title: Functional characterization of a new human melanocortin-4 receptor homozygous mutation (N72K) that is associated with early-onset obesity.
abstract: 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 EC50 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.
authors.affiliation: Departments of Internal Medicine and Pediatric Endocrinology, Erasmus MC, Rotterdam, The Netherlands, p.delhanty@erasmusmc.nl.
authors.forename: Patric J D
authors.initials: PJ
authors.lastname: Delhanty
authors.forename: Elise
authors.initials: E
authors.lastname: Bouw
authors.forename: Martin
authors.initials: M
authors.lastname: Huisman
authors.forename: Resie M L
authors.initials: RM
authors.lastname: Vervenne
authors.forename: Axel P N
authors.initials: AP
authors.lastname: Themmen
authors.forename: Aart Jan
authors.initials: AJ
authors.lastname: van der Lely
authors.forename: Erica L T
authors.initials: EL
authors.lastname: van den Akker
mesh_terms: D005260:Female; D057809:HEK293 Cells; D006720:Homozygote; D006801:Humans; D006963:Hyperphagia / Q000235:genetics; D007223:Infant; D020125:Mutation, Missense; D063766:Pediatric Obesity / Q000235:genetics*; D044105:Receptor, Melanocortin, Type 4 / Q000235:genetics*; D000521:alpha-MSH / Q000494:pharmacology
chemical_list: C470072:MC4R protein, human; D044105:Receptor, Melanocortin, Type 4; D000521:alpha-MSH