Functional interaction of the carboxylic acid group of agonists and the arginine residue of the seventh transmembrane domain of prostaglandin E receptor EP3 subtype

Prostaglandin (PG) E₂ binds to PGE receptor EP3 subtype and induces G_i activity. To assess the role of the interaction of the carboxylic acid group of agonists and its putative binding site, Arg-309 in the seventh transmembrane domain of EP3α receptor, in receptor activation, we have mutated the positively charged Arg-309 to the polar but uncharged Gln (EP3α-R309Q) and Asn (EP3α-R309N), and to the non-polar Leu (EP3α-R309L). Wild-type, EP3α-R309Q and EP3α-R309N receptors showed high-affinity binding for PGE₂, but the EP3α-R309L receptor showed very-low-affinity binding. Guanosine 5´-[γ-thio]triphosphate increased the PGE₂ binding to the wild-type receptor, decreased the binding to EP3α-R309Q and EP3α-R309N receptors, but did not affect that to the EP3α-R309L receptor. Furthermore we examined the G_i activities of two types of EP3 agonist, TEI-3356 with a negatively charged carboxylic acid, and TEI-4343, a methyl ester of TEI-3356 with an uncharged but polar group, towards those receptors. Both agonists inhibited the forskolin-stimulated cAMP formation in wild-type, EP3α-R309Q and EP3α-R309N receptors in the same concentration-dependent manner, but the agonists showed a very low inhibition of EP3α-R309L receptor. These findings demonstrate that the hydrogen-bonding interaction of EP3 agonists and residue 309 in the seventh transmembrane domain of the EP3α receptor is sufficient for the functional activation of the EP3α receptor.