A conserved threonine in the second extracellular loop of the human EP2 and EP4 receptors is required for ligand binding

Abstract G protein coupled receptors for prostaglandins are activated when agonists are bound to a binding pocket formed in part by the seven transmembrane domains. Recent studies have determined that substitution of a conserved threonine in the second extracellular loop of the prostaglandin EP 3 receptor resulted in increased affinity for ligands with a C1 methyl ester moiety. The homologous threonine in the second extracellular loop of the human prostaglandin EP 2 and EP 4 receptors was mutated to alanine. When expressed in COS1 cells, detectable radioligand binding at both of these receptors bearing the threonine to alanine substitution (EP 2 T185A; EP 4 T168A) was abolished, as well as the receptors' ability to stimulate intracellular [cAMP]. In contrast, EP 2 and EP 4 receptors bearing conservative threonine to serine mutations (EP 2 T185S; EP 4 T168S) displayed K d values for [ 3 H ]prostaglandin E 2 similar to wild type receptors: 8.8±0.7 nM for EP 2 T185S compared to 12.9±1.2 nM for EP 2 wild type; 2.0±0.8 nM for EP 4 T168S compared to 0.9±0.3 nM for the EP 4 wild type receptor. The EC 50 values for cAMP stimulation were 1.3±0.6 nM for EP 2 wild type; 2.7±1.3 nM for EP 2 T185S; 1.1±0.3 nM for EP 4 wild type; and 1.4±0.33 nM for EP 4 T168S. These studies suggest a critical role for the hydroxyl moiety on these conserved threonine residues at position 168/185 of the second extracellular loop in prostaglandin receptor–ligand interactions.