A Cluster of Aromatic Amino Acids in the i2 Loop Plays a Key Role for Gs Coupling in Prostaglandin EP2 and EP3 Receptors

Yukihiko Sugimoto, Toshiyuki Nakato, Ayumi Kita, Yuko Takahashi, Noriyuki Hatae, Hiroyuki Tabata, Satoshi Tanaka, Atsushi Ichikawa

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Abstract

To assess the structural requirements for Gs coupling by prostaglandin E receptors (EPs), the Gs-coupled EP2 and Gi-coupled EP3β 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 EP3β 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 EP3β 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 Trp141 to Ala but not at the following Tyr142 weakened the acquired ability to stimulate cAMP levels in the EP3 mutant. Mutation of EP2 at adjacent Phe144 to Ala but not at Tyr145 reduced the efficiency of agonist-induced cAMP formation. In Chinese hamster ovary cells stably expressing Gs-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 Gs coupling.

Original languageEnglish
Pages (from-to)11016-11026
Number of pages11
JournalJournal of Biological Chemistry
Volume279
Issue number12
DOIs
Publication statusPublished - Mar 19 2004
Externally publishedYes

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ASJC Scopus subject areas

  • Biochemistry

Cite this

Sugimoto, Y., Nakato, T., Kita, A., Takahashi, Y., Hatae, N., Tabata, H., Tanaka, S., & Ichikawa, A. (2004). A Cluster of Aromatic Amino Acids in the i2 Loop Plays a Key Role for Gs Coupling in Prostaglandin EP2 and EP3 Receptors. Journal of Biological Chemistry, 279(12), 11016-11026. https://doi.org/10.1074/jbc.M307404200