SELECTIVE COUPLING OF PROSTAGLANDIN-E RECEPTOR EP3D TO G(I) AND G(S) THROUGH INTERACTION OF ALPHA-CARBOXYLIC ACID OF AGONIST AND ARGININE RESIDUE OF 7TH TRANSMEMBRANE DOMAIN

Prostaglandin (PG) E receptor EP3D is coupled to both G(i) and G(s). To examine the roles of the interaction of alpha-carboxylic acid of PGE(2) and its putative binding site, the arginine residue in the seventh transmembrane domain of EP3D, in receptor-G protein coupling, we have mutated the arginine residue to the noncharged glutamine. PGE(2) with a negatively charged alpha-carboxylic acid and sulprostone, an EP3 agonist with a noncharged modified alpha-carboxylic acid, inhibited the forskolin-stimulated adenylate cyclase activity via G(i) activation in the EP3D receptor in the same concentration-dependent manner, In contrast, the adenylate cyclase stimulation via G(s) activation by sulprostone was much lower than that by PGE(2). On the other hand, both PGE(2) and sulprostone showed potent G(i) activity but failed to show G(s) activity in the mutant receptor. EP3D receptor showed a high affinity binding for PGE(2) in the form coupled to either G(i) or G(s). Although the mutant receptor showed high affinity binding when coupled to G(i), it lost high affinity binding in the condition of G(s) coupling. Furthermore, sulprostone bound to the G(i)-coupled EP3D receptor with higher affinity than the G(s)-coupled receptor. Among various EP3 agonists, ly-carboxylic acid-unmodified agonists showed both G(i) and G(s) activities, but the modified agonists showed only G(i) activity. These findings suggest that the interaction between the alpha-carboxylic acid of PGE(2) and the arginine residue of the receptor regulates the selectivity of the G protein coupling.