RT Journal Article SR Electronic T1 Extracellular Loop II Modulates GTP Sensitivity of the Prostaglandin EP3 Receptor JF Molecular Pharmacology JO Mol Pharmacol FD American Society for Pharmacology and Experimental Therapeutics SP mol.112.080473 DO 10.1124/mol.112.080473 A1 Chandramohan Natarajan A1 Aaron N. Hata A1 Heidi E. Hamm A1 Roy Zent A1 Richard M. Breyer YR 2012 UL http://molpharm.aspetjournals.org/content/early/2012/10/18/mol.112.080473.abstract AB Unlike the majority of G-protein coupled receptors, the prostaglandin E2 (PGE2) EP3 receptor binds agonist with high affinity that is insensitive to the presence of GTPγS. Here, we report the identification of mutations that confer GTPγS sensitivity to agonist binding. Seven point mutations were introduced into the conserved motif in the second extracellular loop (ECII) of EP3, resulting in acquisition of GTP-sensitive agonist binding. One receptor mutation W203A was studied in detail. Loss of agonist binding was observed on intact HEK293 cells expressing the W203A receptor, conditions where high GTP levels are present; however, high affinity binding [3H]PGE2 was observed in broken cell preparations washed free of GTP. The [3H]PGE2 binding of W203A in broken cell membrane fractions was inhibited by addition of GTPγS (IC50 21 ± 1.8 nM). Taken together, these results suggest that the wild type EP3 receptor displays unusual characteristics of the complex coupled equilibria between agonist-receptor and receptor - G protein interaction. Moreover, mutation of ECII can alter this coupled equilibrium from GTP insensitive agonist binding to more conventional GTP sensitive binding. This suggests that for the mutant receptors, ECII plays a critical role in linking the agonist bound receptor conformation to the G-protein nucleotide bound state.