Prostaglandin E2 receptor EP3alpha subtype: the role of N-glycosylation in ligand binding as revealed by site-directed mutagenesis.
Functional mouse prostaglandin E2 (PGE2) receptor EP3alpha subtype has been expressed in insect cells using a baculovirus system (Huang C. and Tai H.-H. Biochem J 1995; 307: 493-498). EP3alpha receptor has two potential sites (Asn-X-Ser/Thr), Asn 16 and Asn 193, for N-glycosylation. The role of glycosylation in ligand binding of the EP3alpha receptor was investigated by site-directed mutagenesis. Asn was mutated to Gln in each of the two potential glycosylation sites in the EP3alpha receptor. Recombinant wild-type and mutant EP3alpha receptors were expressed in insect cells using baculovirus. Ligand binding assay indicated that the affinity of PGE2 binding was reduced by 50% in the Gln 193 mutant EP3alpha receptor, while the specificity of ligand binding was unaltered. The affinity for PGE2 binding was not affected in the Gln 16 mutant EP3alpha receptor. However, its specificity was partially changed as the EP3-specific agonist became less effective in displacing the [3H]-PGE2 binding to the mutant receptor. These results indicated that N-glycosylation of the EP3alpha receptor could partially affect the affinity and specificity of the ligand binding.
Huang
C
C
Division of Medicinal Chemistry and Pharmaceutics, College of Pharmacy, University of Kentucky, Lexington 40536, USA.
Tai
H H
HH
D000818:Animals; D016367:Baculoviridae / Q000235:genetics; D006031:Glycosylation; D008024:Ligands; D051379:Mice; D016297:Mutagenesis, Site-Directed / Q000235:genetics*; D009154:Mutation / Q000235:genetics; D011485:Protein Binding; D018078:Receptors, Prostaglandin E / Q000235:genetics*; D011994:Recombinant Proteins / Q000235:genetics; D018411:Spodoptera / Q000235:genetics
D008024:Ligands; D018078:Receptors, Prostaglandin E; D011994:Recombinant Proteins