Endocrine Abstracts

Introduction: Melanocortin-4 receptor (MC4R) is a G-protein-coupled receptor expressed in regions of the hypothalamus regulating appetite and energy expenditure. Extensive evidence from genetic and biological studies show that MC4R is a key player in the homeostatic regulation of body weight. MC4R loss of function (LoF) variants are the most common cause of monogenic obesity. We have identified 13 heterozygous MC4R variants in patients with obesity 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, and the cell surface expression was measured using HiBiT Detection System.

Results: Thirteen variants were identified by sequencing the MC4R gene of adult and pediatric patients with obesity using an obesity gene panel. Eight of these variants have not been previously reported in literature. The median age of onset obesity for the adult patients was 1.0 year and for the pediatric patients was 1.9 years. Furthermore, the median BMI of the adult patients was 48.8 kg/m²(range 36 - 58.8) and the median BMI-SD of the pediatric group was +4.2 SD (range +3 SD - +7 SD). Eleven out of 13 patients presented with hyperphagia. MC4R variants had differential effects on cAMP production, βarr2 recruitment and cell surface expression. Eight of the 13 MC4R variants caused partial or complete LoF for both cAMP production and βarr2 recruitment; out of those eight variants, three showed no cell surface expression. Surprisingly, two of the 13 MC4R variants resulted in a gain of function for cAMP production, and two other variants showed a normal cAMP response as well as βarr2 recruitment.

Conclsion: We show that the MC4R variants identified in our patients with obesity affect MC4R signaling differently, through modulation of cell surface expression, cAMP and/or βarr2 signaling pathways. Therefore, this study demonstrates the value of examining 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.