FIG. 1.
Mouse CAR activity requires the same structural motifs as ligand-dependent activity by other nuclear receptors. (A) CV-1 cells were cotransfected with different CAR expression plasmids, a luciferase reporter construct containing two copies of the βRE2 response element, and CMX-βgal. Cells were treated with ligands (TCPOBOP at 10 μM and androstanol at 5 μM) for 40 h before luciferase and β-galactosidase activities were measured. (B) Coactivator recruitment assay was performed by mixing wild-type (WT) or mutant CAR, RXR, a 32P-labeled βRE probe, and the bacterially expressed RIDs of SRC-1. TCPOBOP (10 μM) was added to the mix where indicated. Heterodimer formation and migration were not changed by the various receptor mutants. (C) A mammalian two-hybrid experiment was performed by transfecting CV-1 cells with expression plasmids for Gal-SRC-1, VP-CAR LBD, the RXR LBD, and a luciferase reporter containing four copies of a Gal4 response element (UASGx4). Cells were treated for 40 h with ligands before luciferase and β-galactosidase activities were measured. Gal-SRC-1 refers to a fusion between the Gal4 DNA-binding domain and the three RIDs of SRC-1 (wild-type RIDs [WT] or mutations in either of the three LXXLL motifs [RID1m, RID2m, and RID3m]). Western blot analysis indicated that all mutants were expressed to levels equivalent to those of the wild-type proteins (data not shown). (D) Same as panel C except that VP-VDR LBD, VP-TRβ LBD, or VP-RARα LBD was used and the cells were treated with the corresponding ligands, as indicated. WT, wild type. Reporter activity refers to the luciferase value divided by the β-galactosidase value for each point. Fold activation represents the reporter activity of the receptor in the presence of ligand divided by the reporter activity of the same receptor in the absence of ligand.