Pharmacological characterization of [H-3]-prostaglandin E-2 binding to thecloned human EP4 prostanoid receptor

1 Prostaglandin (PG) E-2 (PGE(2)) is a potent prostanoid derived from arach idonic which can interact with EP1, EP2, EP3 and EP4 prostanoid receptor su btypes. 2 Recombinant human EP4 receptors expressed in human embryonic kidney (HEK- 293) cells were evaluated for their binding characteristics using [H-3]-PGE (2) and a broad panel of natural and synthetic prostanoids in order to defi ne their pharmacological properties. 3 [H-3]-PGE(2) binding was optimal in 2-[N-Morpholino]ethanesulphonic acid (MES) buffer (pH 6.0) yielding 98 +/- 0.7% specific binding. The receptor d isplayed high affinity (K-d = 0.72 +/- 0.12 nM; n = 3) for [H-3]-PGE(2) and interacted with a saturable number of binding sites (B-max = 6.21 +/- 0.84 pmol mg(-1) protein). 4 In competition studies, PGE(2) (K-i = 0.75 +/- 0.03 nM; n = 12) and PGE(1 ) (K-i = 1.45 +/- 0.24 nM; n = 3) displayed high affinities, as did two der ivatives of PGE(1), namely 11-deoxy-PGE(1) (K-i = 1.36 +/- 0.34 nM) and 13, 14-dihydro-PGE(1) (K-i = 3.07 +/- 0.29 nM). 5 Interestingly, synthetic DP receptor-specific agonists such as BW245C (K- i = 64.7 +/- 1.0 nM; n = 3) and ZK118182 (K-i = 425 +/- 42 nM; n = 4), and the purported EP3 receptor-specific ligand enprostil (K-i = 43.1 +/- 4.4 nM ), also displayed high affinity for the EP4 receptor. 6 Two known EP4 receptor antagonists were weak inhibitors of [H-3]-PGE(2) b inding akin to their known functional potencies, thus: AH23848 (K-i = 2690 +/- 232 nM); AH22921 (K-i = 31,800 +/- 4090 nM). 7 These studies have provided a detailed pharmacological characterization o f the recombinant human EP4 receptor expressed in HEK-293 cells.