Abstract

A high degree of homology between the four G_s-coupled prostaglandin (PG) receptors [EP₂, EP₄, prostacyclin (IP), PGD₂ (DP)] and the four G_q/G_i-coupled receptors [EP₁, EP₃, PGF_2α (FP), thromboxane A₂(TP)] suggests that prostaglandin receptors evolved functionally from an ancestral EP receptor before the development of distinct binding epitopes. If so, ligand selectivity should be determined by a limited number of amino acids. EP₂ receptor transmembrane domain residues that are similar to those in the EP₄ receptor but differ from those in the IP receptor were mutated to the corresponding IP receptor residue. Activation of the mutant receptors by PGE₂ (EP₂ ligand), iloprost (stable prostacyclin analog), and PGE₁ (EP₂/IP ligand) was determined using a cAMP-dependent reporter gene assay. A Leu304-to-tyrosine substitution in the seventh transmembrane domain enhanced iloprost potency approximately 100-fold. A glycine substitution at Ser120 in the third transmembrane domain had no effect on drug potency but improved the response of the Tyr304 mutant. The potency of the natural prostaglandins PGF_2α and PGD₂ was not enhanced by the mutations. In contrast, the potency of all prostaglandins was reduced 10- to 100-fold when arginine 302, which is thought to be a counterion for the prostaglandin carboxylic acid, was mutated. Thus, a single amino acid change resulted in a selective gain of function for iloprost, which is consistent with the proposed phylogeny of the prostaglandin receptors.