Substitution of charged amino acid residues in transmembrane regions 6 and 7 affect ligand binding and signal transduction of the prostaglandin EP3 receptor.

Expression of the rabbit EP3 receptor isoform 77A in COS1 and HEK293tsA201 cells demonstrated specific binding of [3H]prostaglandin (PG)E2 and receptor-evoked decreases in intracellular cAMP levels. Competition binding with PGE2, PGE2 methyl ester, misoprostol-free acid, misoprostol, and sulprostone suggested that a negative charge at the C1 position is essential for high affinity ligand binding and that the charge at this position is more important than steric bulk. Charged amino acid residues within the transmembrane (TM) domains of the receptor were mutated, and the resulting receptor proteins were analyzed for the effects of these mutations on receptor structure and/or function. Positively charged TM residues are candidates for interaction with the C1 carboxylic acid moiety of prostanoid ligands. Substitution of R329 (TM VII) with either alanine or glutamate resulted in a loss of both detectable [3H]PGE2 binding and receptor activation despite expression of the receptor protein as determined by immunoprecipitation and immunofluorescence. Substitution of K300 (TM V) with alanine had no effect on binding or signal transduction. Substitution of the conserved aspartic acid at position 338 (TM VII) with alanine caused a loss of detectable receptor-evoked inhibition of cAMP generation, although this mutation did not appreciably affect ligand binding. These studies suggest that R329 but not K300 is a key determinant in receptor/ligand interaction. Furthermore, D338 plays a critical role in G1 activation by the EP3 receptor.