Ser-268 plays an important role in ligand binding of prostaglandin E(2) receptor EP3 alpha subtype

Functional mouse prostaglandin Eg (PGE(2)) receptor EP(3 alpha) subtype has been expressed in insect cells using the baculovirus system (C. Huang and H. H. Tai, 1995, Biochem. J, 307, 493-498). Site-directed mutagenesis was carried out at Thr-221, Ser-268, and Ser-272. These hydroxy amino acid residues are highly conserved among prostaglandin receptors of many species and could potentially form hydrogen bonds with the hydroxyl groups or the keto group of the receptor ligand PGE,. The mutant EP(3 alpha) receptors again were expressed in insect cells and were studied by [H-3]PGE(2) binding assay. The results indicated that (1) replacement of Ser-272 by alanine did not cause any significant change in PGE(2)-binding activity, (2) replacement of Thr-221 by alanine also did not cause any significant change in PGE(2)-binding activity, and (3) replacement of Ser-268 by alanine almost abolished the PGE(2)-binding activity, while replacement by a threonine did not cause significant change in the PGE(2)-binding activity but did alter subtype specificity. These results suggest that Ser-268 of EP(3 alpha) receptor plays an important role in ligand binding. (C) 1996 Academic Press, Inc.