Extracellular loop II modulates GTP sensitivity of the prostaglandin EP3 receptor.
Unlike the majority of G protein-coupled receptors, the prostaglandin E(2) (PGE(2)) E-prostanoid 3 (EP3) receptor binds agonist with high affinity that is insensitive to the presence of guanosine 5[prime]-O-(3-thio)triphosphate (GTPγS). 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 human embryonic kidney 293 cells expressing the W203A receptor, conditions where high GTP levels are present; however, high affinity binding [(3)H]PGE(2) was observed in broken cell preparations washed free of GTP. The [(3)H]PGE(2) binding of W203A in broken cell membrane fractions was inhibited by addition of GTPγS (IC(50) 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.
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CHEMICAL CHEMICAL CHEMICAL CHEMICAL CHEMICAL CHEMICAL CHEMICAL CHEMICAL GENE-N GENE-N CHEMICAL GENE-N GENE-N GENE-N GENE-N GENE-N GENE-Y GENE-N GENE-Y GENE-N GENE-N CHEMICAL GENE-Y GENE-N GENE-Y CHEMICAL CHEMICAL CHEMICAL CHEMICAL CHEMICAL CHEMICAL CHEMICAL
[(3)H]PGE(2) PGE(2) GTP [(3)H]PGE(2) E-prostanoid GTPγS GTP prostaglandin G protein ECII GTP ECII G protein G protein-coupled receptors second extracellular loop ECII EP3 W203A prostaglandin E(2) (PGE(2)) E-prostanoid 3 (EP3) receptor W203A W203A GTP EP3 Extracellular loop II prostaglandin EP3 receptor nucleotide guanosine 5[prime]-O-(3-thio)triphosphate GTPγS GTPγS GTP prostaglandin E(2) GTP