The melanocortin 4 receptor (MC4R) is expressed in the hypothalamus and is essential for regulation of appetite and energy expenditure. MC4R dysfunction in humans causes hyperphagia, impaired satiety and obesity. We have identified a novel c.216C>A (N72 K) homozygous mutation in MC4R in a girl with severe obesity. The patient presented with early-onset obesity and hyperphagia indicating an effect of the homozygous mutation on her phenotype. In silico analyses indicate a damaging effect on receptor function, and the mutation is unusual in occurring in the first intra-cellular loop of the receptor. Site-directed mutagenesis was used to generate plasmid constructs expressing wild-type and mutant MC4R. These were transfected into HEK293 cells and assessed for cAMP responsiveness to α-MSH. Cells expressing N-terminal HA and C-terminal GFP-tagged MC4R were assessed by immunofluorescence confocal microscopy and flow cytometry for correct cell-surface localization. The maximal response of the mutant MC4R to α-MSH was decreased to 20 ± 1 % of the wild type receptor response, and the EC<inf>50</inf> was increased from 16.5 ± 5.4 nM to 37.0 ± 8.3 nM. Localization of N- and C-terminally tagged MC4R by confocal microscopy and flow cytometry showed aberrant retention of the mutant receptor in the cytoplasm. Our data describe a rare homozygous inactivating mutation in the first intra-cellular loop of MC4R that markedly impairs its function and is associated with early-onset obesity and hyperphagia.

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Keywords MC4R, Mutation, Obesity
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Journal Molecular Biology Reports
Delhanty, P.J.D, Bouw, E, Huisman, M, Vervenne, R.M.L, Themmen, A.P.N, van der Lely, A-J, & van den Akker, E.L.T. (2014). Functional characterization of a new human melanocortin-4 receptor homozygous mutation (N72K) that is associated with early-onset obesity. Molecular Biology Reports, 41(12), 7967–7972. doi:10.1007/s11033-014-3691-7