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

Abstract

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 γ (PPARγ). Binding studies using the PPARγ ligand binding domain showed that [32P]AGP and [3H]rosiglitazone (Rosi) both specifically bind to PPARγ and compete with each other. [ 32P]AGP bound PPARγ with an affinity (Kd(app) 60 nM) similar to that of Rosi. However, AGP displaced ∼40% of bound [ 3H]Rosi even when applied at a 2000-fold excess. Activation of PPARγ reporter gene expression by AGP and Rosi showed similar potency, yet AGP-mediated activation was ∼40% that of Rosi. A complex between AGP and PPARγ was generated using molecular modeling based on a PPARγ crystal structure. AGP-interacting residues were compared with Rosi-interacting residues identified within the Rosi-PPARγ 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 PPARγ 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 PPARγ but that it binds via interactions distinct from those involved in Rosi binding. These distinct interactions are likely responsible for the partial PPARγ agonism of AGP. © 2006 by The American Society for Biochemistry and Molecular Biology, Inc.

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Journal of Biological Chemistry

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