Structural basis of coactivation of liver receptor homolog-1 by β-catenin

Abstract

We report the three-dimensional structure of a β-catenin armadillo repeat in complex with the liver receptor homolog-1 (LRH-1) ligand binding domain at 2.8 Å resolution as the first structure of β-catenin in complex with any nuclear receptor. The surface of β-catenin that binds LRH-1 partly overlaps defined contact sites for peptide segments of β-catenin partners, including T-cell factor-4. The surface of LRH-1 that engages β-catenin is comprised of helices 1, 9, and 10 and is distinct from known interaction surfaces of LRH-1, including corepressor and coactivator binding sites. Targeted mutagenesis of amino acids forming both sides of the LRH-1/β-catenin interface reveals that they are essential for stable interactions between these proteins in solution. The LRH-1 binding site in β-catenin is also required for association with androgen receptor, providing evidence that the observed LRH-1/β-catenin interaction may be prototypic.

Fig. 1.

Structure of the LRH-1/β-catenin complex. β-catenin is represented in green, and hLRH-1 is in cyan. The ligand of the LRH-1 is in blue (ball and stick).

Fig. 2.

Superposition of the β-catenin structures of TCF-4 complex (PDB: 1JDH) and of LRH-1 LBD complex (this work). The β-catenin surface is shown in green, and the TCF-4 peptide is colored red. LRH-1 LBD is colored in cyan.

Fig. 3.

(A) Interfacial residues of the complex of β-catenin and LRH-1 LBD. β-catenin is colored in green and Y306, K345, and W383 in the β-catenin are shown in ball and stick model. LRH-1 is colored in cyan and D483, M486, and T493 in the LRH-1 are shown ball and stick. (B) 2F_o-F_c omit map of LRH-1 LBD and β-catenin interface. The map is contoured at 1.77σ, visualized with model in Coot (Fig. S4).

Fig. 4.

Point mutations diminish the LRH-1/β-catenin LBD interaction. (A) ³⁵S-labeled β-catenin or β-catenin mutants pulled down by bacterially expressed GST-LRH-1 LBD. In this figure, and similar figures below, input refers to 10% of the total amount of radiolabeled input protein included in the initial binding reaction and control refers to the amount of material retained on beads that contain GST protein without an in frame fusion. (B)³⁵S-labeled full length LRH-1 or LRH-1 mutants pulled down by bacterially expressed GST-β-catenin armadillo repeat (138–663). (C) ³⁵S-labeled full length LRH-1 pulled down by bacterially expressed GST-β-catenin armadillo repeat +/- increasing doses of TCF-4 or BCL9 derived peptide.

Fig. 5.

(A) Results of luciferase assays performed on extracts of cells transfected with a GAL-responsive reporter and expression vectors for a GAL-LRH-1 fusion protein (pM is the empty vector) and wild type or mutant β-catenin. Number of the data point, n = 6. (B) Luciferase assays performed on cells transfected with a TCF-4 driven expression vector and expression vectors for TCF-4 +/- wild-type or mutant β-catenin. n = 3 (C) As in Fig. 5A except that experiment utilized wild type or mutant versions of GAL-LRH-1 LBD expression vector +/- wild-type β-catenin. n = 5. This is another independent experiment from the one in Fig. 5A. (A–C) Bar represents mean + /-SEM *P < 0.05; **P < 0.01. N.S., not significant.

Fig. 6.

Mutations of β-catenin amino acids that bind LRH-1 also disrupt AR interactions. (A) Results of luciferase assays using GAL-AR LBD +/- DHT and wild type or mutant β-catenin expression vector, as in Fig. 6D. n = 4. Bar represents mean + /-SEM *P < 0.05; **P < 0.01 (B) ³⁵S-labeled β-catenin or β-catenin mutants pulled down by bacterially expressed GST-AR LBD. (C) ³⁵S-labeled full length AR pulled down by bacterially expressed GST-β-catenin armadillo repeat +/- TCF-4 or BCL9 derived peptide.