Oligodeoxynucleotide-directed mutagenesis was carried out as described previously [31] on the hMOPR cDNA with the overlap polymerase chain reaction method. The A118/N40- or G118/D40-hMOPR cDNA (NcoI/XbaI), tagged with 3HA (Hind III/NcoI) 5′ to the initiation codon, was subcloned into HindIII and XbaI sites of the mammalian expression vector pcDNA3. The cDNA sequences were determined to confirm the presence of desired mutations and the absence of unwanted mutations.
