SUBSTITUTION OF CHARGED AMINO-ACID-RESIDUES IN TRANSMEMBRANE REGION-6AND REGION-7 AFFECT LIGAND-BINDING AND SIGNAL-TRANSDUCTION OF THE PROSTAGLANDIN EP(3) RECEPTOR

Expression of the rabbit EP(3) receptor isoform 77A in COS1 and HEK293 tsA201 cells demonstrated specific binding of [H-3]prostaglandin (PG)E (2), and receptor-evoked decreases in intracellular cAMP levels. Compe tition binding with PGE(2), PGE(2) 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 th e charge at this position is more important than steric bulk. Charged amino acid residues within the transmembrane (TM) domains of the recep tor were mutated, and the resulting receptor proteins were analyzed fo r the effects of these mutations on receptor structure and/or function . Positively charged TM residues are candidates for interaction with t he C1 carboxylic acid moiety of prostanoid ligands. Substitution of R3 29 (TM VII) with either alanine or glutamate resulted in a loss of bot h detectable [H-3]PGE(2) binding and receptor activation despite expre ssion of the receptor protein as determined by immunoprecipitation and immunofluorescence. Substitution of K300 (TM VI) with alanine had no effect on binding or signal transduction. Substitution of the conserve d aspartic acid at position 338 (TM VII) with alanine caused a loss of detectable receptor-evoked inhibition of cAMP generation, although th is mutation did not appreciably affect ligand binding. These studies s uggest that R329 but not K300 is a key determinant in receptor/ligand interaction. Furthermore, D338 plays a critical role in G(i) activatio n by the EP(3) receptor.