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 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 PGE₂  = PGE₁  ≈#62;  PGF_2α =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 GTPγS 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 R*G, a high affinity G protein coupled state.

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