Introduction

Melanocortin-4 receptor (MC4R) is a G-protein coupled receptor expressed in regions of the hypothalamus that regulate appetite and energy expenditure. It signals via cAMP after activation by α-melanocyte stimulating hormone (MSH). Subsequent recruitment of β-arrestins by MC4R then represses this signal. MC4R loss of function (LoF) variants are the most common cause for monogenetic obesity.

We have identified 13 MC4R variants of uncertain significance in obese patients at our academic Obesity Center CGG (Centrum Gezond Gewicht), Erasmus MC. However, it is unknown whether these variants impact MC4R signaling and are causing obesity. Our aim was to functionally characterize these variants by analyzing the effects on MSH-induced cAMP production and β-arrestin2 recruitment.

Methods

HEK293 cells were transiently transfected with expression plasmids encoding WT or variant MC4R and stimulated with MSH. Cyclic AMP responses were measured using GloSensor cAMP bioluminescence assay (Promega) and, in separate experiments, NanoBiT complementation luminescence assay (Promega) was used to measure β-arrestin2 recruitment.

Results

We identified differential effects of the MC4R variants on cAMP production and β-arrestin2 recruitment. Seven out of 13 MC4R variants caused partial or complete LoF for both cAMP production and β-arrestin2 recruitment compared to WT MC4R. Surprisingly, two of the variants showed a gain of function for cAMP production. The other MC4R variants showed a normal cAMP production, but were defective in β-arrestin2 recruitment.

Conclusion

We have demonstrated that the MC4R variants identified in our patients with obesity impact MC4R signaling. However, since these variants affected cAMP and β-arrestin2 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.