The C-terminus of the prostaglandin-E-receptor EP3 subtype is essential for activation of GTP-binding protein.
Three isoforms of the mouse prostaglandin-E-receptor EP3 subtype (EP3), EP3 alpha, EP3 beta and EP3 gamma, with different C-termini, which are produced through alternative splicing, showed different efficiencies with respect to heterotrimeric GTP-binding protein activation and adenylate cyclase inhibition [Sugimoto, Y., Negishi, M., Hayashi, Y., Namba, T., Honda, A., Watabe, A., Hirata, M., Narumiya, S. & Ichikawa, A. (1993) J. Biol. Chem. 268, 2712-2718; Irie, A., Sugimoto, Y., Namba, T., Harazono, A., Honda, A., Watabe, A., Negishi, M., Narumiya, S. & Ichikawa, A. (1993) Eur. J. Biochem. 217, 313-318]. To assess the role of the C-terminus in GTP-binding protein coupling, we truncated the C-terminus of EP3 at an alternative splicing site and expressed the mutant receptor. The truncated receptor retained the ability to physically associate with Gi2, forming an agonist/receptor/Gi2 ternary complex, and to undergo the characteristic conversion of its agonist-binding affinity, mediated by a guanine nucleotide from a low-affinity state to a high-affinity state. However, sulprostone, an EP3 agonist, failed not only to inhibit the forskolin-induced cAMP accumulation in the mutant receptor-expressing cells but also to stimulate the GTPase activity in the mutant receptor-expressing cell membrane. These results indicated that the C-terminus of EP3 is essential for the activation of GTP-binding protein.
Irie
A
A
Department of Physiological Chemistry, Kyoto University, Japan.
Sugimoto
Y
Y
Namba
T
T
Asano
T
T
Ichikawa
A
A
Negishi
M
M
D000067956:Adenylyl Cyclase Inhibitors; D000595:Amino Acid Sequence; D000818:Animals; D016466:CHO Cells; D005576:Colforsin / Q000378:metabolism / Q000494:pharmacology; D006224:Cricetinae; D000242:Cyclic AMP / Q000378:metabolism; D015232:Dinoprostone / Q000378:metabolism; D020558:GTP Phosphohydrolases / Q000378:metabolism; D019204:GTP-Binding Proteins / Q000378:metabolism*; D008969:Molecular Sequence Data; D009154:Mutation; D011233:Precipitin Tests; D018078:Receptors, Prostaglandin E / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism
D000067956:Adenylyl Cyclase Inhibitors; D018078:Receptors, Prostaglandin E; D005576:Colforsin; D000242:Cyclic AMP; D020558:GTP Phosphohydrolases; D019204:GTP-Binding Proteins; D015232:Dinoprostone