Pharmacological characterization of [3H]‐prostaglandin E2 binding to the cloned human EP4 prostanoid receptor

Prostaglandin (PG) E2 (PGE2) is a potent prostanoid derived from arachidonic which can interact with EP1, EP2, EP3 and EP4 prostanoid receptor subtypes. Recombinant human EP4 receptors expressed in human embryonic kidney (HEK‐293) cells were evaluated for their binding characteristics using [3H]‐PGE2 and a broad panel of natural and synthetic prostanoids in order to define their pharmacological properties. [3H]‐PGE2 binding was optimal in 2‐[N‐Morpholino]ethanesulphonic acid (MES) buffer (pH 6.0) yielding 98±0.7% specific binding. The receptor displayed high affinity (Kd=0.72±0.12 nM; n=3) for [3H]‐PGE2 and interacted with a saturable number of binding sites (Bmax=6.21±0.84 pmol mg−1 protein). In competition studies, PGE2 (Ki=0.75±0.03 nM; n=12) and PGE1 (Ki=1.45±0.24 nM; n=3) displayed high affinities, as did two derivatives of PGE1, namely 11‐deoxy‐PGE1 (Ki=1.36±0.34 nM) and 13,14‐dihydro‐PGE1 (Ki=3.07±0.29 nM). Interestingly, synthetic DP receptor‐specific agonists such as BW245C (Ki=64.7±1.0 nM; n=3) and ZK118182 (Ki=425±42 nM; n=4), and the purported EP3 receptor‐specific ligand enprostil (Ki=43.1±4.4 nM), also displayed high affinity for the EP4 receptor. Two known EP4 receptor antagonists were weak inhibitors of [3H]‐PGE2 binding akin to their known functional potencies, thus: AH23848 (Ki=2690±232 nM); AH22921 (Ki=31,800±4090 nM). These studies have provided a detailed pharmacological characterization of the recombinant human EP4 receptor expressed in HEK‐293 cells. British Journal of Pharmacology (2000) 130, 1919–1926; doi:10.1038/sj.bjp.0703525