Melanocortin receptor 4 (MC4) Redistribution Assay

• Designed to assay compounds for their ability to modulate internalization of the MC4 receptor
• Coupled to EGFP for easy monitoring of the cellular translocation event
• Robust cell-based assay for use in high content analysis and fluorescence microscope applications

• Biologically relevant data: Compounds tested in a cellular environment
• Validated: Functionally tested cells provided with an optimized assay protocol
• Easy to use: Just plate cells, add compounds, and image

Melanocortins are peptides that regulate eating behavior, metabolism, and possibly also emotional states. The melanocortin receptors are expressed in the central nervous system and are divided into five subtypes (MC1-MC5). They are members of the family of G-protein coupled receptors (GPCRs) and signal through the Gαs subunit that stimulates production of cAMP by adenylate cyclase. Activation of the MC4 receptor also initiates other regulatory mechanisms such as receptor internalization. The role of MC4 in body weight control is evident from studies in mice, where loss of function mutations are associated with obesity. Moreover, such MC4 mutations are the most common monogenic defect in human obesity. Thus, MC4 is a possible therapeutic target in control of body weight. The natural ligand for MC4 is the alpha-melanocyte-stimulating hormone α-MSH that is released from synthesizing neurons.

Figure 1. Figure 4. Internalization of MC4-EGFP. Cells were untreated (DMSO control, left panel) or treated with 10 nM NDP-α-MSH for 2 hr (right panel). Arrow indicates the MC4 internalization detected by the image analysis algorithm.

Figure 2.  Concentration response curves in the MC4 assay: A) NDP-α-MSH concentration response in the MC4 agonist assay (n=16). The EC₅₀ is approximately 0.6 nM. Concentration response was measured in 9 point half log dilution series. Cells were treated with NDP-α-MSH for 2 hr. Cells were then fixed and receptor internalization was measured using the IN Cell Analyzer 3000 (GE Healthcare). % activity was calculated relative to the positive (10 nM NDP-α-MSH) and negative control (0.25% DMSO). 

B) HS014 concentration response in the MC4 antagonist assay (n=16). The EC₅₀ is approximately 5 nM. Cells were treated with NDP-α-MSH in the presence of a half log dilution series of HS014 for 2 hr. Cells were then fixed and receptor internalization was measured using the Cellomics ArrayScan V^TI Reader and the SpotDetectorV3 BioApplication. % activity was calculated relative to the positive (100 nM HS014) and negative control (0.25% DMSO).