EXPRESSION AND SITE-DIRECTED MUTAGENESIS OF MOUSE PROSTAGLANDIN E(2) RECEPTOR EP(3) SUBTYPE IN INSECT CELLS

A cDNA encoding for mouse prostaglandin E(2) (PGE(2)) receptor EP(3) subtype was cloned from a mouse kidney cDNA library by PCR using terminal primers derived from the known sequence of mouse lung EP(3) receptor cDNA. The cloned cDNA was confirmed by sequencing and was expressed in Trichoplusia ni (MG1) insect cells using a baculovirus expression system. A specific protein of 60 kDa was detected by immunoblot with antibodies generated against a unique decapeptide sequence present in the second extracellular loop of the EP(3) receptor. Specific binding of [H-3]PGE(2) with a K-d of 3 nM was also found in the membrane fraction of the insect cells. Ligand binding of the receptor was further studied by site-directed mutagenesis. Arg-309 of the receptor was separately mutated to lysine, glutamate and valine. cDNAs of the wild-type and mutant EP(3) receptors were respectively expressed and studied in MG1 insect cells. Binding studies indicated that both glutamate and valine mutant EP(3) receptors had no binding of [H-3]PGE(2). On the contrary, the lysine mutant receptor exhibited an even tighter binding (K-d = 1.3 nM) than the wild-type EP(3) receptor. Immunoblot studies indicated that these receptors were expressed in a comparable amount in MG1 insect cells. These results suggest that Arg-309 of EP(3) receptor may be essential in ligand binding through ionic interaction.