Site-Directed Mutagenesis of Human MC4R

Receptor mutagenesis was performed as previously described.^{47 (link)–49 (link), 54} The human WT N-terminal FLAG-tagged MC4R cDNA (Supplemental Figure 1A) was generously provided by Dr. Robert Mackenzie^{59 (link)} and was sub-cloned into the pBluescript plasmid (Stratagene) for subsequent mutagenesis. Site directed hMC4R mutagenesis was performed using a polymerase chain reaction (PCR) based strategy, using the Pfu turbo polymerase (Stratagene). Complementary sets of primers were designed containing nucleotide base pair changes resulting in the modified amino acids (Supplemental Figure 1B). Upon completion of the PCR reaction (95 ºC 30 s, 12 cycles of 95 ºC 30 s, 55 ºC 1 min, 68 ºC 9 min), the product was purified (Qiaquick PCR Purification Kit, Qiagen) and eluted in water. Subsequently, the sample was cut with Dpn1 (Invitrogen) to eliminate any methylated WT DNA, leaving only nicked circularized mutant DNA. The mutant hMC4R DNA was transformed into competent DH5α E. coli cells and single colonies were selected. The presence of the desired mutation was verified by DNA sequencing. The plasmid DNA containing the mutant was excised and sub-cloned into the HindIII/XbaI restrictions sites of the pCDNA₃ expression vector (Invitrogen). Complete FLAG-MC4R sequences were confirmed free of PCR nucleotide base errors by DNA sequencing (University of Florida sequencing core facilities).