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 EP3 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 EP2 and EP4 receptors was mutated to alanine. When expressed in COS1 cells, detectable radioligand binding at both of these receptors bearing the threonine to alanine substitution (EP2T185A; EP4T168A) was abolished, as well as the receptors' ability to stimulate intracellular [cAMP]. In contrast, EP2 and EP4 receptors bearing conservative threonine to serine mutations (EP2T185S; EP4T168S) displayed Kd values for [3H]prostaglandin E2 similar to wild type receptors: 8.8 +/- 0.7 nM for EP2T185S compared to 12.9 +/- 1.2 nM for EP2 wild type; 2.0 +/- 0.8 nM for EP4T168S compared to 0.9 +/- 0.3 nM for the EP4 wild type receptor. The EC50 values for cAMP stimulation were 1.3 +/- 0.6 nM for EP2 wild type; 2.7 +/- 1.3 nM for EP2T185S; 1.1 +/- 0.3 nM for EP4 wild type; and 1.4 +/- 0.33 nM for EP4T168S. 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.