Different nuclear hormone receptors interact with different domains of SMRT.
We previously demonstrated that both T3Rs and RARs bind to the C-terminal portion of SMRT; dissection of this phenomenon by yeast two-hybrid analysis (40
) suggested that more than one receptor interaction domain (RID) may exist within this SMRT C terminus. To confirm and extend this observation, we used an in vitro binding assay. Defined portions of SMRT were expressed as GST fusions in E. coli
(Fig. ) and were tested for the ability to bind to radiolabeled receptors synthesized by transcription-translation in vitro. Receptor molecules bound to the immobilized GST-SMRT constructs were subsequently eluted and analyzed by SDS-PAGE (Fig. ).
FIG. 2 Different receptors preferentially interact with different domains of SMRT in an in vitro assay. Different nuclear hormone receptors were synthesized as radiolabeled proteins by transcription and translation in vitro and were tested for the ability to (more ...)
Two distinct domains within the SMRT C terminus were able to bind to T3R in vitro; these were denoted RID-1 (SMRT codons 1055 to 1291) and RID-2 (SMRT codons 1291 to 1495) (Fig. and ). In contrast, little or no binding of T3R was detected with a nonrecombinant GST construct or with GST fusion proteins representing more N-terminal SMRT domains (Fig. and data not shown). Intriguingly, T3R exhibited nearly equal interactions with both RID-1 and RID-2 of SMRT (with a slight preference for RID-2), whereas RAR preferentially interacted with RID-1 (Fig. ). T3Rs (and RARs) repress transcription primarily in the absence of hormone, whereas the addition of hormone causes release of the corepressor and conversion of the receptor into a transcriptional activator (1
). Notably, this hormone-mediated release of the corepressor was observed with either the RID-1 or the RID-2 construct, indicating that the binding of hormone concurrently destabilizes receptor interactions with both RIDs in SMRT (Fig. ).
Although T3Rs and RARs exemplify the receptors known to function as transcriptional repressors, we wished to determine if SMRT might also participate in the transcriptional functions of other members of the nuclear hormone receptor family. We determined that SMRT also interacted with RXRs in vitro (Fig. ), consistent with previous observations of an RXR-SMRT interaction by two-hybrid analysis in yeast (40
). Although this RXR-SMRT interaction was significantly weaker than that between SMRT and T3R or RAR, it was highly reproducible and clearly above the background observed with nonrecombinant GST or with GST fusions containing the SMRT N terminus (Fig. ). Unlike RAR or T3R, RXR interacted exclusively with RID-2 of SMRT, and an RXR ligand (9-cis
retinoic acid) actually slightly stimulated rather than inhibited the RXR-SMRT interaction (45
) (Fig. and data not shown). Extending these experiments to other receptors revealed moderate to strong interactions between PPARγ and the RID-2 region of SMRT, whereas no interaction above the background could be detected between any of our SMRT constructs and VDR or the glucocorticoid receptor in either the presence or the absence of the cognate hormone (Fig. and data not shown).
We next used a mammalian two-hybrid assay to determine if the SMRT-nuclear hormone receptor interactions observed in vitro extended to a more physiological context in vivo. For this assay, different portions of SMRT were fused to GAL4DBD and inserted into a mammalian expression vector, pSG5. In parallel, relevant portions of the nuclear hormone receptors were fused to GAL4AD and placed into the same pSG5 vector. In this fashion, interactions between SMRT and the receptors should lead to a functional reconstitution of the GAL4 transcriptional activator, assayed as the stimulation of a GAL4 (17-mer)-luciferase reporter, when all three constructs are cointroduced into mammalian CV-1 cells.
Consistent with our in vitro data, both RARα and T3Rα strongly interacted with SMRT in our mammalian two-hybrid assay, whereas no stimulation of the reporter was observed if either the GAL4DBD-SMRT or the GAL4AD-receptor construct was replaced by an equivalent nonrecombinant GAL4 vector (Fig. A and B). RXRα also demonstrated a reproducible interaction with SMRT in the two-hybrid assay, although, again, at a much lower level than T3R or RAR (Fig. C; note the change in scale), whereas VDR exhibited no detectable interaction with SMRT in this assay (Fig. D). In vivo, as in vitro, T3R interacted with both RID-1 and RID-2 (although it exhibited a preference for RID-2), whereas RAR interacted almost exclusively with RID-1 and RXR interacted almost exclusively with RID-2 (Fig. ). Also paralleling our in vitro experiments, the two-hybrid interaction between SMRT and RAR or T3R was virtually abolished by the addition of the cognate hormone, whereas the interaction between SMRT and RXR was actually enhanced by 9-cis retinoic acid (Fig. ). We conclude that the two RIDs in SMRT are nonequivalent in their interactions with different members of the nuclear hormone receptor family and that this nonequivalence can be observed in both in vivo and in vitro assays.
FIG. 3 Different receptors preferentially interact with different domains of SMRT in a two-hybrid assay in vivo. pSG5 vectors expressing GAL4DBD (DBD) only or GAL4DBD fused with different domains of SMRT (as indicated below each panel) were introduced into CV-1 (more ...)
We wished to extend these interaction studies to N-CoR, a second member of the SMRT corepressor family that exhibits approximately 50% amino acid relatedness to SMRT over regions of overlap. T3Rα displayed a pattern of interactions with N-CoR similar to that observed with SMRT, interacting independently with two distinct C-terminal domains of N-CoR, denoted here as nRID-1 and nRID-2, that correspond in general location to RID-1 and RID-2 of SMRT, respectively (compare Fig. A and A). Intriguingly, RARα also interacted with both nRID-1 and nRID-2 of N-CoR (Fig. B). This result is in marked contrast to the strong specificity that RARα exhibited for RID-1 of SMRT (compare Fig. B and B). It should be noted that the nRID-2 construct used here is 235 amino acids long; the RID-2 SMRT construct is 204 amino acids long. Thus, the precise limits of the N-CoR and SMRT constructs used in these experiments were similar but not identical. Nonetheless, within the limits of our experimental methodology, we conclude that the RIDs of SMRT and N-CoR possess distinguishable, although overlapping, specificities for the different nuclear hormone receptors.
FIG. 4 Different receptors interact with different domains of N-CoR in a two-hybrid assay in vivo. pSG5 vectors expressing GAL4DBD only (DBD) or GAL4DBD fused with the domains of N-CoR indicated below the panels (corresponding to the schematic in Fig. (more ...) The three different RAR isoforms diverge in their ability to interact with SMRT.
RARs are encoded by three different loci in the mammalian genome, resulting in the synthesis of three major subclasses, or isoforms, of RARs (denoted RARα, RARβ, and RARγ; reviewed in references 6
). Although believed to serve distinct, if partially redundant, functions in vivo, these three different RAR isoforms are virtually indistinguishable in most of their biochemical properties in vitro. Unexpectedly, RARβ exhibited a severely limited ability to interact with SMRT in the mammalian two-hybrid assay compared to either the RARα or the RARγ isoforms (Fig. A); notably, all three GAL4AD-RAR isoform fusions were nonetheless expressed and exhibited nearly equal abilities to interact with a GAL4DBD-RXR fusion used in the same assay as a positive control (data not shown). This relative inability of the RARβ isoform to associate with the SMRT corepressor could also be observed in analogous two-hybrid experiments with N-CoR (Fig. B) and was also evident in our in vitro binding assay (Fig. C). Analysis of chimeras of RARα and RARβ localized the sequences responsible for this isoform-specific SMRT interaction to a small region within the central domain of the receptor, in between the DNA-binding and hormone-binding domains (Fig. C). RAR derivatives that possess the α sequence in this region bound to SMRT very strongly in vitro and in vivo, whereas receptor derivatives that contain the equivalent β sequences exhibited a greatly reduced ability to interact with SMRT (Fig. C). This region contains a cluster of amino acids that are present in RARα or RARγ but divergent in RARβ and that are likely to account for the different SMRT interaction phenotypes (Fig. D). Notably, this isoform-specific amino acid cluster is adjacent to an N-CoR box previously proposed to be necessary for the interaction of the receptor with the corepressor (23
FIG. 5 Different RAR isoforms differ in their abilities to interact with SMRT. (A) Interactions of different RAR isoforms with SMRT, as determined with a mammalian two-hybrid assay in vivo. RARα, RARβ, and RARγ were expressed as GAL4AD (more ...)
When mapping the determinants of RAR involved in these isoform-specific SMRT interactions, we observed that deletions of the AF-2 domain at the C terminus of either RARα (denoted RARα 403-t) or RARβ resulted in a modestly enhanced interaction of these receptor derivatives with SMRT (Fig. and data not shown). This enhanced interaction appeared to be mediated, at least in part, by an increased ability of the receptor to interact with RID-2 of SMRT; notably, however, the vast majority of the RAR-SMRT interaction remained mediated through RID-1 (Fig. ). We suggest that there are cryptic interaction sites for SMRT that, within native RARs, are obscured by the extreme C terminus of the receptor. Also, as reported previously, removal of the RAR C-terminal domain renders the receptor-SMRT interaction refractory to hormone (Fig. ). These results are consistent with proposals that changes in the conformation of the receptor C terminus can regulate the association of the receptor with SMRT (2
FIG. 6 Deletion of the RAR C terminus enhances binding to SMRT. Full-length wild-type RARα (wt-RAR) or a C-terminal truncation (RAR-403t) were synthesized as radiolabeled proteins by transcription and translation in vitro and were tested for the ability (more ...) Heterodimer formation by different nuclear hormone receptors can result in novel modes of SMRT interaction.
Many nuclear hormone receptors can form heterodimers with other receptors, resulting in novel DNA and hormone recognition properties (6
). We examined whether receptor heterodimerization could also be manifested as an altered interaction with SMRT. We first tested RXR heterodimers, given the proposed preeminent role of RXRs as a partner for RARs and T3Rs. Although radiolabeled RXR interacted only weakly with immobilized GST-SMRT, the addition of unlabeled RAR (obtained from recombinant baculovirus-infected Sf9 cell extracts) greatly enhanced the binding of RXR to SMRT: 0.8% of the input RXR bound to SMRT in the absence of RAR, but 28.4% bound in the presence of RAR (Fig. A; Sf9 + RARα). In contrast, extracts of Sf9 cells infected with nonrecombinant baculovirus had no effect (Fig. A; Sf9). The addition of all-trans
retinoic acid greatly reduced the amount of RXR retained by the GST-SMRT matrix, consistent with the participation of the RAR partner in tethering radiolabeled RXR to SMRT. The hormone 9-cis
retinoic acid is a high-affinity ligand for RARs as well as for RXRs (6
); similar to the effects of all-trans
retinoic acid, the addition of 9-cis
retinoic acid led to the dissociation of the presumptive RXR-RAR heterodimer from the GST-SMRT matrix.
FIG. 7 Enhancement of the RXR-SMRT interaction by the addition of RAR or T3R in vitro. Radiolabeled RXRα was synthesized by transcription and translation in vitro and tested for the ability to bind to a GST-SMRT (RID-1 plus RID-2) polypeptide (A and (more ...)
We also repeated these experiments with constructs of SMRT limited to the individual RID-1 or RID-2 regions (Fig. B and C). The ability of RARα to enhance the binding of RXRα to SMRT was also clearly observed with these individual RID constructs, if at a somewhat reduced level compared to the effects seen when SMRT constructs containing both RIDs were used (Fig. A). Given the specificity, when tested alone, of RXRα for SMRT RID-2 and of RARα for SMRT RID-1 (Fig. ), these results suggest that the presumptive RAR-RXR heterodimer can be tethered to SMRT by either receptor moiety in the dimer.
The addition of unlabeled T3Rα was also able to enhance the binding of RXRα to SMRT; approximately 0.6% of input RXR bound to SMRT (RID-1 plus RID-2) in the absence of T3R (Fig. D; Sf9), compared to 6.0% in the presence of T3R (Fig. D; Sf9 + T3Rα). A similar enhancement was observed when the individual SMRT RIDs were tested separately (Fig. E and F). The addition of a T3R ligand, Triac, interfered with this enhancement when either type of SMRT construct (RID-1 plus RID-2 or RID-1 alone) was used, consistent with the participation of T3R in the tethering of radiolabeled RXR to SMRT under these conditions (Fig. D and E). In contrast, Triac did not inhibit the binding of the presumptive RXR-T3R heterodimer to the SMRT (RID-2) construct (Fig. F), perhaps suggesting that the abstracted SMRT RID-2 interacts primarily with the RXR moiety under these conditions. Intriguingly, 9-cis retinoic acid, which stabilized the interactions of RXR with the corepressor in the absence of T3R (Fig. C), inhibited the binding of the RXR-T3R heterodimer to the SMRT (RID-1 plus RID-2) and SMRT (RID-1) constructs. A similar paradoxical effect, suggestive of differences in the effects of hormone ligands on heterodimers versus homodimers, was also observed in our two-hybrid studies (see below).
These heterodimeric interactions could be mimicked in the mammalian two-hybrid assay. As previously noted, the GAL4AD-RXR fusion by itself exhibited only a weak interaction with GAL4DBD-SMRT (RID-1 plus RID-2) in the two-hybrid assay, whereas GAL4AD-RAR exhibited a moderate interaction with GAL4DBD-SMRT (Fig. A). The simultaneous introduction of both GAL4AD-RAR and GAL4AD-RXR, however, resulted in a stronger interaction with GAL4DBD-SMRT that was much greater than the sum of the interactions of the two receptor constructs introduced separately (Fig. A). An analogous synergistic interaction of GAL4AD-T3R and GAL4AD-RXR with GAL4DBD-SMRT was also observed (Fig. B). In vivo as in vitro, the combined interaction of RXR and RAR with SMRT was abolished by RAR ligands (all-trans or 9-cis retinoic acid), whereas the combined interaction of RXR and T3R with SMRT was slightly reduced by the addition of 9-cis retinoic acid and strongly inhibited by the addition of Triac.
FIG. 8 Combinatorial interactions of receptors with SMRT in a two-hybrid analysis in vivo. A mammalian two-hybrid protocol similar to that in Fig. was used, but with a pSG5 GAL4DBD-SMRT (amino acids 751 to 1495) construct in all cases and with (more ...)
Although VDR did not exhibit an autonomous ability to associate with SMRT, vitamin D3 signal transduction in vivo is thought to be primarily mediated by RXR-VDR heterodimers. We therefore tested if RXR-VDR heterodimers displayed novel interactions with SMRT not observed when these receptors were tested individually. Indeed, RXRs and VDRs cointroduced as GAL4AD fusions exhibited a clear and robust interaction with GAL4DBD-SMRT, in contrast to the much weaker, or undetectable, SMRT interaction observed when either of these receptors was introduced individually (Fig. C). An analogous enhancement of the abilities of VDR and RXR to interact with SMRT was also observed when these receptors were tested in combination in vitro (data not shown). Intriguingly, the addition of either vitamin D3 or 9-cis retinoic acid destabilized the RXR-VDR two-hybrid interaction with SMRT (Fig. C). Thus, it appears that heterodimerization with RXR can enhance an otherwise cryptic ability of VDR to interact with SMRT and that attachment of a hormone ligand to either receptor partner can, at least in vivo, partially disrupt this interaction. In contrast to these RXR heterodimers, the cointroduction of T3R-RAR, T3R-VDR, or T3R-PPAR into the mammalian two-hybrid system yielded largely additive interactions with SMRT, without any indication of a synergistic or combinatorial outcome (e.g., Fig. D and data not shown). We conclude that certain receptor heterodimers are capable of conferring a variety of interactions with corepressors that are not observed with the parental receptors tested individually and that the effects of hormones on these interactions differ in the homodimeric and heterodimeric contexts.