A cluster of aromatic amino acids in the i2 loop plays a key role for Gs coupling in prostaglandin EP2 and EP3 receptors.
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.
Sugimoto
Yukihiko
Y
Department of Physiological Chemistry, Graduate School of Pharmaceutical Sciences, Kyoto University, Sakyo-ku, Kyoto 606-8501, Japan.
Nakato
Toshiyuki
T
Kita
Ayumi
A
Takahashi
Yuko
Y
Hatae
Noriyuki
N
Tabata
Hiroyuki
H
Tanaka
Satoshi
S
Ichikawa
Atsushi
A
D000595:Amino Acid Sequence; D024322:Amino Acids, Aromatic / Q000737:chemistry / Q000378:metabolism*; D000818:Animals; D016466:CHO Cells; D002460:Cell Line; D006224:Cricetinae; D019205:GTP-Binding Protein alpha Subunits, Gs / Q000737:chemistry / Q000378:metabolism*; D006801:Humans; D008969:Molecular Sequence Data; D018078:Receptors, Prostaglandin E / Q000819:agonists / Q000737:chemistry / Q000378:metabolism*; D058307:Receptors, Prostaglandin E, EP2 Subtype; D058308:Receptors, Prostaglandin E, EP3 Subtype; D017386:Sequence Homology, Amino Acid
D024322:Amino Acids, Aromatic; C545644:PTGER2 protein, human; C545647:PTGER3 protein, human; D018078:Receptors, Prostaglandin E; D058307:Receptors, Prostaglandin E, EP2 Subtype; D058308:Receptors, Prostaglandin E, EP3 Subtype; D019205:GTP-Binding Protein alpha Subunits, Gs