Stable expression of human melanocortin 3 receptor fused to EGFP in the HEK293 cells

Among the melanocortins alpha-MSH is known to be involved in feeding behavior. These hormones mediate their effects through G protein-coupled receptors by stimulating adenylate cyclase. In this study, we have developed an in vitro expression model for human melanocortin 3 receptor (hMC3R) tagged at its C terminus with EGFP. The corresponding chimeric cDNA was stably expressed in HEK293 cells. The selected clones expressing the hMC3R-EGFP exhibited cell surface fluorescence and responded to NDP-MSH stimulation by producing cAMP in a dose-dependent manner (EC(50): 0.3 nM). Binding studies revealed a single class of binding sites with a K(D) of 2.24 nM. Moreover, Agouti-related protein was also demonstrated to be an antagonist of the hMC3R-EGFP. Thus, the hMC3R tagged with EGFP stably expressed in HEK293 cells, exhibiting the same characteristics than the wild-type hMC3R, is the only model of expression of this receptor allowing its direct localization inside living cells.