A single amino-acid substitution in the EP2 prostaglandin receptor confers responsiveness to prostacyclin analogs.

A high degree of homology between the four G s -coupled prostaglandin (PG) receptors [EP 2 , EP 4 , prostacyclin (IP), PGD 2 (DP)] and the four G q /G i -coupled receptors [EP 1 , EP 3 , PGF 2α (FP), thromboxane A 2 (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. EP 2 receptor transmembrane domain residues that are similar to those in the EP 4 receptor but differ from those in the IP receptor were mutated to the corresponding IP receptor residue. Activation of the mutant receptors by PGE 2 (EP 2 ligand), iloprost (stable prostacyclin analog), and PGE 1 (EP 2 /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 PGD 2 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.