FIG. 2.

Mouse CAR requires the RXR LBD, but not its coactivator interaction domains, for full activity. (A) A mammalian two-hybrid assay was performed as described for Fig. 1C except that no ligands were added. (B) A mammalian two-hybrid assay was performed as for Fig. 1C. (C) CV-1 cells were cotransfected with the indicated RXR expression plasmids, a luciferase reporter construct containing three copies of an RXRE, and CMX-βgal. Cells were treated for 40 h with 100 nM LG268 before luciferase and β-galactosidase activities were measured. (D) The coactivator recruitment assay was performed by mixing wild-type (WT) or mutant RXR, a ³²P-labeled DR-1 probe, and the bacterially expressed RIDs of SRC-1. LG268 (100 nM) was added to the mix where indicated. Only the bound DNA complexes are shown. Heterodimer formation and migration were not changed by the various receptor mutants. (E) A mammalian two-hybrid experiment was performed by transfecting CV-1 cells with expression vectors for Gal-SRC-1, VP-CAR LBD, the indicated RXR LBD mutants, and a luciferase reporter containing four copies of a Gal4 response element (UAS_Gx4). No ligands were added. (F) Coactivator recruitment assay was performed by mixing CAR, wild-type or mutant RXR, a ³²P-labeled βRE probe, and the bacterially expressed RIDs of SRC-1. Only the bound DNA complexes are shown.