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.

Functional Characterization and Structural Modeling of Obesity Associated Mutations in the Melanocortin 4 Receptor

“…The I269N substitution may change the packing of the extracellular ends of TM6 and TM5, and, thus, may affect the receptor stability, leading to misfolding and loss-of-function. This mechanism is supported by a recent observation that the I194T mutation of a contacting residue from TM5 caused impaired MC4R expression and signaling and an obese phenotype in mice (28). The presence of a polar residue in the lipid-exposed surface of TM6 may also alter the orientation of the whole receptor relative to the membrane plane that would affect protein-protein interaction in membranes.…”

A novel melanocortin-4 receptor gene mutation in a female patient with severe childhood obesity

“…The current trend indicated by several recent studies is to test any identified MC4R mutation for loss-of-function, although loss-offunction mutations in the MC4R do not necessarily lead to obesity as the penetrance of MC4R mutations in causing obesity is not 100% [30]. Nevertheless, it has been established that MSHs, signaling through Gs protein/adenylyl cyclase, activate MC4R, a critical component of the hypothalamic melanocortin system involved in energy homeostasis, leading investigators to deduce that mutations in MC4R could be responsible for monogenic obesity in humans [12,13] and mice [8,31]. MC4R loss-of-function can be characterized as decreased cell surface expression, decreased ligand binding, and/or disturbed receptor signaling.…”

Mouse models for the central melanocortin system

“…Only one report demonstrated a lower potency of orexigenic AGRP on V103I corresponding to the distribution of this allele in population screens [52]. Several point mutations in the Mc4r gene introduced by chemical mutagenesis have been shown to cause different severity of obesity in mice [40]. Mouse lines encoding certain point mutations of interest are required to elucidate the effects of certain alleles on metabolism in vivo.…”