Fig. 5.

Leu-100 is predicted to contact 3Cl-AHPC within the SHP ligand binding pocket and is important for the enhancement of SHP repression of LRH-1 transactivation by 3Cl-AHPC. A, Predicted structure of the ligand binding pocket of mouse SHP occupied by 3Cl-AHPC with amino acid residues that contact 3Cl-AHPC indicated. The molecular model for the interaction of 3Cl-AHPC with the SHP was built by using the Prime program as described in Materials and Methods. Residues interacting with the ligand and 3Cl-AHPC are shown as stick models. B and C, Two amino acids, Leu-100 and Ala-148, identified from the molecular modeling as ligand interacting residues (A), were selected for functional cell-based reporter assays. HepG2 cells were cotransfected with Gal4-TATA-luc (B) or CYP7A1 promoter-luc (C) reporters along with expression plasmids, including the SHP mutants L100E or A148D as indicated (for plasmid amounts, see Materials and Methods). Twenty-four hours later, cells were treated with 200 nm 3Cl-AHPC or vehicle overnight, and reporter assays were performed. The values for firefly luciferase activities were normalized by dividing by the values for β-galactosidase activities. The mean and sem are plotted (n = 3). Statistical significance was measured using the Student's t test. *, P < 0.05. NS, Nonsignificant. D, Expression levels of flag-SHP wild type (WT) and mutants were detected by Western blot analysis (WB).