Abstract
To assess the structural requirements for G(s) coupling by prostaglandin E receptors (EPs), the G(s)-coupled EP2 and G(i)-coupled EP3beta receptors were used to generate hybrid receptors. Interchanging of the whole i2 loop and its N-terminal half (i2N) had no effect on the binding of both receptors expressed in HEK293 cells. Agonist-induced cAMP formation was observed in wild type EP2 but not in the i2 loop- or i2N-substituted EP2. Wild type EP3beta left cAMP levels unaffected, whereas i2 loop- and i2N-substituted EP3 gained agonist-induced adenylyl cyclase stimulation. In EP2, the ability to stimulate cAMP formation was lost by mutation of Tyr(143) into Ala but retained by mutations into Phe, Trp, and Leu. Consistent with this observation, substitution of the equivalent His(140) enabled EP3beta to stimulate cAMP formation with the rank order of Phe > Tyr > Trp > Leu. The point mutation of His(140) into Phe was effective in another EP3 variant in which its C-terminal tail is different or lacking. Simultaneous mutation of the adjacent Trp(141) to Ala but not at the following Tyr(142) weakened the acquired ability to stimulate cAMP levels in the EP3 mutant. Mutation of EP2 at adjacent Phe(144) to Ala but not at Tyr(145) reduced the efficiency of agonist-induced cAMP formation. In Chinese hamster ovary cells stably expressing G(s)-acquired EP3 mutant, an agonist-dependent cAMP formation was observed, and pertussis toxin markedly augmented cAMP formation. These results suggest that a cluster of hydrophobic aromatic amino acids in the i2 loop plays a key role for G(s) coupling.
Highlights
Individual members of the superfamily of G protein-coupled receptors (GPCRs)1 efficiently interact only with a subset of the many structurally similar G protein heterotrimers [1,2,3]
For every mutant receptor used in this study, the expression of receptor proteins in HEK293 cells was examined by immunofluorescent analysis using antibodies against the N-terminal region of the mouse EP2 and EP3 receptors under nonpermeabilized conditions, and membrane surface expression and the expression levels of each mutant receptor were found to be comparable with those of wild-type receptors (Fig. 1A and data not shown)
In the current system, we could hardly detect cAMP increases with any significant difference in wildtype EP3␥ and T335 even in the presence of 10Ϫ5 M agonist. These results suggested that the effects of i2 loop mutation on Gs coupling of EP3 are independent of C-terminal structure, which is likely to govern the balance of constitutive and agonist-induced G protein activation as observed in the Gi activity of the EP3 isoforms
Summary
Materials—Sulprostone and butaprost were generous gifts from Dr M. PGE2-binding Assay—The harvested HEK293 cells expressing each receptor were homogenized using a Potter-Elvehjem homogenizer in 20 mM Tris-HCl (pH 7.5), containing 10 mM MgCl2, 1 mM EDTA, 20 M indomethacin, and 0.1 mM phenylmethylsulfonyl fluoride. The receptor-expressing HEK293 cells cultured in 24-well plates (2 ϫ 105 cells/well) were washed with HEPES-buffered saline containing 140 mM NaCl, 4.7 mM KCl, 2.2 mM CaCl2, 1.2 mM MgCl2, 1.2 mM KH2PO4, 11 mM glucose, 10 M indomethacine, and 15 mM HEPES, pH 7.4, and preincubated for 10 min. Statistical analysis was carried out by Student’s t test. p values of Ͻ0.005 were considered to indicate a significant difference
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