A Cluster of Aromatic Amino Acids in the i2 Loop Plays a Key Role for G_s Coupling in Prostaglandin EP2 and EP3 Receptors

To assess the structural requirements for G_s coupling by prostaglandin E receptors (EPs), the G_s-coupled EP2 and Gi-coupled EP3β receptors were used to generate hybrid receptors. Interchanging of the whole i2 loop and its N-terminal half (i2N) had no effect on the binding of both receptors expressed in HEK293 cells. Agonist-induced cAMP formation was observed in wild type EP2 but not in the i2 loop- or i2N-substituted EP2. Wild type EP3β left cAMP levels unaffected, whereas i2 loop- and i2N-substituted EP3 gained agonist-induced adenylyl cyclase stimulation. In EP2, the ability to stimulate cAMP formation was lost by mutation of Tyr ¹⁴³ into Ala but retained by mutations into Phe, Trp, and Leu. Consistent with this observation, substitution of the equivalent His ¹⁴⁰ enabled EP3β to stimulate cAMP formation with the rank order of Phe > Tyr > Trp > Leu. The point mutation of His ¹⁴⁰ into Phe was effective in another EP3 variant in which its C-terminal tail is different or lacking. Simultaneous mutation of the adjacent Trp¹⁴¹ to Ala but not at the following Tyr¹⁴² weakened the acquired ability to stimulate cAMP levels in the EP3 mutant. Mutation of EP2 at adjacent Phe¹⁴⁴ to Ala but not at Tyr¹⁴⁵ reduced the efficiency of agonist-induced cAMP formation. In Chinese hamster ovary cells stably expressing G_s-acquired EP3 mutant, an agonist-dependent cAMP formation was observed, and pertussis toxin markedly augmented cAMP formation. These results suggest that a cluster of hydrophobic aromatic amino acids in the i2 loop plays a key role for G_s coupling.