title: Pharmacological characterization of [(3)H]-prostaglandin E(2) binding to the cloned human EP(4) prostanoid receptor.
abstract: Prostaglandin (PG) E(2) (PGE(2)) is a potent prostanoid derived from arachidonic which can interact with EP(1), EP(2), EP(3) and EP(4) prostanoid receptor subtypes. Recombinant human EP(4) receptors expressed in human embryonic kidney (HEK-293) cells were evaluated for their binding characteristics using [(3)H]-PGE(2) and a broad panel of natural and synthetic prostanoids in order to define their pharmacological properties. [(3)H]-PGE(2) 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 (K(d)=0.72+/-0.12 nM; n=3) for [(3)H]-PGE(2) and interacted with a saturable number of binding sites (B(max)=6.21+/-0.84 pmol mg(-1) protein). 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 derivatives 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). 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 EP(3) receptor-specific ligand enprostil (K(i)=43.1+/-4.4 nM), also displayed high affinity for the EP(4) receptor. Two known EP(4) receptor antagonists were weak inhibitors of [(3)H]-PGE(2) binding akin to their known functional potencies, thus: AH23848 (K(i)=2690+/-232 nM); AH22921 (K(i)=31,800+/-4090 nM). These studies have provided a detailed pharmacological characterization of the recombinant human EP(4) receptor expressed in HEK-293 cells.
authors.affiliation: Molecular Pharmacology Unit, Alcon Research, Ltd., (R2-19) 6201 South Freeway, Fort Worth, Texas, TX 76134, USA.
authors.forename: T L
authors.initials: TL
authors.lastname: Davis
authors.forename: N A
authors.initials: NA
authors.lastname: Sharif
mesh_terms: D001667:Binding, Competitive / Q000187:drug effects; D002460:Cell Line; D002462:Cell Membrane / Q000187:drug effects / Q000378:metabolism; D004274:DNA, Recombinant; D015232:Dinoprostone / Q000378:metabolism* / Q000494:pharmacology; D004305:Dose-Response Relationship, Drug; D006801:Humans; D008024:Ligands; D011453:Prostaglandins / Q000378:metabolism / Q000494:pharmacology; D011869:Radioligand Assay; D018078:Receptors, Prostaglandin E / Q000235:genetics / Q000378:metabolism*; D058309:Receptors, Prostaglandin E, EP4 Subtype; D014316:Tritium
chemical_list: D004274:DNA, Recombinant; D008024:Ligands; C545650:PTGER4 protein, human; D011453:Prostaglandins; D018078:Receptors, Prostaglandin E; D058309:Receptors, Prostaglandin E, EP4 Subtype; D014316:Tritium; D015232:Dinoprostone