The key amino acid residue of prostaglandin EP3 receptor for governing G protein association and activation steps

To assess the role of the conserved DPWXY motif of the seventh transmembran e domain in prostanoid receptor-mediated G protein activation, we have muta ted the negatively charged Asp-318 in this motif of the Gi-coupled mouse pr ostaglandin EP3 receptor to uncharged but polar Asn (EP3-D318N) and to the nonpolar Leu (EP3-D318L). The EP3 agonist and antagonist showed similar bin ding affinities for the mild-type and two mutant receptors. The wild-type a nd EP3-D318N receptors but not EP3-D318L receptor associated with Gi in gua nine nucleotide- and pertussis toxin-sensitive manners. On the other hand, the wild-type receptor but not two mutant receptors had the ability to stim ulate GTPase activity and to inhibit the adenylate cyclase. These findings demonstrate that the chemical nature of the amino acid residue at position 318 of the seventh transmembrane domain of the EP3 receptor dissociates the step of Gi association from that of subsequent Gi activation in the proces s of the EP3 receptor-Gi coupling (C) 1999 Academic Press.