title: Characterization of [3H]-prostaglandin E2 binding to prostaglandin EP4 receptors expressed with Semliki Forest virus.
abstract: 1. The human prostaglandin EP4 receptor has been expressed by use of the Semliki Forest virus system. 2. In cell membranes [3H]-prostaglandin E2 ([3H]-PGE2) bound to a high affinity site with a Kd of 1.12 +/- 0.3 nM and a Bmax of 3.1 +/- 0.3 pmol mg-1 protein. 3. In competition studies the rank order of potency for prostaglandins was PGE2 = PGE1 > > PGE2 alpha = PGI2. 4. The binding of [3H]-PGE2 to cell membranes was inhibited by approximately 60% by the addition of guanylnucleotides, suggesting that this proportion of the receptors was G-protein coupled. 5. [3H]-PGE2 binding was increased by greater than 200% by the addition of divalent cations, with little change in the IC50 of PGE2. 6. In saturation studies removal of divalent cations and addition of GTP gamma S resulted in a 65% reduction in the Bmax with no change in the Kd. These results are consistent with the ligand labelling two states of the receptor R*, a high affinity state and R*G, a high affinity G protein coupled state.
authors.affiliation: Receptor Systems Unit, Glaxo Wellcome Medicines Research Centre, Hertfordshire.
authors.forename: F H
authors.initials: FH
authors.lastname: Marshall
authors.forename: K
authors.initials: K
authors.lastname: Patel
authors.forename: K
authors.initials: K
authors.lastname: Lundstrom
authors.forename: J
authors.initials: J
authors.lastname: Camacho
authors.forename: S M
authors.initials: SM
authors.lastname: Foord
authors.forename: M G
authors.initials: MG
authors.lastname: Lee
mesh_terms: D001667:Binding, Competitive; D015232:Dinoprostone / Q000378:metabolism*; D016244:Guanosine 5'-O-(3-Thiotriphosphate) / Q000494:pharmacology; D006801:Humans; D007700:Kinetics; D008274:Magnesium / Q000494:pharmacology; D018078:Receptors, Prostaglandin E / Q000378:metabolism*; D011994:Recombinant Proteins / Q000378:metabolism; D012672:Semliki forest virus / Q000235:genetics
chemical_list: D018078:Receptors, Prostaglandin E; D011994:Recombinant Proteins; D016244:Guanosine 5'-O-(3-Thiotriphosphate); D008274:Magnesium; D015232:Dinoprostone