Posttranslational modifications (PTMs) of human melanocortin receptor 4 (hMC4R) plays a fundamental role in receptor regulation by agonists. We have examined the effects of melanocortin peptide and nonpepeide agonists on the level of phosphorylation using FLAG epitope tagging in stably transfected human embryonic kidney (HEK293) cells which stably expressed hMC4R. Stably transfected HEK293 cell lines that expressed hMC4R resulted in a KD value in the nanonmolar range when the super potent agonist MTII was competed with [125I]NDP-á-MSH. Treatment of the tagged receptors in the HEK293 cells with agonist resulted in down-regulation which indicates that these tagged receptors retain their biological functions. The hMC4R was solubilized from cell membranes with n-dodecyl-â-D-maltoside and purified at a Nickel chelating resin and a newly constructed ligand affinity column. The purified hMC4R was a glycoprotein that migrated on SDS/PAGE with a molecular mass of 60 kDa. The results from Matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) mass spectrometry was used to identify and characterize peptides and non peptide treated purified receptor which derived from the hMC4R following in-gel digestion with chymotrypsin. The phosphorylation sites were identified on the purified hMC4R with agonists (peptide vs small molecule) treatment. In addition, the effects of agonists (peptide vs nonpeptide) mediated endocytosis give more evidence of cell signaling of human melanocortin 4 receptor.

Supported by Grants from the USPHS DK 17420 and DA 06284