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Published on: November 13, 2012
[Cloning and functional analysis of melanocortin 4 receptor mutation gene F261S]
Xin-Yu Shao1, Wei-Ping Jia, Shu-Bing Cai
1Shanghai Diabetes Institute, Shanghai Jiaotong University Affiliated Sixth Hospital, Shanghai 200233, China.
Objective:
To evaluate the function change of the melanocortin 4 receptor (MC4R) protein with mutation of F261S.
Methods:
Human embryonic cells of the HEK293 line were cultured. Wild-type genomic DNA and F261S mutation human melanocortin 4 receptor genes from the genomic DNA of aproband of homozygotic F612 mutation were amplified and cloned into a topo-TA eukaryotic expression plasmid vector. After the wild-type and F261S mutated proteins were expressed in HEK293 cells, alpha-MSH (10(-11) approximately 10(-5) mmol/L) was added, then the intracellular cAMP was detected with dual luciferase reporter assay system.
Results:
When the concentration of alpha-MSH added was 10(-9) approximately 10(-8) mmol/L, the intracellular alpha-MSH concentration of the cells transfected with wild-type MC4R gene was significantly higher than that of the cells transfected with F261S mutation gene (P < 0.05). When the concentration of alpha-MSH added went to 10(-7) approximately 10(-5) mmol/L, the differences became even more significant (all P < 0.01).
Conclusion:
The novel MC4R mutation F261S undermines the signal transduction. It may be the possible reason leading to monogenic mutation obesity in Chinese.
Insights
The F261S mutation in the melanocortin 4 receptor (MC4R) protein impairs its function, potentially causing monogenic obesity in Chinese individuals. This MC4R variant shows reduced signal transduction capabilities.
Area of Science:
- Molecular biology
- Genetics
- Biochemistry
Context:
- The melanocortin 4 receptor (MC4R) plays a crucial role in regulating energy balance.
- Mutations in MC4R are a known cause of monogenic obesity.
- Understanding the functional impact of specific MC4R mutations is essential for genetic diagnostics and therapeutic development.
Purpose:
- To investigate the functional consequences of the F261S mutation in the MC4R protein.
- To determine if this specific mutation affects MC4R signaling pathways.
- To explore the potential link between the F261S MC4R mutation and obesity in the Chinese population.
Summary:
- HEK293 cells were transfected with wild-type or F261S mutated MC4R genes.
- Cells were stimulated with alpha-MSH, and intracellular cAMP levels were measured using a dual luciferase reporter assay.
- The F261S mutation significantly reduced MC4R-mediated signaling compared to the wild-type receptor.
Impact:
- The F261S MC4R mutation demonstrably impairs signal transduction.
- This functional deficit suggests that the F261S mutation is a potential cause of monogenic obesity in Chinese patients.
- Findings contribute to the genetic understanding of obesity and may inform future diagnostic and therapeutic strategies.
