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Functional characterization of mutations in melanocortin-4 receptor associated with human obesity
1Department of Cell Biology, Parke-Davis Pharmaceutical Research, Ann Arbor, Michigan 48105, USA.
Abstract:
Melanocortin-4 receptor (MC4R) is a G protein-coupled receptor implicated in the regulation of body weight. Genetic studies in humans have identified two frameshift mutations of MC4R associated with a dominantly inherited form of obesity. We have generated and expressed the corresponding MC4R mutants in 293T cells and found that cells transfected with the truncation mutants failed to exhibit agonist binding or responsiveness despite retention of structural motifs potentially sufficient for binding and signaling. Immunofluorescence studies showed that the mutant proteins were expressed and localized in the intracellular compartment but absent from the plasma membrane, suggesting that these mutations disrupted the proper cellular transport of MC4R. Further studies identified a sequence in the cytoplasmic tail of MC4R necessary for the cell surface targeting. We further investigated a possible dominant-negative activity of the mutants on wild-type receptor function. Co-transfection studies showed that the mutants affected neither signaling nor cell surface expression of wild-type MC4R. We also characterized three human sequence variants of MC4R, but these exhibited identical affinities for peptide ligands and identical agonist responsiveness. Thus, unlike the obesity-associated MC4R truncation mutants, the polymorphisms of MC4R are unlikely to be contributors to human obesity.
Insights
Obesity-linked Melanocortin-4 receptor (MC4R) truncation mutants are retained intracellularly, failing to reach the cell surface. These MC4R mutations do not impact wild-type receptor function or cell surface expression.
Area of Science:
- Molecular biology
- Genetics
- Cell biology
Background:
- The Melanocortin-4 receptor (MC4R) is a G protein-coupled receptor crucial for regulating body weight.
- Genetic studies link MC4R frameshift mutations to a hereditary form of obesity.
Purpose of the Study:
- To investigate the cellular mechanisms underlying MC4R mutations associated with obesity.
- To determine if MC4R mutants exhibit dominant-negative effects on wild-type receptors.
- To assess the role of MC4R polymorphisms in human obesity.
Main Methods:
- Expression of MC4R truncation mutants and wild-type receptors in 293T cells.
- Agonist binding and responsiveness assays.
- Immunofluorescence studies for protein localization.
- Co-transfection studies to assess dominant-negative effects.
- Characterization of human MC4R sequence variants.
Main Results:
- MC4R truncation mutants failed to bind agonists or elicit a response, despite possessing structural binding motifs.
- Mutant MC4R proteins were localized intracellularly and not detected at the plasma membrane.
- A specific sequence in the MC4R cytoplasmic tail was identified as essential for cell surface targeting.
- MC4R mutants did not impair the signaling or cell surface expression of co-transfected wild-type MC4R.
- Human MC4R sequence variants showed no significant differences in ligand affinity or agonist responsiveness compared to wild-type.
Conclusions:
- Obesity-associated MC4R truncation mutations disrupt proper receptor transport, leading to intracellular retention.
- These MC4R mutants do not exhibit dominant-negative effects on wild-type MC4R.
- MC4R polymorphisms are unlikely to be significant contributors to human obesity, distinguishing them from the truncation mutants.