FIG. 6.

Agonist activity and helix 12 are required for ligand-stimulated degradation. (A) CV1 cells were transfected with an expression plasmid for hRXRα, and cells were incubated for 36 h in the absence (lane 1) or presence of 1.0 μM LGD1268 (RXR-selective agonist; lane 2) or 100 nM LG100754 (RXR-specific antagonist; lane 3). After incubation with ligands, RXR protein levels were examined by Western blotting with anti-RXR and anti-TBP antibodies. (B) CV1 cells were transfected with an expression plasmid for hRXRα and incubated for 36 h in the absence (lane 1) or presence of 10 nM LGD1268 (RXR-selective agonist; lane 2), 1.0 μM LG100754 (RXR-specific antagonist; lane 3), 10 nM LGD1268 plus 1.0 μM LG100754 (lane 4). RXR levels were analyzed as described for panel A. (C) CV1 cells were transfected with an expression plasmid for hRXRα (lanes 1 and 2) or the helix 12 (AF-2) deletion mutant RXR443 (lanes 3 and 4). Cells were incubated for 36 h in the absence (lanes 1 and 3) or presence (lanes 2 and 4) of 1.0 μM LGD1268. RXR levels were analyzed as described for panel A.