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.