Human MC4R Stable Cell Line - HEK293

Melanocortin-4 receptor (MC4R) plays a crucial role in regulating appetite, food intake, and energy homeostasis. Setmelanotide, an MC4R agonist, is currently approved for weight loss in obese adults and children with mutations in components of the leptin-melanocortin pathway. Here, researchers compared the structural and functional properties of the physiological MC4R agonist α-melanocyte-stimulating hormone (α-MSH) with setmelanotide. The data showed that setmelanotide exhibited higher cAMP generation potency but weaker effects on ERK1/2 phosphorylation compared to α-MSH, indicating functional selectivity, also known as biased agonism. Furthermore, the researchers provided structural data showing that setmelanotide has a higher binding affinity to MC4R than α-MSH. Finally, the study found that two loss-of-function and two gain-of-function MC4R variants not only altered the conformation of the receptor ligand-binding pocket but also changed the conformation of the peptide upon binding to the receptor, as both the interaction network and the residues involved in binding were altered.

The classic signaling pathway of MC4R begins with its coupling to Gαs, which activates adenylyl cyclase, thereby generating cAMP (cyclic adenosine monophosphate). To study the coupling of MC4R to Gαs, researchers used a cAMP-based assay performed in MC4R-expressing HEK293 cells. Compared to NDP-α-MSH, the dose-response curve shifted to the left when using setmelanotide (Figure 1A). After calculating the EC50 values, the researchers found that setmelanotide was more than twice as potent as NDP-α-MSH (Figure 1B). Compared to using the same concentration of NDP-α-MSH, stimulation of cells with 100 nM setmelanotide resulted in higher cAMP production over time (Figure 1C). In addition to activating Gαs, MC4R can also recruit β-arrestin. To assess the ability of setmelanotide to activate G protein-independent pathways downstream of MC4R, researchers measured the phosphorylation levels of ERK1/2 in MC4R-expressing HEK293 cells, as shown in Figures 1D and 1E. Cells were stimulated with 1 μM of NDP-α-MSH or setmelanotide and measured at different time points. Both agonists rapidly produced a signal at 2 minutes, peaked at 5 minutes, and then gradually decreased due to receptor internalization. The same pattern was observed for both agonists, but the overall stimulatory effect of setmelanotide was weaker (Figure 1D). Quantitative analysis of the blots revealed that setmelanotide induced significantly less ERK1/2 activation at 2 minutes compared to NDP-α-MSH, and this reduction was borderline significant at 10 and 30 min (Figure 1E).

Figure 1. MC4R signaling pathways activated by α-MSH and setmelanotide. (Hammad M M, et al., 2022)