title: Point mutations in the melanocortin-4 receptor cause variable obesity in mice.
abstract: Mutations in the melanocortin-4 receptor (MC4R) are associated with early-onset obesity in humans. Furthermore, a null Mc4r allele in mice leads to severe obesity due to hyperphagia and decreased energy expenditure. As part of independent N-ethyl- N-nitrosourea (ENU) mutagenesis screens, two obesity mutants, Fatboy and Southbeach, were isolated. Mapping revealed linkage to the melanocortin-4 receptor (Mc4r) and sequencing found single amino acid changes in Mc4r for each line. Expression of the mutant receptors in HEK 293 cells revealed defects in receptor signaling. The mutated Fatboy receptor (I194T) shows an increase in the effective concentration necessary for 50% of maximal signaling (EC(50)) when stimulated with alpha-MSH. Based on competitive binding, I194T is expressed on the cell surface at lower levels than the nonmutated receptor. In contrast, Southbeach (L300P) displays minimal receptor signaling when stimulated with the natural ligand alpha-MSH or the synthetic agonist NDP-alpha-MSH. Cell surface binding is absent, which usually indicates a lack of cell surface expression. However, antibody binding to Flag-tagged receptors by flow cytometry analysis and immunofluorescence demonstrates that L300P is translocated to the plasma membrane at a level comparable to the wild-type receptor. These results indicate a correlation with remaining receptor activity and the severity of the obesity in the mice homozygous for the mutations. Southbeach has less receptor activity and becomes more obese. These mutants will serve as good models for the variability in phenotype in humans carrying mutations in the MC4R gene.
authors.affiliation: Department of Molecular and Human Genetics, Baylor College of Medicine, Houston, Texas 77030, USA.
authors.forename: Thomas P
authors.initials: TP
authors.lastname: Meehan
authors.forename: Koichi
authors.initials: K
authors.lastname: Tabeta
authors.forename: Xin
authors.initials: X
authors.lastname: Du
authors.forename: Lanette S
authors.initials: LS
authors.lastname: Woodward
authors.forename: Karen
authors.initials: K
authors.lastname: Firozi
authors.forename: Bruce
authors.initials: B
authors.lastname: Beutler
authors.forename: Monica J
authors.initials: MJ
authors.lastname: Justice
mesh_terms: D000477:Alkylating Agents / Q000633:toxicity; D019943:Amino Acid Substitution; D000818:Animals; D001667:Binding, Competitive; D002462:Cell Membrane / Q000378:metabolism; D002478:Cells, Cultured; D000242:Cyclic AMP / Q000378:metabolism; D004734:Energy Metabolism; D005038:Ethylnitrosourea / Q000633:toxicity; D005260:Female; D005434:Flow Cytometry; D005455:Fluorescent Antibody Technique; D006720:Homozygote; D006801:Humans; D007668:Kidney / Q000378:metabolism; D008297:Male; D051379:Mice; D008810:Mice, Inbred C57BL; D009765:Obesity / Q000235:genetics* / Q000378:metabolism; D017354:Point Mutation / Q000235:genetics*; D021381:Protein Transport; D044105:Receptor, Melanocortin, Type 4 / Q000235:genetics*; D011956:Receptors, Cell Surface / Q000378:metabolism; D015398:Signal Transduction; D000521:alpha-MSH / Q000031:analogs & derivatives / Q000378:metabolism
chemical_list: D000477:Alkylating Agents; D044105:Receptor, Melanocortin, Type 4; D011956:Receptors, Cell Surface; D000521:alpha-MSH; C027756:MSH, 4-Nle-7-Phe-alpha-; D000242:Cyclic AMP; D005038:Ethylnitrosourea