Introduction
Melanocortin-4 receptor (MC4R) is a G-protein coupled receptor expressed in regions of the hypothalamus regulating appetite and energy expenditure. It signals via cAMP upon α-melanocyte stimulating hormone (MSH) activation. Recruitment of β-arrestins by MC4R then represses this signal. MC4R loss of function (LoF) variants are the most common cause of monogenic obesity.
We have identified 13 MC4R variants in obese patients at our academic Obesity Center CGG, Erasmus MC. However, it is unknown whether these variants affect MC4R signaling and are causing obesity. Here, we functionally characterized these variants by analyzing the effects on cell surface expression, MSH-induced cAMP production and β-arrestin 2 (βarr2) recruitment.
Methods
HEK293 cells were transiently transfected with expression plasmids encoding WT or variant MC4R and stimulated with MSH. cAMP response was measured using GloSensor cAMP bioluminescence assay. NanoBiT complementation luminescence assay was used to measure βarr2 recruitment. Cell surface expression was measured using HiBiT Detection System.
Results
The MC4R variants had differential effects on cAMP production, βarr2 recruitment and cell surface expression. Seven out of 13 MC4R variants caused partial or complete LoF for both cAMP production and βarr2 recruitment, and 3 of these variants showed no cell surface expression. Surprisingly, 2 of the variants were gain of function for cAMP production. The other MC4R variants showed a normal cAMP response, but were defective for βarr2 recruitment.
Conclusion
We have demonstrated that the MC4R variants identified in our patients with obesity affect MC4R signaling. However, since these variants modulated cell surface expression, cAMP and βarr2 signaling pathways differently, our study demonstrates that it is essential to examine different aspects of MC4R signaling to understand possible biased effects of mutations on these pathways. Overall, our results show the clinical importance of assessing the function of MC4R variants as these studied variants are likely to be causative of obesity.