Identification of determinants of ligand binding affinity and selectivity in the prostaglandin D2 receptor CRTH2.

Article Details

Citation

Copy to clipboard

Hata AN, Lybrand TP, Breyer RM

Identification of determinants of ligand binding affinity and selectivity in the prostaglandin D2 receptor CRTH2.

J Biol Chem. 2005 Sep 16;280(37):32442-51. Epub 2005 Jul 19.

PubMed ID

16030019 [ View in PubMed

]

Abstract

The chemoattractant receptor-homologous molecule expressed on Th2 cells (CRTH2) is a G protein-coupled receptor that mediates the pro-inflammatory effects of prostaglandin D(2) (PGD(2)) generated in allergic inflammation. The CRTH2 receptor shares greatest sequence similarity with chemoattractant receptors compared with prostanoid receptors. To investigate the structural determinants of CRTH2 ligand binding, we performed site-directed mutagenesis of putative mCRTH2 ligand-binding residues, and we evaluated mutant receptor ligand binding and functional properties. Substitution of alanine at each of three residues in the transmembrane (TM) helical domains (His-106, TM III; Lys-209, TM V; and Glu-268, TM VI) and one in extracellular loop II (Arg-178) decreased PGD(2) binding affinity, suggesting that these residues play a role in binding PGD(2). In contrast, the H106A and E268A mutants bound indomethacin, a nonsteroidal anti-inflammatory drug, with an affinity similar to the wild-type receptor. HEK293 cells expressing the H106A, K209A, and E268A mutants displayed reduced inhibition of intracellular cAMP and chemotaxis in response to PGD(2), whereas the H106A and E268A mutants had functional responses to indomethacin similar to the wild-type receptor. Binding of PGE(2) by the E268A mutant was enhanced compared with the wild-type receptor, suggesting that Glu-268 plays a role in determining prostanoid ligand selectivity. Replacement of Tyr-261 with phenylalanine did not affect PGD(2) binding but decreased the binding affinity for indomethacin. These results provided the first details of the ligand binding pocket of an eicosanoid-binding chemoattractant receptor.

DrugBank Data that Cites this Article

Drug Targets

Drug	Target	Kind	Organism	Pharmacological Action	Actions
Indomethacin	Prostaglandin D2 receptor 2	Protein	Humans	Unknown	Other/unknown	Details