|Coordinate||66,859,142 bp (GRCm38)|
|Base Change||A ⇒ G (forward strand)|
|Gene Name||melanocortin 4 receptor|
|Chromosomal Location||66,857,715-66,860,472 bp (-)|
FUNCTION: This gene encodes a member of the melanocortin receptor family. Melanocortin receptors are transmembrane G-protein coupled receptors, which respond to small peptide hormones and exhibit diverse functions and tissue type localization. As part of the central nervous melanocortin system, the encoded protein is competitively bound by either melanocyte stimulating hormone or agouti-related protein to regulate energy homeostasis. Disruption of this gene promotes hyperphagia and obesity, and is associated with increased cholesterol levels and insulin resistance. [provided by RefSeq, Dec 2012]
PHENOTYPE: Mutations in this gene result in hyperglycemia and weight gain. [provided by MGI curators]
|Amino Acid Change||Leucine changed to Proline|
|Institutional Source||Beutler Lab|
|Gene Model||not available|
AA Change: L300P
|Predicted Effect||probably damaging
PolyPhen 2 Score 1.000 (Sensitivity: 0.00; Specificity: 1.00)
|Meta Mutation Damage Score||Not available|
|Is this an essential gene?||Probably nonessential (E-score: 0.100)|
|Candidate Explorer Status||CE: no linkage results|
Linkage Analysis Data
|Alleles Listed at MGI|
|Mode of Inheritance||Autosomal Semidominant|
|Local Stock||Embryos, gDNA|
|Last Updated||2019-05-25 7:24 AM by Diantha La Vine|
The Southbeach phenotype was identified among ENU-induced homozygous G3 mutant mice. Homozygous Southbeach mice develop severe obesity but not diabetes (1). By 20 weeks of age, homozygous male Southbeach mutants weigh approximately 65% more than wild type mice (Figure 1). Heterozygotes display an intermediate weight between homozygotes and controls, demonstrating that the Southbeach phenotype is codominant.
Upon identification of the Southbeach mutation in the gene encoding the melanocortin 4 receptor (MC4R), Mc4rSouthbeach mutant receptors expressed in HEK 293 cells were tested for ligand binding and intracellular signaling properties (1). Ligand binding to two MC4R agonists, α-melanocyte stimulating hormone (α-MSH) and NDP-MSH, was decreased to 0.6 ± 0.1% of wild type receptor levels, despite normal to increased numbers of cells with surface expression of the mutant receptor. Signaling by Southbeach MC4R, as measured by maximal cAMP accumulation stimulated by α-MSH or NDP-MSH, was reduced to approximately 14% of wild type MC4R.
|Nature of Mutation|
The Southbeach mutation was mapped to Chromosome 18, and corresponds to a T to C transition at position 1331 of the Mc4r transcript. Mc4r contains one exon.
The mutated nucleotide is indicated in red lettering, and results in a conversion of leucine to proline at residue 300 of the MC4R protein.
A pure modeling approach to deduce the three-dimensional structure of MC4R was also performed based on the crystal structure of the bovine G-protein rhodopsin (6). When a minimal inhibitory 3-mer peptide (RFF) from the sequence of the endogenous inhibitor Agouti-related protein (Agrp) was modeled as a ligand, residues in TM3, TM4, TM5 and TM6 were predicted to flank the ligand binding site, consistent with data from mutational analysis. Extracellular loops 2 and 3 also participate in docking of ligand. Interestingly, TM1 and TM7 were not predicted to contribute to ligand binding, although F284 was found at the edge of the ligand-binding pocket (6). The third intracellular loop of MC4R is predicted to form an α-helical segment, and play an important role in coupling the receptor to Gs. Finally, the model predicts two conserved proline kinks at P260 and P299, although the role of these kinks in protein function is unknown (6).
The Southbeach mutation results in the substitution of leucine 300 by a proline residue.
Genetic factors can contribute a predisposition to obesity, and several single spontaneous mutations in both mice and humans result in obesity (8). In all cases, the mutated gene products function within a defined hypothalamic neural network that controls energy balance through the regulation of central melanocortinergic systems. A main mechanism of regulation involves the control of signaling by the central melanocortin receptors (MCRs), MC3R and MC4R. These receptors are now known to be activated or inhibited by the hormonally controlled production and release of neuropeptide ligands, based on both long- and short-term energy requirements of the body.
The search for brain melanocortin receptors was triggered by the discovery that the yellow, hyperphagic (having increased feeding behavior) and obese phenotype of mice carrying the dominant agouti lethal yellow (Ay) mutation is caused by ectopic expression of Agouti protein [reviewed in (9)]. Agouti protein, a paracrine signaling molecule normally limited to the skin, controls hair color by inhibiting the melanocyte-specific Mc1r. Obesity was hypothesized to result from aberrant inhibition of a related receptor controlling body weight. RT-PCR screening of brain tissue soon identified MC3R and MC4R (2;10), and significantly, Agouti-related protein (Agrp) was discovered as a hypothalamus-specific antagonist of MC3R and MC4R (11). The endogenous MC4R agonist was shown to be α-melanocyte stimulating hormone (α-MSH), a proteolytic product of pro-opiomelanocortin (Pomc), from which all melanocortin receptor agonists arise (2). Agrp stimulates food intake and weight gain; α-MSH inhibits food intake and promotes weight loss. Both Agouti protein and Agrp antagonize MC3R and MC4R signaling, but they are truly inverse agonists rather than antagonists of the receptors. Inverse agonists stabilize the inactive conformation of a receptor, while antagonists bind but favor neither the active nor inactive form of the receptor. Agrp was shown to be an inverse agonist by its reduction of the intrinsic, constitutive activity of MC4R (12). Both Agouti protein and Agrp decrease the affinity of the ligand-receptor complex for the regulatory subunit of the G-protein, decreasing signaling from the receptor.
The physiological function of MC4R in the central regulation of energy balance was confirmed by the phenotype of Mc4r-/- mice, which develop obesity like that caused by overexpression of Agouti protein in Ay mice (20). Consistent with the antagonistic function of Agouti protein and Agrp towards MC4R, transgenic overexpression of Agouti protein or Agrp in mice both result in obesity (11). Pharmacological activation of MC4R by administration of α-MSH or a synthetic agonist suppresses feeding, and conversely, inhibition of MC4R by Agrp or a synthetic antagonist promotes feeding behavior in mice (21). In addition to the control of appetite, central melanocortin receptors also regulate insulin signaling. One week-long central infusion of α-MSH enhanced the actions of insulin on both glucose uptake and production, while a synthetic antagonist had the opposite effects (22). MC4R-null mice develop hyperinsulinemia and are hyperglycemic (20).
In humans, mutations in MC4R are associated with obesity (OMIM #601665; *155541). By late 2005, 58 MC4R mutations had been reported [reviewed in (23)], and such mutations are the most common cause of monogenic obesity in humans. It is estimated that 4% of single gene mutations causing severe childhood-onset obesity occur in MC4R (24;25). Human patients with MC4R mutations exhibit increased body mass index, increased appetite, increased height, increased lean mass, increased bone mineral density and hyperinsulinemia (24). With the exception of increased bone mineral density, these phenotypes are recapitulated in Mc4r null mice (20). Notably, in contrast to knockout mice which exhibit defects in basal energy expenditure, humans with MC4R mutations have apparently normal basal energy expenditure, suggesting that in humans, obesity is caused primarily by hyperphagia (20;24).
Analysis of the properties of MC4R protein containing the Southbeach mutation indicates that this receptor is correctly targeted to the plasma membrane, but fails to bind its ligands, resulting in a drastic reduction in signaling from the receptor (1). Of the 24 amino acids in TM7, all but two (295 and 296) are identical between mouse and human MC4R. Leucine 300 is conserved in humans, although no mutations in this residue have been reported among human patients. However, mutations of both proline 299 and isoleucine 301 occur in obese patients. The P299H mutation is reported to cause intracellular retention of MC4R, resulting in surface expression levels of 18 ± 7% relative to wild type receptor (26). In contrast, I301T reduces ligand binding by over 80% compared to control cells in an [125I] NDP-αMSH binding assay using cells transiently expressing MC4R I301T (25). Either surface expression and/or ligand affinity may be reduced for the I301T receptor mutant (25). The di-isoleucine sequence at position 316/317 of human MC4R is required for cell surface expression of the receptor (27), but effects on surface expression can result from mutations throughout the sequence of the protein (23). It is not clear why the adjacent mutations L300P (1) and P299H (26) result in different effects on receptor surface expression.
The majority of human MC4R loss-of-function mutations follow an autosomal dominant inheritance pattern, consistent with the intermediate obesity phenotype observed in Mc4r+/- mice compared to their homozygous and wild type littermates (20;24;25). Reports indicate dominant negative effects (28), and others a lack thereof (29), for various MC4R mutants, suggesting that haploinsufficiency and dominant negative effects are both plausible causes for mutant phenotypes which must be evaluated for each specific case. The Southbeach phenotype is codominant, but haploinsufficiency has not been rigorously tested.
|Primers||Primers cannot be located by automatic search.|
Southbeach genotyping is performed by amplifying the region containing the mutation using PCR, followed by sequencing of the amplified region to detect the single nucleotide change.
Primers for PCR amplification
South(F): 5’-AGCGCAGCCTCCCAACTTCTACAG -3’
South(R): 5’-TCAGATCGGGCCAGAGTGACAAAG -3’
1) 94°C 2:00
2) 94°C 0:15
3) 60°C 0:30
4) 68°C 1:20
5) repeat steps (2-4) 35X
6) 68°C 5:00
7) 4°C ∞
Primers for sequencing
South_seq(F): 5’- TCCGCCAGGGTACCAACATGAAGG -3’
South_seq(R): 5’- CAGATCGGGCCAGAGTGACAAAG -3’
The following sequence of 1177 nucleotides (from Genbank genomic region NC_000084 for linear DNA sequence of Mcr4) is amplified:
317 agcg cagcctccca acttctacag gcatacagac tgggagagaa
361 tcactcggag cttccctgac ccaggaggtt ggatcagttc aaggaggact caaatccagc
421 tgctgcagga agatgaactc cacccaccac catggcatgt atacttccct ccacctctgg
481 aaccgcagca gctacgggct gcacggcaat gccagcgagt cgctggggaa gggccacccg
541 gacggaggat gctatgagca actttttgtt tcccccgagg tgtttgtgac tctgggtgtc
601 ataagcctgt tggagaacat tctagtgatc gtggcgatag ccaagaacaa gaacctgcac
661 tcacccatgt actttttcat ctgtagcctg gctgtggcag atatgctggt gagcgtttcg
721 aatgggtcgg aaaccatcgt cattaccctg ttaaacagta cggatacgga tgcccagagc
781 ttcaccgtga acattgataa tgtcattgac tctgtgatct gtagctcctt gctcgcatcc
841 atttgcagcc tgctttccat tgcggtggac aggtatttca ctatctttta cgcgctccag
901 taccataaca tcatgacggt taggcgggtc gggatcatca taagttgtat ctgggcagct
961 tgcactgtgt caggcgtcct cttcatcatt tactcggaca gcagcgctgt catcatctgc
1021 ctcatttcca tgttcttcac tatgctagtt ctcatggcct ctctctatgt ccacatgttc
1081 ctgatggcga ggcttcacat taagaggatt gctgtcctcc caggcacagg gaccatccgc
1141 cagggtacca acatgaaggg ggcgattacc ttgaccatcc tgattggagt ctttgttgtc
1201 tgctgggccc cgttctttct ccatttactg ttctacatct cttgccctca gaatccatac
1261 tgcgtgtgct tcatgtctca ttttaatttg tatctcatac tgatcatgtg taacgccgtc
1321 atcgaccctc tcatttatgc cctccggagt caagaactga ggaaaacttt caaagagatc
1381 atctgtttct atcctctggg aggcatctgt gagttgtcta gcaggtatta agtgggggac
1441 agagtgcaaa ctaggtagat acctgcagac tttgtcactc tggcccgatc tga
PCR primer binding sites are underlined; sequencing primer binding sites are highlighted in gray; the mutated T is shown in red text.
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|Science Writers||Eva Marie Y. Moresco|
|Illustrators||Diantha La Vine|
|Authors||Koichi Tabeta, Bruce Beutler|