Wyatt Rachael A, Jamaluddin Aqfan, Mistry Vinesh, Quinn Caitlin, Gorvin Caroline M
Department of Metabolism and Systems Science, University of Birmingham, Birmingham, B15 2TT, United Kingdom.
Centre of Membrane Proteins and Receptors (COMPARE), Universities of Birmingham and Nottingham, Birmingham, B15 2TT, United Kingdom.
Hum Mol Genet. 2025 Mar 7;34(6):533-546. doi: 10.1093/hmg/ddaf005.
The melanocortin-4 receptor (MC4R) is a G protein-coupled receptor expressed at hypothalamic neurons that has an important role in appetite suppression and food intake. Mutations in MC4R are the most common cause of monogenic obesity and can affect multiple signaling pathways including Gs-cAMP, Gq, ERK1/2, β-arrestin recruitment, internalization and cell surface expression. The melanocortin-2 receptor accessory protein 2 (MRAP2), is a single-pass transmembrane protein that interacts with and regulates signaling by MC4R. Variants in MRAP2 have also been identified in overweight and obese individuals. However, functional studies that have only measured the effect of MRAP2 variants on MC4R-mediated cAMP signaling have produced inconsistent findings and most do not reduce MC4R function. Here we investigated the effect of twelve of these previously reported MRAP2 variants and showed that all variants that have been identified in overweight or obese individuals impair MC4R function. When expressed at equal concentrations, seven MRAP2 variants impaired MC4R-mediated cAMP signaling, while nine variants impaired IP3 signaling. Four mutations in the MRAP2 C-terminus affected internalization. MRAP2 variants had no effect on total or cell surface expression of either the MRAP2 or MC4R proteins. Structural models predicted that MRAP2 interacts with MC4R transmembrane helices 5 and 6, and mutations in two MRAP2 residues in putative contact sites impaired the ability of MRAP2 to facilitate MC4R signaling. In summary, our studies demonstrate that human MRAP2 variants associated with obesity impair multiple MC4R signaling pathways and that both Gs-cAMP and Gq-IP3 pathways should be assessed to determine variant pathogenicity.
黑皮质素-4受体（MC4R）是一种在下丘脑神经元表达的G蛋白偶联受体，在食欲抑制和食物摄入中起重要作用。MC4R突变是单基因肥胖最常见的原因，可影响包括Gs-cAMP、Gq、ERK1/2、β-抑制蛋白募集、内化和细胞表面表达在内的多种信号通路。黑皮质素-2受体辅助蛋白2（MRAP2）是一种单次跨膜蛋白，可与MC4R相互作用并调节其信号传导。在超重和肥胖个体中也发现了MRAP2的变体。然而，仅测量MRAP2变体对MC4R介导的cAMP信号传导影响的功能研究结果并不一致，且大多数并未降低MC4R功能。在此，我们研究了12种先前报道的MRAP2变体的作用，结果显示在超重或肥胖个体中鉴定出的所有变体均损害MC4R功能。当以相同浓度表达时，7种MRAP2变体损害MC4R介导的cAMP信号传导，而9种变体损害IP3信号传导。MRAP2 C末端的4个突变影响内化。MRAP2变体对MRAP2或MC4R蛋白的总量或细胞表面表达均无影响。结构模型预测，MRAP2与MC4R跨膜螺旋5和6相互作用，假定接触位点的两个MRAP2残基发生突变会损害MRAP2促进MC4R信号传导的能力。总之，我们的研究表明，与肥胖相关的人类MRAP2变体损害多种MC4R信号通路，应评估Gs-cAMP和Gq-IP3两条信号通路以确定变体的致病性。
