Crystal structure of K-80003-bound RXRα-LBD

To understand how K-80003 (Supplementary Fig. 1a) modulates the biological activity of tRXRα, we determined the crystal structure of RXRα-LBD in complex with K-80003 to a resolution of 2.6 Å (Table 1). We found that the RXRα-LBD/K-80003 complex adopts a tetrameric structure, similar to those observed in crystals of apo-RXRα or RXRα-LBD/tRA-isomer complex22 (Supplementary Fig. 1b), in which two RXRα-LBD canonical homodimers (labelled A1/B1 and A2/B2) pack in a bottom-to-bottom manner (Fig. 1a) with point group symmetry D2. Besides the canonical dimer interface32, there are two symmetry-related interfaces involving subunits A1/B2 and B1/A2. These ‘tetramer’ interfaces comprise three sub-regions: parallel packing between symmetry-related H3 helices; ‘end-to-end’ packing between H11 that reduces their length by two helical turns (compared with the agonist-bound structure); and the invasion of each H12 helix into its apposing domain, where it binds to the coregulator-binding groove, consisting of elements of H3 and H4 (Fig. 1a). The apposing domain reciprocates the process in a pairwise ‘exchange of C-terminal arms’. An LMEML motif near the C terminus of H12 binds to the coregulator groove by mimicking the LxxLL/LxxIL motif of coregulators (Supplementary Fig. 2). The tetramer interfaces create two substantial symmetry-related interfacial cavities that are readily accessible to solvent and small molecules.

Table 1 Data collection and refinement statistics. Full size table

Figure 1: Crystal structure of the RXRα-LBD tetramer in complex with K-80003. (a) The RXRα-LBD tetramer in complex with K-80003. The two biological dimers (A1/B1 and A2/B2) are shown as wheat/yellow and green/cyan cartoons, respectively. The bound K-80003 molecules are shown as brown sticks and blue sticks. Helices contributing to the tetramer interfaces are marked. (b) The orthogonal view showing the hydrophobic void (semitransparent grey) at the interface between the subunits A1 and B2. The smaller pockets and water channels are removed for clarity. 3 K-80003 molecules (K-80003A, B and K-80003C) filling the cavity are shown as blue and brown balls. Full size image

Refinement of the structure and careful inspection of difference Fourier maps revealed six molecules of K-80003-bound per tetramer, with three molecules per A1/B2 or A2/B1 interfacial cavity (Fig. 1a; Supplementary Fig. 3). This stoichiometry is consistent with values derived by isothermal titration calorimetry (ITC) (Supplementary Fig. 5a). The 2 A1/B2 and A2/B1 interfacial cavities are symmetry-related and the three bound molecules in each cavity (K-80003A, K-80003B and K-80003C, respectively, Fig. 1b) appear to play distinct but complementary roles in stabilizing the tetramer. Thus, K-80003A and K-80003B are arranged about the A1/B2 pseudo-dyad, making similar but distinct interactions (Figs 1b and 2b). They bind in a region where the H12 is located in the agonist-bound RXRα-LBD structure (Fig. 2a) and thus distinct from the canonical ligand-binding region. The indene ring of K-80003A makes many interactions with monomer B2, including parallel aromatic stacking with W305, and hydrophobic binding with L276 and L309, and R302. Its isopropylphenyl ring contacts K-80003C. On the other face of K-80003A, there are several hydrophobic/aromatic interactions, including with the side-chains of F439, I447 and L451 from the A1 monomer (Fig. 2b). On the fourth side, to complete the ‘cage’ around K-80003C, there are numbers of polar and ionic interactions. Notably, the carboxylate of K-80003A makes a bifurcated salt-bridge with K440 from A1 and R302 from B2, as well as an H-bond with the indole N-H of W305 (B2) (Fig. 2b). Thus, K-80003A is firmly encased on all sides at a unique location: by side-chains from A1 and B2 at the top and bottom, by molecule K-80003C on one side, and by elements of the invading H11–H12 turn and H12 helix on the other.

Figure 2: Locations of the bound K-80003 molecules and their interactions with RXRα-LBD. (a) Superposition of a monomer subunit (green cartoon) with bound K-80003 molecules K-80003A (brown sticks) and K-80003C (blue/red sticks) on the agonist-binding RXRα-LBD (magenta cartoon, PDB code 1FBY, ligand 9-cis-RA in yellow/red stick). (b) Binding of the three K-80003 molecules (blue and brown sticks) at the interface between subunits A1 and B2 of the RXRα-LBD tetramer. Selected side-chains of RXRα-LBD subunits that interact with the ligands are shown as sticks. Full size image

For K-80003B, the symmetry-related packing is similar but distinct with fewer interactions. The ionic interactions with R302 of A1 and K440 of B2 are much weaker, which may explain why K-80003B is less ordered than K-80003A, and with less well-defined electron density. Nevertheless, it makes similar aromatic stacking interactions—in this case, parallel with F439, but a less-optimal (45° stacking angle) with W305 (Fig. 2), as well as hydrophobic interactions with L433 of A1 and L436 of B2.

K-80003C binds in the pocket of monomer B2, in a mode that resembles a canonical ligand (Fig. 2a). Unlike K-80003A and K-80003B, it makes strong contacts only with the B2 subunit (Fig. 2b). Its carboxyl motif makes a salt-bridge with R316, while the indene ring sits in a broad hydrophobic cavity, and both are well-defined in the electron density map. However, the isopropylbenzene moiety sticks out of the pocket and into the cavity, making only weak contacts with other residues; it presumably adopts multiple conformations, consistent with the weak electron density (Supplementary Fig. 3).

Taken together, the interactions between the three bound K-80003 molecules and the protein suggest that K-80003 stabilizes RXRα-LBD tetramer by a unique combination of distinct and canonical-binding mechanisms acting like a ‘3-pronged’ binding mode: K-80003A is tightly packed in a hydrophobic, aromatic and polar cage that strengthens the tetramer and provides additional glue to hold H12 in its place to prevent coregulator binding. K-80003B also contributes to tetramer stabilization, but appears less tightly packed; significantly, however, it blocks the entrance to the unoccupied pocket in monomer A1, which might be otherwise favored by ligands such as 9-cis-RA. K-80003C binds in the pocket in monomer B2 interacting with K-80003A and B2.

To understand why K-80003 binds asymmetrically to the tetramer, we closely examined the shape of the tetramer’s interfacial cavity. Superposition of A1/B2 subunit on to itself reveals that A1/B2 is asymmetric (Supplementary Fig. 4a) with a smaller pocket in monomer A1 than in the B2 monomer (Supplementary Fig. 4b). This asymmetry seems to be resulted from the shifts of H3 and H11 into the pocket in A1 (Supplementary Fig. 4c) and provides an explanation for why small molecules bind asymmetrically to the tetramer22,33. In the previously reported RXRα-LBD/tRA-isomer tetramer structure22, tRA-isomer binds to the narrower pocket in monomer A1 where K-80003 molecule is too big to fit in (Supplementary Fig. 4b). In our structure, the large size of the K-80003C molecule presumably selects the wider LBP pocket of the A1/B2 dimer. Despite these differences in ligand binding, the two tetramers have similar overall structures (r.m.s. deviation for Cα atoms is 1.20 Å), and the largest changes are observed at the tetramer interface (Supplementary Fig. 1b), presumably induced by the non-canonical binders, K-80003A and K-80003B.

K-80003 promotes tetramerization of tRXRα but not RXRα

The unique binding of K-80003 observed in the tetrameric form of K-80003-bound RXRα-LBD crystal structure prompted us to determine whether K-80003 binding could promote RXRα-LBD tetramerization. In non-denaturing polyacrylamide gel electrophoresis, purified RXRα-LBD protein existed as two distinct bands corresponding to homodimer and homotetramer, respectively (Fig. 3a). As expected, incubation of RXRα with 9-cis-RA known to induce homodimerization18 resulted in a shift of RXRα-LBD from tetramers to dimers. In contrast, incubation with K-80003 induced an accumulation of tetramers. L433 near the C terminus of H10 packs directly against the indene ring of K-80003B, and its mutation to D would destabilize key interactions with K-80003 due to loss of existing hydrophobic interaction and introduction of repulsive charge–charge interaction with the carboxyl motif of K-80003B. Indeed, K-80003 failed to bind to RXRα-LBD/L433D (Supplementary Fig. 5a) and promote its tetramerization (Fig. 3a). Substitution of Q275 in H3 with E, or F439 in H11 with A, which are involved in the binding of K-80003 (Fig. 2), impaired the tetramerization of respective mutants by K-80003, confirming the role of H3 and H11 in the K-80003-induced stabilization of RXRα-LBD tetramers. Interestingly, mutating R316 in LBP, which is essential for 9-cis-RA binding12 and involved in K-80003C binding (Fig. 2b), with E, resulted in a mutant (RXRα-LBD/R316E) that exhibited mainly as a tetramer independent of the presence of either 9-cis-RA or K-80003, in agreement with a previous report25. The effect of K-80003 on stabilizing RXRα-LBD tetramers appeared overpower the 9-cis-RA-induced homodimerization as RXRα-LBD was mainly found as a tetramer in the presence of both K-80003 and 9-cis-RA (Fig. 3a).

Figure 3: Induction of RXRα tetramerization by K-80003 and its regulation by the N-C intramolecular interaction. (a) Equal amounts of purified RXRα-LBD or mutant protein were incubated with DMSO, 9-cis-RA, and/or K-80003, and separated by non-denaturing polyacrylamide gel electrophoresis followed by Coomassie Bright Blue staining. The percentage of tetramer and dimer of RXRα-LBD or mutants was quantitated by densitometric analysis of the corresponding blots. One of four similar experiments is shown. (b) RXRα-LBD incubated with K-80003 or 9-cis-RA was subject to gel filtration chromatogram assay. Results showed that 9-cis-RA-induced RXRα-LBD was mostly in dimer (D), while K-80003-induced RXRα-LBD was mostly in tetramer (T). One of three similar experiments is shown. (c) HepG2 cells transfected with RXRα, tRXRα or RXRα-LBD were treated with 9-cis-RA or K-80003. Cell lysates prepared were then subjected to BS3 crosslinking, and analysed by western blotting using ΔN197 anti-RXRα antibody. One of more than five similar experiments is shown. (d) Schematic representations of RXRα and mutants. A/B, C, D, E/F domains in RXRα are indicated. (e) HepG2 cells transfected with HA-RXRα-A/B and Myc-RXRα-LBD were treated with 9-cis-RA, and analysed by coIP with anti-HA antibody. One of two similar experiments is shown. (f) Inhibition of K-80003-induced tRXRα tetramerization by A/B domain. HEK293T cells transfected with tRXRα together with RXRα-A/B were treated with K-80003. Cell lysates were subjected to BS3 crosslinking, and analysed by western blotting using ΔN197 anti-RXRα antibody. One of three similar experiments is shown. (g) RXRα-A/B interaction with RXRα N-terminal deletion mutants. HA-RXRα-A/B and Myc-tagged RXRα N-terminal deletion mutants were transfected in to HEK293T cells, and their interaction was analysed by coIP. (h) Interaction of RXRα-A/B with RXRα mutants. HA-RXRα-A/B and Myc-tagged RXRα-mutants were transfected together in to HEK293T cells, and their interaction was analysed by coIP. One of three similar experiments is shown. (i) Mutation of Trp305 does not affect N/C interaction. Myc-tagged RXRα-LBD or RXRα-LBD/W305Q was transfected together with HA-RXRα-A/B into HEK293T cells in the presence or absence of 9-cis-RA (10−7M). Cell lysates were prepared and analysed by coIP. Full size image

The ability of K-80003 to stabilize RXRα-LBD tetramer was also illustrated by gel filtration chromatography showing that purified RXRα-LBD protein mainly existed as a tetramer in the presence of K-80003, while it displayed predominantly as a homodimeric complex in the presence of 9-cis-RA (Fig. 3b). Evaluation of a panel of chemical crosslinkers identified homobifunctional N-hydroxysuccimide-based chemical cross-linker BS3 as the most efficient one to crosslink the RXRα-LBD tetramer (Supplementary Fig. 5b). Thus, BS3 was subsequently used to study the effect of K-80003 on RXRα tetramerization. Extracts from cells transfected with RXRα-LBD and treated with either K-80003 or 9-cis-RA were prepared and subsequently incubated with BS3. Figure 3c showed that the treatment of cells with K-80003 produced crosslinked species on SDS-PAGE gels of 25, 50 and 100 kDa, corresponding to monomer, dimer and tetramer of RXRα-LBD, respectively. For comparison, treatment with 9-cis-RA resulted in only monomer and homodimer. Interestingly, RXRα or mutants expressed in cells mainly existed as a monomer even after crosslinking, which is different from purified RXRα proteins. Examination of the effect of K-80003 on tetramerization of tRXRα and RXRα revealed that K-80003 effectively promoted the formation of tRXRα tetramers. Unexpectedly, K-80003 failed to promote the tetramerization of the full-length RXRα, indicating that the N-terminal A/B domain interferes with its tetramerization.

The RXRα N/C interaction inhibits its tetramerization

The above observation prompted us to determine how the N-terminal A/B domain of RXRα inhibited its tetramerization (Fig. 1). Thus, the interaction between RXRα-A/B domain and RXRα-LBD (Fig. 3d) was studied by cell-based coimmunoprecipitation (coIP) assays. Immunoprecipitation of the RXRα-A/B protein resulted in a strong coIP of the RXRα-LBD protein, demonstrating their interaction. The interaction was inhibited by 9-cis-RA in a dose-dependent manner (Fig. 3e), and was confirmed by immunostaining showing extensive colocalization of transfected RXRα-A/B with RXRα-LBD but not RXRα in the cytoplasm of cells (Supplementary Fig. 6a). Cotransfection of the RXRα A/B domain completely suppressed the effect of K-80003 on promoting tRXRα tetramerization (Fig. 3f). These data reveal an extensive intramolecular interaction between the N terminus and the C terminus (N/C) in RXRα and its critical role in regulating RXRα tetramerization, and also provides a molecular explanation for the differential effect of K-80003 on the tetramerization of tRXRα and RXRα.

To further study the N/C interaction, we first conducted deletion analysis of the N-terminal A/B region to narrow down the region required for the interaction. Deletion of either the N-terminal 40 or 60 amino acids could not confer the ability of the resulting mutants to interact with the A/B domain (Fig. 3g). Deletion of additional 20 amino acids resulted in a mutant (tRXRα), which strongly interacted with the RXRα-A/B protein. RXRα-Δ60, like tRXRα, interacted strongly with RARγ (Supplementary Fig. 6b), demonstrating that RXRα-Δ60 is still active in heterodimerization with RARγ. These results suggested that amino acids from 60 to 80 are critical for the N/C interaction. We next determined region in RXRα-LBD required for binding A/B domain. RXRα-A/B could interact with RXRα mutants lacking N-terminal sequences (tRXRα, RXRα-Δ100 and RXRα-LBD) but not with mutant lacking C-terminal LBD (RXRα-1-235) or mutants lacking AF2/H12 region (RXRα-ΔAF2 and RXRα-ΔA/BΔAF2) (Fig. 3h). tRXRα without AF2/H12 also failed to interact with RXRα-A/B (Supplementary Fig. 6c). Thus, the N/C intramolecular interaction involves the N-terminal A/B domain and the C-terminal AF2/H12. The conclusion was supported by data showing the inability of the A/B domain to bind to the full-length RXRα, likely due to the unavailability of the C-terminal binding site masked by its own N-terminal A/B domain. The AF2/H12 is involved in the formation of the hydrophobic coactivator-binding groove that was shown to mediate the N/C interaction of some nuclear receptors34,35. To determine whether the N/C interaction involves the coactivator-binding groove, we tested whether W305, which is located in H5 and was shown to play a critical role in the formation of the coactivator-binding groove12, was involved in the N/C interaction. Our results showed that RXRα-LBD/W305Q with W305 mutated to Q, which failed to bind to an LxxLL-containing protein (see below), could still bind to the RXRα-A/B protein in a 9-cis-RA sensitive manner similar to the wild-type RXRα-LBD (Fig. 3i). These results preclude the involvement of the coactivator-binding groove in the N/C interaction.

An LxxLL motif in p85α mediates p85α interaction with RXRα

tRXRα differs from RXRα in its ability to reside in the cytoplasm and interact with cytoplasmic p85α protein, an event that is inhibited by K-80003 (ref. 29). To address the role of tetramerization in modulating tRXRα interaction with p85α, we first determined how p85α binds to tRXRα. Thus, several p85α mutants (Fig. 4a) were constructed and analysed for their interaction with tRXRα. CoIP experiments showed that the N-terminal region of p85α, including the N-terminal SH3 and BCR domains, is sufficient for interacting with tRXRα, whereas the C-terminal region including NSH2, iSH2 and CSH2 domains was dispensable (Fig. 4b). We further observed that a p85α mutant encompassing only the BCR domain could interact with tRXRα and RXRα-LBD but not RXRα-A/B (Fig. 4c).

Figure 4: Identification of an LxxLL-like motif in p85α and its interaction with the coactivator-binding groove in tRXRα. (a) Schematic representations of p85α and mutants. SH3, BCR, nSH2, iSH2, cSH2 domains in p85α and the location of the BCR-LxxLL motif are indicated. BCR and mutant peptides are shown. (b) Interaction of tRXRα with p85α mutants. Myc-tRXRα expression vector was transfected into HEK293T cells along or together with p85α mutant tagged with HA epitope, and their interaction was analysed by coIP using anti-Myc antibody. (c) BCR domain interaction with RXRα mutants. HA-p85α-BCR was cotransfected with Myc-tagged RXRα mutants into HEK293T cells, and their interaction was analysed by coIP using anti-HA antibody. (d) The BCR-LxxLL peptide (structure was extracted from the BCR crystal structure of PDB code 1PBW) docks to the coactivator-binding groove on RXRα (PDB code 3FUG). (e) SPR analysis of BCR-peptide binding to RXRα-LBD in the presence of 9-cis-RA. (f) Enhancing effect of 9-cis-RA on p85α-BCR interaction with RXRα-LBD and tRXRα. Cells transfected with Myc-tRXRα or Myc-RXRα-LBD together with HA-p85α-BCR were treated with 9-cis-RA (10−7M), and analysed by coIP. One of two similar experiments is shown. (g) Inhibitory effect of LxxLL-containing BCR peptide on RXRα-LBD interaction with p85α-BCR. HEK293T cells transfected with HA-p85α-BCR and Myc-RXRα-LBD were exposed to the indicated peptide and compound for 12 h. Cell lysates were prepared and analysed by coIP. (h) Mutation of the LxxLL motif in BCR impairs its interaction with RXRα-LBD. BCR and BCR-LxxLL mutant were tagged with Flag epitope and transfected into A549 cells with Myc-RXRα-LBD. Cell lysates were prepared and analysed for interaction of BCR and its mutant with RXRα-LBD by coIP. (i) Trp305 is essential for RXRα-LBD binding to p85α-BCR. Myc-tagged RXRα-LBD or RXRα-LBD/W305Q was transfected together with HA-p85α-BCR into HEK293T cells in the presence or absence of 9-cis-RA (10−7M). Cell lysates were prepared and analysed by coIP. For western blotting, one of three or four similar experiments is shown. Full size image

In an effort to understand the molecular basis for BCR interaction with tRXRα, we noticed the presence of two LxxLL motifs, 161LRQLL165 and 240LQYLL244, in BCR, which are commonly found in coactivators that mediates the transactivation of nuclear receptors10,13,14,15,16. Inspection of both motifs in the published BCR structure36 revealed that the 240LQYLL244 motif is buried in the central core of the BCR domain, while the 161LRQLL165 motif is located in a separate helix within a loop region. The 161LRQLL165 motif docks well to the coactivator-binding groove of RXRα (Fig. 4d), suggesting that the motif might be critical for RXRα binding. A peptide (BCR peptide) that encompasses the 161LRQLL165 motif (Fig. 4a) was therefore synthesized and examined for its binding to RXRα-LBD by Biacore assay. The peptide binds strongly to RXRα-LBD in the presence of 9-cis-RA with a K d of 320 nM (Fig. 4e), which is in the range of coregulator peptide binding to nuclear receptor37. The role of the 161LRQLL165 motif was also illustrated by the enhancing effect of 9-cis-RA on p85α-BCR interaction with either RXRα-LBD or tRXRα (Fig. 4f). Furthermore, 9-cis-RA-induced RXRα-LBD interaction with p85α-BCR was inhibited by the LxxLL-containing BCR peptide conjugated with the cell-penetrating peptide derived from trans-activator of transcription (TAT), similar to the effect of K-80003, but not by the corresponding mutant peptide (Fig. 4g). Substitution of L164 and L165 in 161LRQLL165 motif with A also abolished the interaction of BCR with RXRα-LBD (Fig. 4h). Mutating W305 critical for the formation of the coactivator-binding groove12 to Q impaired the binding of RXRα-LBD with p85α-BCR either in the presence or absence of 9-cis-RA (Fig. 4i), even though the same mutation had no effect on RXRα-LBD interaction with RXRα-A/B (Fig. 3i). The interaction of the LxxLL-like motif in p85α with the coactivator-binding groove of tRXRα is biologically relevant as TNFα-induced activation of AKT in cells transfected with tRXRα and p85α was inhibited by cotransfection of BCR but not BCR mutant, similar to the inhibitory effect of K-80003 (Supplementary Fig. 7a). Exposure of cells to BCR peptide also resulted in a similar inhibition (Supplementary Fig. 7b). Altogether, these results demonstrate that the LxxLL motif in p85α can bind to the coactivator-binding groove of RXRα in analogy to the binding of transcriptional coactivators.

Tetramerization prevents tRXRα from interacting with p85α

To further characterize the ‘coactivator-like’ binding of p85α, we studied the requirement of AF2/H12 in tRXRα, which is essential for the formation of the coactivator-binding groove12,38, for tRXRα interaction with p85α. As previously reported29, tRXRα interacted with the full-length p85α in the presence of TNFα. Removing AF2/H12 from tRXRα (tRXRα/ΔAF2) abolished its interaction with p85α (Fig. 5a). Interfering AF2/H12 activity by transfecting RXRα-A/B capable of binding AF2/H12 also inhibited 9-cis-RA-induced interaction of tRXRα with p85α-ΔiSH2 (Fig. 5b). These results further support the ‘coactivator-like’ binding of p85α.

Figure 5: Tetramerization of tRXRα prevents its interaction with p85α. (a) AF2/H12 is required for binding p85α. HEK293T cells transfected with HA-p85α and Myc-tRXRα or mutants were treated with TNFα (10 ng ml−1) for 1 h, and analysed by coIP assay using anti-Myc antibody. (b) Regulation of tRXRα interaction with p85α by ligand and RXRα-A/B region. HEK293T cells transfected with the indicated HA-p85α-ΔiSH2, Myc-tRXRα and RXRα-A/B were treated with or without 9-cis-RA (10−7M) for 6 h, and analysed by coIP assay using anti-Myc antibody. (c) Tetramerization of tRXRα/R316E and tRXRα/F313A was analysed in MCF-7 cells transfected with tRXRα/R316E or tRXRα/F313A, which were then treated with 9-cis-RA (10−7 M) or K-80003 (5 × 10−6 M) for 6 h. Cell lysates were subjected to BS3 crosslinking and analysed by western blotting using ΔN197 anti-RXRα antibody. (d) Interaction of tRXRα/R316E and tRXRα/F313A with p85α. HEK293T cells transfected with the indicated expression plasmids were treated with TNFα (10 ng ml−1) or 9-cis-RA (10−7M) for 1 h, and analysed by coIP assay using anti-Myc antibody. (e) Characterization of p85α interaction with tRXRα/R316E or tRXRα/F313A. HEK293T cells transfected with the indicated expression plasmids were treated with 9-cis-RA (10−7M) or K-80003 (5 × 10−6M) for 6 h., and analysed by coIP assay using anti-HA antibody. (f) Mutation of R316 impairs tRXRα cytoplasmic localization. HEK293T cells cotransfected with Myc-tRXRα/R316E and HA-p85α were pretreated with K-80003 (5 × 10−6M) for 3 h before exposed to TNFα (10 ng ml−1) for 30 min. Cells were immunostained with anti-Myc and anti-p85α antibody, and visualized by confocal microscopy. Scale bar, 10 μm. (g) Tetramerization of tRXRα impairs its interaction with p85α. HepG2 cells transfected with Myc-tRXRα together with HA-p85α-ΔiSH2 were treated with K-80003 and or 9-cis-RA. Cell lysates were then subjected to BS3 crosslinking, and analysed by western blotting using anti-Myc antibody. (h) Tetramerization of RXRα-LBD impairs its interaction with p85α-BCR. HEK293T cells transfected with Myc-RXRα-LBD together with HA-p85α-BCR were treated with K-80003. Cell lysates were subjected to BS3 crosslinking, and analysed by western blotting using anti-HA antibody. For western blotting, one of three or four similar experiments is shown. Full size image

Because the coactivator-binding groove of RXRα was shielded in the tetrameric form of RXRα-LBD (Fig. 1), our observation that the LxxLL-like motif in p85α interacted with the coactivator-binding groove of RXRα implied that the p85α-binding activity of tRXRα is impaired in its tetrameric form. To address this, we evaluated the binding of p85α by two tRXRα mutants, tRXRα/F313A and tRXRα/R316E, which exist in different oligomeric forms25. RXRα/F313A is a transcriptionally constitutively active mutant receptor with H12 adopting an active conformation to form the coactivator-binding groove39. The mutant does not exist as a tetramer as its H12 is not available to contribute to the formation of tetramers25. In contrast, tRXRα/R316E, a transcriptionally inactive mutant, exhibited predominantly as a tetramer25. In agreement with previous observations25, our cross-link experiments detected a strong tetrameric species of tRXRα/R316E but not tRXRα/F313A independent of the presence of 9-cis-RA or K-80003 (Fig. 5c). When both mutants were analysed for their interaction with p85α by coIP experiments, we found that tRXRα/R316E could not bind to p85α either in the absence or presence of TNFα or 9-cis-RA, whereas tRXRα/F313A showed a constitutive and 9-cis-RA-independent interaction with p85α compared to tRXRα (Fig. 5d). K-80003 also failed to modulate the interaction of both mutants with p85α (Fig. 5e). Immunostaining revealed lack of colocalization of the tRXRα/R316E mutant with p85α in the absence or presence of TNFα (Fig. 5f). To further determine the inhibitory effect of tRXRα tetramerization, we examined the interaction of p85α-ΔiSH2 with tRXRα tetramers crosslinked by BS3. Cells transfected with HA-p85α-ΔiSH2 and Myc-tRXRα were treated with 9-cis-RA and/or K-80003, and subsequently exposed to BS3. 9-cis-RA-induced tRXRα interaction with p85α-ΔiSH2 was potently inhibited by BS3 that stabilized tRXRα tetramers (Fig. 5g). Furthermore, the monomeric form but not the tetrameric form of RXRα-LBD interacted with p85α-BCR (Fig. 5h). These results together with our structural information demonstrate that K-80003 inhibits tRXRα interaction with p85α by promoting tRXRα tetramerization that masks the p85α-binding region on tRXRα.

Tetramerization regulates tRXRα subcellular localization

The interaction of tRXRα with p85α occurs in the cytoplasm29. We next determined whether K-80003-induced stabilization of tRXRα tetramers could modulate its subcellular localization. When transfected into cells, both tRXRα and RXRα resided mainly (>80%) in the nucleus. However, upon TNFα treatment tRXRα was found in the cytoplasm of cells (>65%), colocalizing extensively with p85α. In contrast, TNFα had no effect on the nuclear localization of RXRα. When cells were cotreated with K-80003, TNFα-induced colocalization of tRXRα with p85α in the cytoplasm was inhibited, resulting in tRXRα nuclear localization (Fig. 6a). The effect of K-80003 was likely due to its binding to tRXRα as the cytoplasmic colocalization of p85α with tRXRα/L433D mutant defective in K-80003 binding was not affected by K-80003. Thus, TNFα-induced cytoplasmic localization of tRXRα was likely due to its cytoplasmic retention by TNFα-activated p85α through protein/protein interaction, suggesting that tRXRα tetramers incapable of binding p85α might reside in the nucleus. To address this, extracts prepared from cells transfected with tRXRα were subjected to crosslinking by BS3. Nuclear and cytoplasmic fractions were then prepared and analysed. Western blotting showed that K-80003-stabilized tetrameric form of tRXRα was found exclusively in the nuclear fraction, while tRXRα monomer was distributed both in the nuclear and cytoplasmic fractions (Fig. 6b). Thus, K-80003-stabilized tRXRα tetramer is mainly nuclear, likely resulted from tRXRα dissociation from p85α or other cytoplasmic proteins.

Figure 6: Effect of tetramerization on the subcellular localization of tRXRα. (a) Effect of TNFα and K-80003. MCF-7 cells cotransfected with Myc-RXRα, Myc-tRXRα, tRXRα/L433D and p85α were pretreated with or without K-80003 (5 × 10−6M) for 3 h before exposed to TNFα (10 ng ml−1) for 30 min. Cells were immunostained with anti-Myc and anti-p85α antibody, and their subcellular localization revealed by confocal microscopy. (b) Tetrameric tRXRα resides in the nucleus. HEK293T cells cotransfected with Myc-tRXRα were treated with or without K-80003 (5 × 10−6M) for 6 h. Nuclear (N) and cytoplasmic (C) fractions were prepared, subjected to BS3 crosslinking, and analysed by western blotting using anti-Myc antibody. The purity of fractions was examined by analysing the expression of nuclear PARP and cytoplasmic α-tubulin in non-crosslinked fractions. One of three similar experiments is shown. (c) Effect of K-80003 on the growth of MCF-7 cells in mice. Nude mice injected with MCF-7 cells stably transfected with control vector, Myc-RXRα, or Myc-tRXRα were administered with K-80003 (20 mg kg−1) for 12 days. *P<0.05; **P<0.01. (d) Effect of K-80003 on PARP cleavage and AKT activation in MCF-7 xenograft tumours. Lysates prepared from tumours from nude mice treated with vehicle or K-80003 were analysed by western blotting. (e) K-80003 alters the subcellular localization of tRXRα in MCF-7 xenograft tumour cells. Tumour sections prepared from nude mice treated with vehicle or K-80003 were immunostained with anti-Myc antibody. (f) Effect of K-80003 on the growth of MMTV-PyMT mammary tumour. Four-week old MMTV-PyMT mice were fed with or without diet containing K-80003 (100 mg kg−1) for 4 weeks, and the appearance of tumour was determined. **P<0.01. (g) Effect of K-80003 on PARP cleavage and cyclin D1 expression in MMTV-PyMT mammary tumour cells. Lysates prepared from tumours from MMTV-PyMT mice fed with vehicle or K-80003 were analysed by western blotting. (h) K-80003 induces RXRα nuclear localization in MMTV-PyMT tumour cells. Tumour sections from MMTV-PyMT mice fed with or without K-80003 (100 mg kg−1) for 4 weeks were immunostained with ΔN197 anti-RXRα antibody. Scale bar, 10 μm. Full size image

To study whether the anti-tumour effect of K-80003 could be attributed to its effect on the subcellular localization of tRXRα in vivo, we stably transfected tRXRα or RXRα into MCF-7 cells, and the resulting stable clones (Supplementary Fig. 8a) were inoculated into nude mice. Overexpression of tRXRα but not RXRα in MCF-7 breast cancer cells enhanced AKT activation in vitro (Supplementary Fig. 8b) and promoted the growth of MCF-7 tumour in animals, which was suppressed when animal were treated with K-80003 (Fig. 6c; Supplementary Fig. 8c). Inhibition of the growth of MCF-7/tRXRα tumour by K-80003 was accompanied with reduced AKT activation and enhanced PARP cleavage (Fig. 6d). To determine whether the anti-cancer activity of K-80003 was associated with its modulation of tRXRα subcellular localization, tumour specimens from nude mice were analysed by RXRα immunostaining (Fig. 6e). While RXRα was nuclear, tRXRα was predominantly cytoplasmic. However, a significant amount of tRXRα was found in the nucleus when animals were administered with K-80003. We also used polyomavirus middle T antigen (PyMT) transgenic mice40 to study the anti-cancer effect of K-80003 and its modulation of the subcellular localization of tRXRα, which was highly expressed in PyMT mammary tumour developed in these mice (Supplementary Fig. 9a). K-80003 potently inhibited the growth of PyMT mammary tumour in this animal model (Fig. 6f), accompanied with induction of PARP cleavage and inhibition of cyclin D expression (Fig. 6g) as well as inhibition of tumour cell proliferation (Supplementary Fig. 9b). Immunostaining of PyMT tumour specimens using Δ197 anti-RXRα antibody that recognizes both RXRα and tRXRα revealed a predominant cytoplasmic RXRα staining (Fig. 6h). In contrast, RXRα staining was mainly found in the nucleus when mice were dosed with K-80003. Thus, K-80003 induction of tRXRα nuclear localization through its modulation of tRXRα tetramerization likely represents a major mechanism by which the compound exerts its potent therapeutic effect.