INTRODUCTION

An important feature of DNA binding by nuclear receptors for thyroid hormone (TR),¹ retinoic acid (RAR), vitamin D (VDR), eicosanoids (PPAR), and a number of orphan receptors is heterodimerization with retinoid X receptor (RXR) (1, 2), which is itself a receptor for 9-cis-retinoic acid (3, 4). In some heterodimers, such as those involving the orphan receptors Nurr1 and LXR (5, 6, 7), ligand binding by RXR contributes to transactivation, whereas in others, such as the RXR·TR and RXR·RAR heterodimer, ligand binding by RXR has a lesser effect on transcription potential (8, 9). In all cases, however, a major role of heterodimerization with RXR is to regulate the affinity and specificity of DNA binding (2).

RXR as well as the nuclear receptors with which it heterodimerizes have inherent affinity for the hexameric sequence AGGTCA. This sequence is recognized by amino acids collectively referred to as the P box within the classical DNA-binding domain (DBD) (10, 11, 12). Both N- (13, 14) and C-terminal (15) regions of the nuclear receptors have additional, important effects upon half-site affinity and specificity. In particular, amino acids immediately adjacent to the carboxyl end of the DBD also play a role in DNA recognition. These T and A boxes (16), also called the C-terminal extension of the DBD (17), increase specificity by making minor groove contacts with DNA sequences 5 to the hexameric core. This is particularly important for orphan receptors that have high affinity for monomeric sites, such as NGFI-B (16), SF1 (18), Rev-Erb (19) and ROR (20). It is also important for monomeric binding by TR, which greatly prefers the dinucleotide TA preceding the AGGTCA motif (21).

RXR heterodimers have selectively increased affinity for direct repeats of the AGGTCA motif (DRs), such that RXR·TR heterodimers have the highest affinity for DRs spaced by 4 base pairs (DR4), while RXR·PPAR, RXR·VDR, and RXR·RAR prefer DRs spaced by 1, 3, and 5 base pairs, respectively (22). Thus, heterodimerization with RXR is one of the most important determinants of the target gene specificity of receptor action. Furthermore, since DRs are inherently asymmetric, RXR heterodimers bind with nonrandom polarity. In the case of TR and RAR, the RXR binds to the upstream half-site, while the receptor partner binds preferentially downstream (23, 24, 25, 26, 27). At least two domains within the nuclear receptors contribute to heterodimerization with RXR. One domain is located within the C terminus of the receptors, in a region that has been referred to as the ninth heptad (28, 29, 30), and is now recognized as helix 11 in the structure of the TR ligand-binding domain (31, 32). This domain within TR and RAR is sufficient for RXR interaction irrespective of DNA binding. A second domain is located within the DBD of the receptors (25, 27, 33). This DBD interaction is relatively weak, but it serves an important function as the molecular determinant of the spacer requirements for selective binding to direct repeats.

TRs are responsible for tissue-specific and developmentally regulated gene expression in response to thyroid hormone (T3). There are two TR genes, and , with multiple isoforms (34). The TR gene encodes TR1, which is a bona fide TR, as well as TR2 and TR3, which are identical to TR1 for 370 amino acids, including the DBD, before diverging within the sequence of heptad 9 (34, 35, 36). The 40 C-terminal amino acids of TR1 that are lacking in TR2 and TR3 include 1) the C-terminal component of heptad 9, which has been shown to be essential for strong interaction with RXR and is highly conserved among RXR-heterodimerizing receptors, 2) amino acids necessary for T3 binding, and 3) an autonomous activation domain at the very C terminus that is highly conserved in the receptor superfamily and mediates the ligand-dependent transcriptional activation and interaction with putative coactivator proteins (37). In place of this important region of TR1, TR2 contains 122 additional amino acids with a unique sequence. TR3 is the result of splicing to an internal acceptor and contains the last 83 amino acids of the TR2 fused to amino acid 370 (Fig. 1). As a result of their lack of the 40 C-terminal amino acids of TR1, both TR2 and TR3 do not bind T3 or activate transcription (38).

Schematic of TR1 and C-terminal TR variants 2 and 3.

The variant isoforms TR2 and TR3 are highly abundant in brain and developmentally regulated (39) and function as inhibitors of T3 action in vitro (40). TR2 has been more carefully studied, and its dominant negative activity appears to be at least in part due to competition with TRs for binding sites in target genes (41, 42). Although other mechanisms may contribute to the inhibitory effects of TR2 (43), point mutations in the DBD that abolish DNA binding by TR2 abrogate its dominant negative properties.²³ DNA binding by TR2 differs from that of TR1 in some important respects. First, phosphorylation of the C terminus of TR2 reduces its monomeric binding affinity (15). Also, TR1 heterodimerizes with RXR on a variety of binding sites, whereas the ability to demonstrate TR2 heterodimerization with RXR has been more variable. TR2 and RXR do not heterodimerize on inverted repeats of AGGTCA, but they have been reported to heterodimerize on some DR4 sites but not others (15, 30, 43, 44).

We previously hypothesized that the differential heterodimerization of TR1 and TR2 is related to their different ninth heptad sequences (Fig. 1) (44). We have now studied RXR heterodimerization of TR1 and TR2 in the context of different DR4-binding sites. TR2, in contrast to TR1, formed stable heterodimers with RXR on only a subset of DR4 sequences. TR2-RXR heterodimerization on DR4 sequences required a downstream half-site that is an optimized TR-binding site (TNAGGTCA). Although the DNA-binding site determined whether TR2 would heterodimerize with RXR, implicating the heterodimerization domain within the DBD, mutation analysis revealed that even in the case of TR2 the ninth heptad was required for selective heterodimerization. Interestingly, TR3 heterodimerized weakly on even nonoptimal DR4s, suggesting that its ninth heptad heterodimerization domain was intermediate in strength between that of TR1 and TR2. Together, the results demonstrate the roles of the DBD and ninth heptad in heterodimerization of TR isoforms and indicate that RXR heterodimerization and DNA binding may be regulated both by strong, DNA-independent protein-protein interactions and weaker protein-protein interactions that are allosterically favored by specific DNA-binding sites.

MATERIALS AND METHODS

The rat TR1 and TR2 cDNAs in pBluescript have been previously described (35). cDNA corresponding to the C terminus of rat TR3 was cloned by reverse transcription-polymerase chain reaction of rat GH3 cell RNA using the following primers: 5-ACATTGGCCAGTCACC-3 and 5-CAAATAACAAGGGAGCTTGG-3.

The polymerase chain reaction product was cut with EcoNI and ligated into the EcoNI site of the TR2 cDNA to produce full-length TR3 cDNA, which was completely sequenced.⁴ The nonphosphorylatable form of TR2 (TR2-SA) in pBluescript has been described previously (15). For creation of C-terminal deleted proteins, TR1 and TR2 were subcloned into the EcoRI and the XhoI/BamHI site of pCMX, respectively. The cDNAs were then digested with a convenient C-terminal restriction enzyme (as indicated below) and NheI, which cuts immediately upstream of multiple stop codons of pCMX. The vector was then blunt-ended using Klenow enzyme and ligated to produce the following truncated proteins: TR2405 (cut with NsiI), TR2387 (cut with Xma3), TR347 (cut with BspMI), and TR250 (cut with EcoNI). HA epitope-tagged-TR1 was created by inserting the EcoRI fragment of TR1 into the MunI site of a T7 promoter-driven expression vector (pGEM Z) encoding the HA epitope (45). HA·TR2 was made by subcloning the unique C-terminal fragment from TR2 pBluescript into the AccI and BamHI of HA·TR1·pGEM Z. RXR in pBluescript was kindly provided by R. Evans. Gal4 DBD (amino acids 1-147) and the Gal4·TR1-(121-410) in pECE have been previously described (41). To construct the Gal4·TR2-(121-492), the following polymerase chain reaction primers were made to isolate TR2 in pBluescript from amino acids 121-492 (missing the DNA-binding domain): 5-AAAGGATCCAAGCCATGGACCTGGTTCTAGA-3 and 5-GTAAAACGACGGCCAGT-3.

Following the polymerase chain reaction, the TR2 fragment was cloned in frame into the BamHI site of pECE containing the Gal4 DBD-(1-147). VP16-RXR fusion protein was kindly provided by K. Chatterjee. The (Gal4 UAS × 5)-SV40-luciferase reporter contained five copies of the Gal4 17-mer binding site and has been previously described (46). Proteins were translated in vitro and labeled with [³⁵S]methionine, using the T3 polymerase TNT reticulocyte lysate transcription and translation system (Promega).

EMSA was performed by incubating 5 µl of each protein at room temperature for 20 min in a 30-µl binding reaction mixture containing 10 mM HEPES, pH 7.9, 80 mM KCl, 5% glycerol, 30 ng of poly(dI-dC), 75 ng of denatured salmon sperm DNA, 10 mM dithiothreitol, 30 ng of bromphenol blue, and 100,000 cpm of ³²P-labeled double-stranded DNA probe. Following incubation, reaction mixtures were loaded on a 5% polyacrylamide gel, dried, and subjected to autoradiography. For off-rate experiments, 5% gels were prerun for 2 h prior to loading. Proteins were preincubated for 1 h with labeled probe at room temperature, and then the first lane was loaded into a running gel. A 500-fold molar excess of unlabeled probe was added to the remaining reaction mix, and equal aliquots were loaded onto the remaining lanes at times indicated in the appropriate figure. For competition experiments, proteins were preincubated for 1 h at room temperature with unlabeled probe at specified -fold excess. Labeled probe was then added and incubated for 20 min at room temperature.

Complementary single-stranded oligonucleotides containing a single copy of a TR-binding site were annealed and labeled with [-³²P]dCTP and used as probes. The sequences of probes used are as follows (hexameric half-sites underlined): Hex-Hex, CCAAGG; Hex-Oct, CCAATA; Oct-Hex, TAAAAA; Oct-Oct, TAAATA. Also, Oct-Oct probes with different spacers were made by varying the third base pair of the spacer.

293T cells were maintained in Dulbecco's modified Eagle's medium supplemented with 10% bovine calf serum and switched to Dulbecco's modified Eagle's medium plus anion exchange resin and charcoal-treated serum 12 h prior to transfection. 60-mm dishes were transfected by the calcium phosphate precipitation method using 1-2 µg of receptor expression vectors, 1 µg of luciferase reporter, and 0.5 mg of -galactosidase expression vector. Cells transfected were lysed in Triton X-100, and cell lysates were subject to luciferase and -galactosidase assays as described previously (46). Results were expressed as -fold activation relative to the control level and normalized to -galactosidase activity, which served as internal control for transfection efficiency.

In vitro translated proteins were incubated in 75 µl of buffer H (20 mM HEPES, pH 7.9, 50 mM KCl, 2 mM EDTA, 0.1% Nonidet P-40, 10% glycerol, 0.5% nonfat dry milk, 5 mM dithiothreitol) in the absence or presence of DNA (2.5 pmol of probe to 45 fmol of protein) for 20 min at room temperature. Samples were then precleared with 50 µl of protein A-Sepharose twice in buffer H for 15 min at room temperature on a rotator. Precleared supernatants were then incubated with 50 µl of protein A-Sepharose prebound to 4 µl of HA antibody for 1 h on a rotator. Reactions were then microcentrifuged for 10 s and then washed four times with ice-cold buffer, boiled in 1 × SDS loading buffer, and separated by electrophoresis on a 10% polyacrylamide gel. Gels were fixed, incubated with En³Hance, dried, and subjected to autoradiography.

RESULTS

TR1 but Not TR2 Interacts with RXR in Solution

We first studied the ability of TR1 and TR2 to heterodimerize with RXR in the absence of TR-binding sites. We have previously shown biochemically that Gal4·TR1 could interact with RXR in a DNA-independent manner (41). This was confirmed using a mammalian two-hybrid assay. All experiments were performed in the absence of T3. Fig. 2A shows that a chimeric protein consisting of the Gal4 DBD fused to the C terminus (amino acids 120-410) of TR1 did not activate transcription from a reporter gene containing five Gal4-binding sites under these conditions (lane 2). Co-transfection of VP16·RXR activated Gal4·TR1 (lane 3), indicating an in vivo interaction between TR1 and RXR. Thus, the C terminus of TR1 was sufficient for this interaction, since in this fusion protein the TR1 DBD is replaced by the Gal4 DBD. In contrast VP16·RXR was unable to stimulate transcription by Gal4·TR2-(120-492) (Fig. 2A, lane 7), indicating that TR2 and RXR did not interact in vivo. In addition, cotransfection of wild-type TR1 was able to inhibit activation of Gal4·TR1 by RXR·VP16 (lane 4), probably by competition for RXR; wild-type TR2 did not inhibit the interaction of Gal4·TR1 and RXR·VP16 (lane 5), again consistent with its inability to heterodimerize with RXR. The inherent ability of full-length TR1, but not full-length TR2, to heterodimerize with RXR was confirmed biochemically using a coimmunoprecipitation assay. In vitro translated ³⁵S-labeled RXR was incubated with HA epitope-tagged TR1 or TR2 prior to immunoprecipitation with HA antibody. Fig. 2B shows that the labeled RXR coprecipitated with HA·TR1 in the presence of HA antibody, whereas HA·TR2 did not (compare lanes 3 and 5). These results showed that TR1 but not TR2 interacts with RXR in solution due to the strong heterodimerization domain in the C terminus of TR1.

TR1 but not TR2 heterodimerizes with RXR in vivo and in vitro.

TR2 and RXR Heterodimerize on DR4 Sequences That Contain an Optimal TR Monomer-binding Site as the Downstream Half-site

The above results were independent of DNA binding by TR·RXR heterodimers. Since TR2 has been shown to heterodimerize with RXR on some but not all TR-binding sites (15, 42), we systematically studied the role of preferred TR monomer-binding sites as the upstream and downstream half-sites of DR4 elements. For these experiments we created DR4 sites with hexameric half-sites (CC, referred to as the ``Hex'' sequence) or optimal TR monomeric half-sites (TA (21), referred to as the ``Oct'' sequence) as one or both half-sites (referred to as Hex-Hex, Hex-Oct, Oct-Hex, and Oct-Oct). Fig. 3 shows that TR1 bound weakly as a monomer to all four sites and bound much more strongly as a heterodimer with RXR to all four of the DR4s with little dependence upon the sequences flanking the AGGTCA hexamer in either of the half-sites. TR2 also bound as a monomer to all four of the DR4s, even more weakly than TR1 monomers, in keeping with our earlier observations that TR2 is phosphorylated in reticulocyte lysate and that phosphorylated TR2 has less DNA binding affinity than nonphosphorylated TR2 (15). RXR alone displayed virtually no binding to these sites.

Selective DNA-dependent heterodimerization between TR2 and RXR.

The striking result of the experiments shown in Fig. 3 was that TR2 and RXR heterodimerized on a specific subset of the DR4s. The TR2·RXR heterodimer bound strongly to the DR4 in which both half-sites were optimized for TR monomer binding (Oct-Oct) but did not bind to the DR4 in which both half-sites were nonoptimal (Hex-Hex). We hypothesized that since TR binds to the downstream half-site of the DR4 (24, 25, 27, 33), it might be the downstream octamer half-site that was of most importance. Indeed, converting the downstream half-site of Oct-Oct to the hexameric sequence (Oct-Hex) abolished TR2·RXR heterodimer binding. In contrast, the TR2·RXR heterodimer bound strongly to the Hex-Oct site, in which only the downstream half-site is optimized for TR. The TR·RXR interaction was clearly cooperative, since TR2 itself had much lower affinity for either half-site, and RXR alone did not bind at all. Thus, the ability of TR2 to cooperatively bind DNA as heterodimer with RXR was allosterically regulated by the DNA-binding site. The simple rule governing this interaction was the requirement for an optimal TR monomer site as the downstream half-site in the context of the DR4.

DNA-dependent Interaction of TR2 and RXR

The role of the DNA-binding site was confirmed in the coimmunoprecipitation assay. Fig. 4 shows that, as shown earlier, RXR coimmunoprecipitated with HA·TR1 in the absence of DNA, whereas it did not coprecipitate with HA·TR2 under the same conditions. TR1-RXR was mildly stimulated by both the nonoptimized DR4 (Hex-Hex) as well as a DR4 with both half-sites optimized for TR binding (Oct-Oct; compare lanes 5 and 7 with lane 3), consistent with previous reports (47). The effect of including DNA in the coimmunoprecipitation was much more striking for TR2-RXR heterodimerization. Inclusion of the Oct-Oct site increased the affinity of TR2 for RXR such that under these conditions RXR was efficiently coimmunoprecipitated with HA·TR2, almost to the level of TR1 (compare lanes 7 and 13). In contrast, the Hex-Hex oligonucleotide had little effect on the interaction between HA·TR2 and RXR, consistent with the DNA binding studies shown earlier.

DNA-dependent heterodimerization of TR2 and RXR.

Fig. 5 shows that a T as the third nucleotide in the spacer (i.e. 2 base pairs 5 to the downstream AGGTCA) was sufficient to promote heterodimerization between TR2 and RXR. Although any nucleotide immediately adjacent to the downstream half-site (i.e. the fourth nucleotide in the spacer) was permissive for heterodimerization, Fig. 5 also shows that a G in this position resulted in the strongest binding of the TR2·RXR. In contrast, monomer binding by TR1 or TR2 and heterodimer binding by TR1·RXR were not nearly as favored by the TG dinucleotide (e.g. compare TR·RXR heterodimer binding in lanes 2 and 4, or TR2 monomer binding in lanes 9 and 11). Thus, the presence of a G adjacent to the AGGTCA specifically favored TR2·RXR heterodimer formation.

Effect of DR4 spacer sequences on TR2 heterodimerization with RXR.

DNA-dependent RXR Heterodimerization of Nonphosphorylated TR2

Phosphorylation of serines 474 and 475 in the C terminus of TR2 greatly reduces its monomeric DNA binding affinity (15). We tested the role of phosphorylation in the mechanism of TR2·RXR heterodimerization by comparing wild type TR2 with a nonphosphorylatable form of TR2 (TR2-SA). Fig. 6, panel A (as well as panel B and Fig. 7) shows that the binding of TR2-SA·RXR heterodimers to permissive DNA-binding sites was greater than that of the wild type TR2 heterodimers. Two potential explanations for this difference in binding were considered: 1) RXR heterodimerized with phosphorylated TR2 had reduced affinity for the Oct-Oct site or 2) RXR heterodimerized with phosphorylated TR2 had very little affinity for the Oct-Oct site, and the binding seen was due to a fraction of nonphosphorylated TR2 in the in vitro translated preparation. To distinguish between these two possibilities, off-rates were determined for the wild type and TR2-SA preparations bound to the Oct-Oct site with RXR. As shown in Fig. 6B, the half-lives were nearly the same in each case, although the initial binding was again greater for the TR2-SA. These results suggest that phosphorylated TR2 bound very weakly as a heterodimer with RXR even to the Oct-Oct site, and the observed heterodimers actually represented a fraction of the in vitro translated preparation that was nonphosphorylated. This is consistent with our observations of variable degrees of DNA binding by TR2 (compared with TR2-SA) related to different batches of reticulocyte lysate (data not shown). In any case, Fig. 6C shows that, as shown earlier for wild type TR2, nonphosphorylated TR2-SA only bound cooperatively as a heterodimer with RXR to the DR4s with the optimized TR monomer sequence as the downstream site, indicating that phosphorylation is not the key determinant of the DNA site specificity of TR2 heterodimerization with RXR.

Effect of the phosphorylation state of TR2 on heterodimerization with RXR.

Role of the ninth heptad in TR2 heterodimerization with RXR.

The Ninth Heptad of TR2 Is Required for DNA-dependent Heterodimerization with RXR

The results thus far indicate that TR1 interacts with RXR in solution, and this preformed heterodimer binds similarly to the Hex-Hex and Oct-Oct sequences. In contrast, TR2 and RXR only heterodimerized on DNA but did so cooperatively in a manner that was highly dependent on the composition of the spacer between the two half-sites. These data were consistent with a major role of the strong ninth heptad dimerization domain in TR1, with the weaker domain located in the DBD of both TR1 and TR2 being more important in TR2. In order to assess whether there was any role of the ninth heptad of TR2, we created a series of C-terminal deletions of TR2 (Fig. 7A) and assayed their ability to heterodimerize on the Oct-Oct binding site. Fig. 7B confirms that TR2-SA·RXR heterodimers bound more strongly than TR2·RXR heterodimers (compare lanes 3 and 5). C-terminal deletion to amino acid 405, which removes the phosphoserines that reduce the DNA binding affinity of TR2, restored the binding of the TR2·RXR heterodimer to the level seen with TR2-SA (compare lanes 5 and 7). Continued C-terminal deletion to amino acid 387, which still contained the ninth heptad region of TR2, did not affect the DNA-dependent heterodimerization on this site (compare lane 9 with lane 5). Note, however, that further deletion into heptad 9 abolished RXR heterodimerization. Parenthetically, deletion to amino acid 347 enhanced TR homodimer formation (compare lane 12 with other even-numbered lanes in which RXR is not present), consistent with the work of others (48) and suggesting an inhibitory role of the C terminus on homodimerization. Nevertheless, both of the C-terminal deletion mutants that lacked heptad 9 (347 and 250) did not heterodimerize with RXR. Thus, the DBD dimerization domain alone was insufficient for even the DNA-dependent RXR heterodimerization of TR2 (or TR1, since all of the C-terminal deletions past amino acid 370 are identical in TR1 and TR2). It is noteworthy in this context that the identification of the DBD dimerization interface has only been demonstrated, both functionally and structurally, at high concentrations of TR and RXR DNA-binding domains. The lack of the C terminus would obliterate potential cis-acting effects of the C terminus on monomer (e.g. by phosphorylation (15)) and dimer (lane 12, and see Ref. 48) binding. The present data suggest that for full-length receptors at lower (and closer to physiological) protein concentrations, the unique ninth heptad of TR2 creates an increased dependence upon the DNA-dependent heterodimerization domain in the DBD but still is required for heterodimerization between TR2 and full-length RXR.

The DNA Dependence of RXR Heterodimerization with TR3 Is Intermediate between That of TR1 and TR2

Although less well studied, TR3 is also a developmentally regulated C-terminal splice product of the TR gene (36, 39, 49). Given the above results suggesting a less dominant but definite role for the ninth heptad of TR2 in permitting the DNA-dependent heterodimerization with RXR, we were interested in comparing the DNA binding and heterodimerization of TR3, which has yet a third ninth heptad sequence. Fig. 8 shows the DNA-dependence of RXR heterodimerization of TR3. Like both TR1 and TR2, TR3 bound strongly with RXR to DR4 sites containing optimal TR monomer-binding sites as downstream half-sites. However, TR3 weakly heterodimerized with RXR on DR4s that lacked optimal TR half-sites downstream. Thus, the characteristics of RXR heterodimerization by TR3 were intermediate between those of TR1 and TR2 in that it was stimulated but not absolutely dependent upon optimization of the spacer.

TR3 heterodimerization with RXR.

DISCUSSION

Heterodimerization with RXR is a major determinant of DNA binding specificity for TR as well as receptors for retinoids, vitamin D, and eicosanoids. In the simplest model, RXR heterodimerization targets TR to DR4 sites, RAR to DR2 and DR5 sites, VDR to DR3 sites, and PPAR to DR1 (2). However, there are clearly additional layers of complexity in heterodimer binding to DRs. For example, the 5-flanking sequence of the upstream half-site in DR1s is critical for PPAR·RXR binding (50). The preferred flanking sequence converts the half-site into a Rev-Erb monomer-binding site (19), consistent with the similarities in the T and A boxes of PPAR and Rev-Erb. We have now shown that a similar preference rule relating to the 5-flanking sequence of the downstream half-site governs TR2 heterodimerization with RXR.

Our understanding of the mechanisms regulating heterodimerization with RXR comes from biochemical and structural studies. Gel shift analyses using full-length receptors have implicated the ninth heptad of the receptors as being necessary for RXR interaction (29, 51, 52). Evidence for this domain is based on mutational analysis, and the regions of direct protein-protein contact are not definitive because the C termini of the receptors have not been co-crystallized with RXR. In contrast, co-crystallization of the TR and RXR DBDs on a DR4 element revealed the importance of the DBD dimerization interfaces (27). This interaction domain can be demonstrated in gel shift assays at high protein concentration (25, 33) but is weak and generally insufficient for heterodimerization in the absence of the ninth heptad in the context of the full-length receptors (44, 51).

Against this background, the comparison of RXR heterodimerization with TR1, TR2, and TR3 is revealing for a variety of reasons. First, these are naturally occurring forms of TR, which are developmentally and tissue-specifically expressed. Second, these proteins have identical DBDs and divergent ninth heptads. The ninth heptad of TR1 is highly similar to those of TR as well as RAR, VDR, and PPAR (28), whereas the C-terminal portion of the ninth heptad regions of TR2 and TR3 are unique. Thus, our results are of biological as well as mechanistic importance. It is not clear why TR3 is intermediate in its RXR heterodimerization, although the ninth heptad sequence comparison shown in Fig. 1 reveals that the major difference between TR2 and TR3 is that TR3 contains an arginine at position 375 that is conserved in TR1 and other RXR-heterodimerizing receptors but is a glutamine in TR2 (53).

The ability of TR1 to heterodimerize with RXR in solution in the absence of DNA is likely to require the ninth heptad, since TR2 does not form heterodimers under the same conditions. Furthermore, the C termini alone were sufficient for interaction in vivo, indicating that the DBD dimerization interface is not required for the heterodimerization between TR1 and RXR. The existence of preformed TR1·RXR heterodimers in solution probably explains in part the lack of strong requirement for specific 5-flanking or spacer sequences. In addition, TR1·RXR heterodimers bind relatively stably to other DR sites such as DR5 (54), as well as to non-DR sites such as inverted and everted repeats (42, 52, 55, 56, 57).

In contrast to the DNA-independent heterodimerization of TR1 and RXR, TR2 heterodimerization with RXR was not demonstrable in the absence of DNA and in fact only occurred on a specific subset of DR4 sequences. Specifically, the presence of a T two base pairs upstream from the AGGTCA (i.e. in the third position of the DR4 spacer) was required for TR2 heterodimerization with RXR. Significantly, this creates a high affinity TR monomer binding sequence at the downstream site (21). TR has been shown to bind to the downstream half-site in the context of binding with RXR to a DR4 (24, 27), and this polarity of binding probably explains why the same flanking sequences in the context of the upstream half-site were ineffective at binding TR2·RXR.

The heterodimeric DNA binding of TR2 and RXR was clearly cooperative because TR2 bound weakly as a monomer, and monomeric RXR binding was undetectable on the same sites under identical conditions. The receptors must also directly interact, because no heterodimers were observed when the positions of the Hex and Oct half-sites were simply reversed. This indicates that the specific DNA-binding site allosterically favored the interaction of TR2 and RXR. Since the TR monomer site was necessary for heterodimerization, we suggest that DNA binding altered the conformation of the TR DBD interaction domain to favor interaction with RXR bound to an upstream half-site. It is also possible that the conformations of both TR and RXR are altered by binding to the specific DR4. Such an allosteric change in receptor structure upon DNA binding would be consistent with the nonvectorial changes in the TR·RXR·DR4 complex that we observed in circular permutation analysis yet could not be attributed to DNA bending (54). The ability of DNA to allosterically regulate the conformation of the TR would be formally analagous to the ability of T3 ligand to allosterically alter TR conformation (9). DNA binding has also been shown to allosterically regulate interaction with transcriptional cofactors (58). Indeed, DBD-dependent protein-protein interactions have already been documented in the crystal structure of the TR·RXR bound to DR4 (27). The present results suggest that in the absence of a strong C-terminal interaction, the inter-DBD interactions are regulated in part by the sequence bound by the TR.

We propose a model in which TR1 and RXR exist as a heterodimer in solution due to their strong C-terminal interaction. The DBD interaction determines the spacing (DR4) and polarity (TR downstream) requirements of binding but does not depend upon specific DNA sequence of the spacer. In contrast, TR2 and RXR do not form a stable heterodimer in solution due to the altered ninth heptad of TR and thus do not bind as heterodimers to all DR4 sites. Since RXR has little monomeric DNA binding affinity (59), we speculate that the first step in TR2·RXR heterodimerization on selected binding sites is binding of TR2 to the TNAGGTCA downstream site. The affinity of this interaction is reduced by phosphorylation of TR2. We suggest that binding of TR2 causes an allosteric change in conformation, which promotes the interaction with RXR, which binds weakly to the upstream site and stabilizes the TR2·RXR·DNA complex. The interaction between TR2 and RXR requires both the DBD and C-terminal interactions as well as a permissive downstream half-site. Such differential heterodimerization may allow TR2 to inhibit T3 action at selected target genes. Furthermore, allosteric regulation of protein conformation by specific DNA-binding sites may be a general mechanism of regulating DNA binding specificity and gene regulation of nuclear hormone receptors. In addition to regulating heterodimerization with RXR, DNA-dependent conformational changes in receptor structure may also regulate interactions with other proteins. For example, DNA-dependent interactions with transcriptional coactivators such as SRC-1 (60), RIP-140 (61), Trip1 (62), or TIF-1 (63) could explain why receptors such as TR activate transcription only on a subset of high affinity binding sites (64).