Different residues mediate recognition of 1-O-oleyl-lysophosphatidic acid and rosiglitazone in the ligand binding domain of peroxisome proliferator-activated receptor γ

Here we showed that a naturally occurring ether analog of lysophosphatidic acid, 1-O-octadecenyl-2-hydroxy-sn-glycero-3-phosphate (AGP), is a high affinity partial agonist of the peroxisome proliferator-activated receptor gamma (PPAR gamma). Binding studies using the PPAR gamma ligand binding domain showed that [P-32]AGP and [H-3]rosiglitazone (Rosi) both specifically bind to PPAR gamma and compete with each other. [P-32]AGP bound PPAR gamma with an affinity (K-d(app) 60 nM) similar to that of Rosi. However, AGP displaced similar to 40% of bound [H-3]Rosi even when applied at a 2000-fold excess. Activation of PPAR gamma reporter gene expression by AGP and Rosi showed similar potency, yet AGP-mediated activation was similar to 40% that of Rosi. A complex between AGP and PPAR gamma was generated using molecular modeling based on a PPAR gamma crystal structure. AGP-interacting residues were compared with Rosi-interacting residues identified within the Rosi-PPAR gamma co-crystal complex. These comparisons showed that the two ligands occupy partially overlapping positions but make different hydrogen bonding and ion pairing interactions. Site-specific mutants of PPAR gamma were prepared to examine individual ligand binding. H323A and H449A mutants showed reduced binding of Rosi but maintained binding of AGP. In contrast, the R288A showed reduced AGP binding but maintained Rosi binding. Finally, alanine replacement of Tyr-473 abolished binding and activation by Rosi and AGP. These observations indicate that the endogenous lipid mediator AGP is a high affinity ligand of PPAR gamma but that it binds via interactions distinct from those involved in Rosi binding. These distinct interactions are likely responsible for the partial PPAR gamma agonism of AGP.