 10% glycerol, 10 mg/ml E. coli extract, and 0.1% Tween 20). Beads were washed once with binding buffer and incubated for 60 min at 4 C in the same buffer with equivalent amounts of various proteins labeled with [35S]methionine by in vitro translation. Nonbound proteins were removed by three washes with binding buffer without E. coli extract, and specifically bound proteins were eluted with 50 mM reduced glutathione in 0.5 M Tris, pH 8.0. Eluted proteins were resolved by PAGE and visualized by fluorography.


    ACKNOWLEDGMENTS
 
We thank Dr. Kyung-Lim Lee for Flu-antibody and Drs. Wongi Seol and David D. Moore for rat HNF4 cDNAs and critical reading of this manuscript.

This research was supported by grants from KOSEF (96–0401-08–01-3 and Hormone Research Center) and Chonnam National University.


    FOOTNOTES
 
Address requests for reprints to: Jae Woon Lee, Ph.D., College of Pharmacy, Chonnam National University, Kwangju, South Korea 500–757.

Received for publication June 27, 1997. Revision received November 24, 1997. Accepted for publication November 26, 1997.


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 RESULTS
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 MATERIALS AND METHODS
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