Ligand selectivity of the peroxisome proliferator-activated receptor alpha

Peroxisome proliferator-activated receptors (PPAR alpha, beta, and gamma) a re nuclear hormone receptors that play critical roles in regulating lipid m etabolism. It is well established that PPARs are the targets for the hypoli pidemic synthetic compounds known as peroxisome proliferators, and it has b een proposed that various long-chain fatty acids and metabolites of arachid onic acid serve as the physiological ligands that activate these receptors in vivo. However, a persistent problem is that reported values of the equil ibrium dissociation constants (K(d)s) Of complexes of PPARs with these liga nds an in the micromolar range, at least an order of magnitude higher than the physiological concentrations of the ligands. Thus, the identity of the endogenous ligands for PPAR remains unclear. Here we report on a fluorescen ce-based method for investigating the interactions of PPAR with ligands. It is shown that the synthetic fluorescent long-chain fatty acid trans-parina ric acid binds to PPAR alpha with high affinity and can be used as a probe to monitor protein-ligand interactions by the receptor. Measurements of K(d )s characterizing the interactions of PPAR alpha with various ligands revea led that PPAR alpha interacts with unsaturated C:18 fatty acids, with arach idonic acid, and with the leukotriene LTB4 with affinities in the nanomolar range. These data demonstrate the utility of the optical method in examini ng the ligand-selectivity of PPARs, and resolve a long-standing uncertainty in understanding how the activities of these receptors are regulated in vi vo.