FIG. 1.

The second LXXLL motif specifically binds to LXRα and LXRβ. (A) A series of chimeric proteins containing amino acids of the second LXXLL motif region within the context of ASC2-2c (i.e., the ASC-2 residues 849 to 1057) were constructed using two-step PCR procedures and synthesized as labeled proteins expressed by in vitro translation using the TNT-coupled transcription-translation system, with conditions as described by the manufacturer. These proteins were incubated with GST fusion to TRβ and LXRβ in the absence and presence of either 0.1 μM T3 or 10 μM T0901317, as previously reported (20). Twenty percent of the total reaction mixture was loaded as input. The ASC-2 residues 875 to 907 (shaded) and 1479 to 1511 (open), containing the first and second LXXLL motif, respectively, are shown. (B) The indicated B42 and LexA plasmids were transformed into a yeast strain containing an appropriate lacZ reporter gene, as described previously (24). The shaded and solid bars indicate the absence and presence of 1 μM of 22(R)-hydroxycholesterol, respectively. Better ligand responsiveness was observed with higher doses of 22(R)-hydroxycholesterol (data not shown). −, B42 alone.