CHARACTERIZATION OF [H-3] PROSTAGLANDIN E-2 BINDING TO PROSTAGLANDIN EP4 RECEPTORS EXPRESSED WITH SEMLIKI-FOREST-VIRUS

1 The human prostaglandin EP4 receptor has been expressed by use of th e Semliki Forest virus system. 2 In cell membranes [H-3]-prostaglandin E-2 ([H-3]-PGE(2)) bound to a high affinity site with a K-d of 1.12+/ -0.3 nM and a B-max of 3.1+/-0.3 pmol mg(-1) protein. 3 In competition studies the rank order of potency for prostaglandins was PGE(2) = PGE (1) much greater than PGF(2 alpha) = PGI(2). 4 The binding of [H-3]-PG E(2) to cell membranes was inhibited by approximately 60% by the addit ion of guanylnucleotides, suggesting that this proportion of the recep tors was C-protein coupled. 5 [H-3]-PGE(2) binding was increased by gr eater than 200% by the addition of divalent cations, with little chang e in the IC50 of PGE(2). 6 In saturation studies removal of divalent c ations and addition of GTP gamma S resulted in a 65% reduction in the B-max with no change in the K-d. These results are consistent with the ligand labelling two states of the receptor R, a high affinity state and RG, a high affinity G protein coupled state.