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

A high degree of homology between the four Gs-coupled prostaglandin (PG) receptors [EP2, EP4, prostacyclin (IP), PGD2 (DP)] and the four Gq/Gi-coupled receptors [EP1, EP3, PGF2alpha (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 PGF2alpha 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.