THE 2ND EXTRACELLULAR LOOP OF THE PROSTAGLANDIN EP3 RECEPTOR IS AN ESSENTIAL DETERMINANT OF LIGAND SELECTIVITY

The prostaglandin EP3 receptor binds Prostaglandin E-2 in a ligand bin ding pocket formed in part by seven transmembrane alpha-helices. The p resent studies demonstrate that the second extracellular loop of the r eceptor is involved in prostanoid ligand recognition as well. Site-dir ected mutagenesis of seven conserved residues clustered in the amino p ortion of the second extracellular loop was performed. Receptors with single amino acid substitutions at each of these positions were transi ently transfected into HEK293tsA201 cells, their ligand binding profil es assessed, and each receptor was tested for its ability to decrease intracellular cAMP levels, Substitution of Trp(199) or Thr(202) with a lanine resulted in receptors with increases in affinity up to 128-fold for prostanoid compounds with a C1 methyl ester but wild type affinit ies for natural prostanoid ligands that have a carboxylate moiety at t he C1 position. In contrast, substitution of Pro(200) with serine caus ed a loss of selectivity up to 20-fold for naturally occurring prostan oid agonists as compared with the wild type EP3 receptor: the PS200 re ceptor displayed a decrease in affinity for E-ring compounds and an in crease in affinity for F- and D-ring compounds. The EC50 for inhibitio n of cAMP remained unchanged for each receptor tested.