Figure 3

Impaired heterodimerization and DNA binding activities of CAR isoforms (A) Mammalian two hybrid assays were performed in COS1 cells transfected with combinations of an expression plasmid encoding a GAL4-DBD/RXRα–LBD fusion protein and expression plasmids encoding VP16-AD/CAR-LBD fusion proteins of CAR isoforms, as indicated. The columns show the mean activation factors (± S.D.) of the co-transfected GAL4-dependent reporter through interaction of the GAL4-DBD/RXRα–LBD fusion protein with VP16-AD/CAR-LBD fusion proteins of the CAR isoforms SV1 to SV6. The activity of the GAL4-DBD/RXRα–LBD fusion in the presence of empty expression vector pVP16-AD was designated as 1. (B) Gel electrophoretic analysis of [³⁵S]-methionine labeled in vitro synthesized CAR protein isoforms, as indicated. Equal amounts of DNA of the respective expression plasmids were transcribed and translated in vitro and proteins were analyzed, as described in Experimental Procedures. (C) Electrophoretic mobility shift assays were performed using in vitro translated proteins bound to a radiolabeled doublestranded oligonucleotide corresponding to the DR3 motif of the XREM of CYP3A4. Binding reactions contained (+) or lacked (-) the indicated proteins. Complexes of CAR/RXRα heterodimers with the oligonucleotide are marked by an arrow.