Ligand-induced coactivator recruitment to peroxisome proliferator-activated receptorα characterized by fluorescence resonance energy transfer

Publisher Summary This chapter describes the way the affinity of ligands can be measured for peroxisome proliferator-activated receptorα (PPARα) using the fluorescence resonance energy transfer (FRET) assay utilizing a small peptide derived from coactivators containing the LXXLL receptor interaction site. It also compares the results with more traditional methods, such as scintillation proximity assay. The FRET assay has several advantages over conventional assays for determining affinities of PPARα ligands. It is a nonradioactive assay and is more sensitive than the scintillation proximity assay (SPA) for certain ligands, such as gemfibrozil and fenofibric acid. A known, high-affinity (labeled) ligand is not required as in SPA and is therefore ideal for identifying agonist ligands, especially for orphan receptors. The FRET assay can also be performed in the presence of a known agonist to detect receptor antagonists. However, the reagents are relatively expensive. Because the peptide is only a small part of the whole coactivator and the coactivators have multiple receptor interacting domains, the affinities determined with a peptide may be different from that using the whole coactivator protein.