Discrete roles for peroxisome proliferator-activated receptor gamma and retinoid X receptor in recruiting nuclear receptor coactivators

Abstract

Peroxisome proliferator-activated receptor gamma (PPARgamma) plays a major role in adipogenesis. PPARgamma binds to DNA as a heterodimer with retinoid X receptor (RXR), and PPARgamma-RXR can be activated by ligands specific for either receptor; the presence of both ligands can result in a cooperative effect on the transactivation of target genes. How these ligands mediate transactivation, however, remains unclear. PPARgamma is known to interact with both the p160/SRC-1 family of coactivators and the distinct, multisubunit coactivator complex called DRIP. A single DRIP subunit, DRIP205 (TRAP220, PBP), binds directly to PPARgamma. Here we report that PPARgamma and RXR selectively interacted with DRIP205 and p160 proteins in a ligand-dependent manner. At physiological concentrations, RXR-specific ligands only induced p160 binding to RXR, and PPARgamma-specific ligands exclusively recruited DRIP205 but not p160 coactivators to PPARgamma. This selectivity was not observed in interaction assays off DNA, implying that the specificity of coactivator binding in response to ligand is strongly influenced by the allosteric effects of DNA-bound heterodimers. These coactivator-selective effects were also observed in transient-transfection assays in the presence of overexpressed p160 or DRIP coactivators. The results suggest that the cooperative effects of PPARgamma- and RXR-specific ligands may occur at the level of selective coactivator recruitment.

FIG. 1

(A) Inducible interactions between PPARγ and coactivators by various PPAR ligands. GST (lanes 2 to 7) or GST-PPARγ (lanes 8 to 13) was incubated with in vitro-translated ³⁵S-labeled SRC-1 (row 1), ACTR (row 2), GRIP-1 (row 3), and DRIP205 (row 4) in the presence or absence (lanes 2 and 8) of 10⁻⁵ M concentrations of the indicated ligands. The input (10%) of each in vitro-translated protein is indicated in lane 1. (B) Interaction of PPARγ-LBD and the multisubunit DRIP coactivator complex from Namalwa B-cell nuclear extracts. Immobilized GST-PPARγ LBD was incubated with a Namalwa B-cell nuclear extract in the presence of vehicle or 10⁻⁵ M GW1929. Bound proteins were eluted with N-lauroyl Sarkosine. Eluted proteins were separated by SDS–7.5% PAGE and visualized by silver nitrate staining. (C) Ligand-inducible interactions between RXR and coactivators. GST (lanes 2 and 3) or GST-RXR (lanes 4 and 5) was incubated with in vitro-translated ³⁵S-labeled SRC-1 (row 1), ACTR (row 2), GRIP-1 (row 3), or DRIP205 (row 4) in the absence (lanes 2 and 4) or presence (lanes 3 and 5) of 10⁻⁶ M LG268. Lane 1 in each row represents 10% of the input in vitro-translated proteins.

FIG. 2

Ligand-selective binding of coactivators to DNA-bound PPARγ-RXR heterodimers. (A) Baculovirus-expressed RXR and in vitro-translated PPARγ were combined and incubated without ligand (lanes l and 2) or in the presence of indicated concentrations of GW1929 (lane 3 and 4) or rosiglitazone (lanes 5 and 6). Receptors were combined with a radiolabeled PPRE probe and GST-DRIP205 (lanes 2 to 6) and DNA-bound complexes separated by EMSA. The PPARγ-RXR heterodimer and the coactivator-heterodimer complexes are indicated. (B) Baculovirus-expressed RXR and in vitro-translated PPARγ were incubated without ligand (lanes 3, 7, 11, 15, and 20) or in the presence of 10⁻⁵ M GW1929 (lanes 4, 8, 12, 16, and 21 ), 10⁻⁸ M LG268 (lanes 5, 9, 13, 17, and 22), or both ligands (lanes 6, 10, 14, 18, and 23). These samples were then combined with the radiolabeled PPRE probe and either GST-DRIP205 (lanes 3 to 6), GST-SRC-1 (lanes 7 to 10), GST-CBP (lanes 11 to 14), GST-ACTR (lanes 15 to 18), or GST-GRIP-1 (lanes 20 to 23) and subjected to EMSA. The PPARγ-RXR heterodimer and the coactivator-heterodimer complexes are indicated.

FIG. 3

Ligand titrations of PPARγ-RXR coactivator interactions. (A) GW1929 titration of DRIP205 binding to PPARγ-RXR. Purified RXR was mixed with in vitro-translated PPARγ and incubated on ice in the presence of the indicated concentrations of GW1929; the DNA probe and GST-DRIP205, GST-SRC-1, or GST-ACTR were added to samples and subjected to EMSA. (B) LG268 titration. Binding and EMSA conditions were exactly as described for panel A.

FIG. 4

AF2 mutations abolish coactivator binding in a receptor-restrictive manner. (A) RXR AF-2 mutation abolishes SRC-1 and ACTR, but not DRIP205, binding to PPARγ-RXR. In vitro-translated wild-type PPARγ and AF-2 mutated RXR (M454A and L455A) were combined and incubated on ice for 10 min in the absence of ligands (lanes 2, 6, and 10) or in the presence of 10⁻⁵ M GW1929 (lanes 3, 7, and 11), 10⁻⁸ M LG268 (lanes 4, 8, and 12), or both ligands (lanes 5, 9, and 13). DNA probe and GST-DRIP205 (lanes 2 to 5), GST–SRC-1 (lanes 3 to 9), or GST-ACTR (lanes 10 to 13) were added to the samples, and the indicated complexes were resolved by EMSA. (B) The PPARγ AF-2 mutation abolishes DRIP205, but not SRC-1 or ACTR binding to PPARγ-RXR. Purified RXR and in vitro-translated AF-2 mutated PPARγ (L466A and L467A) were combined, and assays were carried out as described for panel A. (C) Comparison of DRIP205 binding to PPARγ-RXR and PPARγ-RXR AF-2 mutant at high LG268 concentrations. PPARγ was incubated with either wild-type RXR (lanes 2 to 5) or AF-2 mutated RXR (lanes 7 to 10), and assays were carried out as described above, except that 10⁻⁶ M LG268 was used.

FIG. 5

NR box 1 of DRIP205 directs the binding of DRIP205 to PPARγ-RXR. Baculovirus-expressed RXR and in vitro-translated PPARγ were combined in the presence of 10⁻⁵ M GW1929 and 10⁻⁸ M LG268. PPRE probe and GST-DRIP205 (residues 527 to 774; lane 2), GST-DRIP205ΔNR1 (residues 604 to 774; lane 3), or GST-DRIP205 ΔNR2 (residues 527 to 604; lane 4) were then added, and the indicated complexes were resolved by EMSA.

FIG. 6

SRC-1 excludes DRIP205 binding to PPARγ-RXR. Baculovirus-expressed RXR and in vitro-translated PPARγ were combined in the absence of ligands (lane 1) or in the presence of both 10⁻⁵ M GW1929 and 10⁻⁸ M LG268 (lanes 2 to 5), together with the indicated amounts of both SRC-1 and DRIP205. Complexes were then resolved by EMSA. Note that a truncated DRIP205 construct was used in this experiment to resolve the two coactivator complexes.

FIG. 7

DRIP205 but not GRIP-1 enhances transactivation in response to a PPARγ-specific ligand, and GRIP-1 but not DRIP205 enhances the response to an RXR-specific ligand. NIH 3T3 cells were transfected with a reporter containing three copies of the acyl-CoA oxidase PPRE cloned upstream of thymidine kinase promoter-luciferase and expression vectors for PPARγ, RXR-α, DRIP205, or GRIP-1. The amounts of coactivators are indicated. Transfected cells were treated with 10⁻⁵ M GW1929, 10⁻⁶ M LG101305, or both ligands for 24 h. Luciferase activity was normalized based on β-galactosidase activity. The results are expressed as the fold induction over the control. Each value represents the mean of three independent experiments. Bars: □, Vehicle; ▧, LG101305; ░⃞, GW1929; ▪, GW1929 plus LG101305.

FIG. 8

Model for the selective recruitment of coactivator complexes by PPARγ- and RXR-specific ligands. See text for details.