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 [EP2, EP4, prostacyclin (IP), PGD(2) (DP)] and the four G(q)/G(i)-coupled receptors [EP1, EP3, PGF(2 alpha) (FP), thromboxane A(2) (TP)] suggests that prostaglandin receptors evolved functionally from an ancestral EP receptor before the development of distinct bind ing epitopes. If so, ligand selectivity should be determined by a limi ted 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 PGE(2) (EP2 ligand), iloprost (stable prostacyclin analog), and PGE(1) (EP2/IP ligand) was determine d using a cAMP-dependent reporter gene assay. A Leu804-to-tyrosine sub stitution in the seventh transmembrane domain enhanced iloprost potenc y approximately 100-fold. A glycine substitution at Ser120 in the thir d transmembrane domain had no effect on drug potency but improved the response of the Tyr304 mutant. The potency of the natural prostaglandi ns PGF(2 alpha) and PGD(2) was not enhanced by the mutations. In contr ast, the potency of all prostaglandins was reduced 10- to 100-fold whe n arginine 302, which is thought to be a counterion for the prostaglan din carboxylic acid, was mutated. Thus, a single amino acid change res ulted in a selective gain of function for iloprost, which is consisten t with the proposed phylogeny of the prostaglandin receptors.