The important role of residue F268 in ligand binding by LXR beta

Liver X receptors (LXRs) are nuclear receptors that regulate the metabolism of cholesterol and bile acids. Despite information on the specificity of t heir natural ligands, oxysterols, relatively little is known about the liga nd binding site in LXRs, The helix 3 region in the ligand binding domain (L BD) of peroxisome proliferator-activated receptors (PPARs) has been implica ted in ligand entry. Sequence alignment of LXRs, farnesoid X receptor (FXR) , and PPARs identified the corresponding helix 3 region in the LXR beta LED . Residues F268 and T272, which are conserved in all the aligned sequences and only in LXRs and FXR, respectively, were replaced with alanine, The eff ects of these mutations on ligand binding and receptor activation were exam ined using an in vitro ligand binding assay and a cell based reporter assay , respectively. The LXR beta mutant F268A did not bind ligand, In contrast, conversion of T272 to alanine has no effect on ligand binding. By transien tly expressing a chimeric receptor containing Escherichia coli tetracycline repressor (TetR) and LXR beta LED and a reporter with a TetR binding site, we show that mutant F268A lost the ability to activate transcription of th e reporter, whereas mutant T272A still has an activity similar to that of t he wild-type LXR beta. These data, consistent with the findings in the in v itro ligand binding assay and our 3D modeling, are the first study that ide ntifies a residue critical for ligand binding in LXR beta, (C) 2000 Federat ion of European Biochemical Societies. Published by Elsevier Science B.V. A ll rights reserved.