Cloning and characterization of chicken prostaglandin E receptor subtypes 2 and 4 (EP2 and EP4)

Abstract

Prostaglandin E2 (PGE2) belongs to the eicosanoids and is an important chemical mediator regulating many vital physiological processes, including muscle relaxation, vascular homeostasis, gastrointestinal function and maintenance of pregnancy. Prostaglandin E receptor subtypes 2 and 4 (EP2 and EP4) are crucial mediators between its ligand, PGE2, and downstream intracellular cyclic AMP/protein kinase A (cAMP/PKA) signaling pathway. Both receptors were identified inmammals and zebrafish, but they have not been cloned in the avian species. In the present study, using reverse transcription-polymerase chain reaction (RT-PCR), the full-length cDNAs for chicken EP2 and EP4 receptors were cloned from adult chicken ovary and testis respectively. The full-length cDNA of EP2 gene encodes a precursor of 475 amino acids with a high degree of amino acid identity to that of mammals, including human (87%), mouse (86%), rat (84%), dog (85%), and cow (83%), and a low sequence identity to zebrash (52%). Chicken EP4 is 356 amino acids in length and also shows high amino acid identity to that of mammals (human, 61%; mouse, 63%; rat, 61%; dog, 58%; cow, 59%) and a lower sequence identity to lower vertebrate (zebrafish, 41%). Using the pGL3-CRE-luciferase reporter system, we also demonstrated that PGE2 strongly induces luciferase activity of CHO/DF-1 cells expressing EP2 in a dose-dependent manner (EC50: <1 nM), conrming the functionality of the cloned EP2 receptor. The same experiment is also under way for EP4. The cloning and characterization of EP2 and EP4 receptors would help us to establish a molecular basis to elucidate the physiological roles of prostaglandin E2 in target tissues including their actions in chicken ovary.