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Cell surface expression of the melanocortin-4 receptor is dependent on a C-terminal di-isoleucine sequence at codons
Donald VanLeeuwen1, Michael E Steffey, Christopher Donahue
1Pfizer Global Research and Development, Ann Arbor, MI 48105, USA.
Abstract:
Loss-of-function mutations in the human melanocortin-4 receptor (MC4R) are associated with obesity. Previous work has implicated a C-terminal di-isoleucine motif at residues 316/317 in MC4R cell surface targeting. It was therefore of interest to examine function and cell surface expression of an MC4R mutation found in an obese proband in which one of these isoleucines was substituted by threonine (I317T). Single mutant (I316T or I317T) and double mutant (I316T,I317T) forms of MC4R were constructed by oligonucleotide-directed mutagenesis and tested for function and cell surface expression in transfected cells. Function was assessed using assays for agonist, [Nle(4)-d-Phe(7)]alpha-melanocyte-stimulating hormone (NDP-alpha-MSH) or forskolin-stimulated cAMP accumulation. Cell surface expression was determined by whole-cell binding of [(125)I]NDP-alpha-MSH, fluorescence immunocytochemistry and fluorescence-activated cell sorting. Maximal cAMP generation of the single mutants was reduced by 40% of wild-type receptor; the double mutant further reduced function to 40% of control, effects that were mirrored by decreases in cell-surface expression. Quantitative RT-PCR showed that, relative to wild-type receptor, transcript levels for the mutated receptors were not reduced. The results further implicate the C-terminal di-isoleucines in cell surface expression of MC4R and suggest that mutations of residues 316 or 317 would predict MC4R hypofunction.
Insights
Mutations in the melanocortin-4 receptor (MC4R) C-terminus, specifically at isoleucine residues 316/317, impair receptor function and cell surface expression, contributing to obesity. These findings highlight the importance of this motif for MC4R activity.
Area of Science:
- Genetics
- Molecular Biology
- Endocrinology
Background:
- Loss-of-function mutations in the human melanocortin-4 receptor (MC4R) are a significant genetic cause of obesity.
- A C-terminal di-isoleucine motif (residues 316/317) is crucial for MC4R cell surface targeting and function.
Purpose of the Study:
- To investigate the functional consequences of a specific MC4R mutation (I317T) found in an obese individual.
- To examine the role of the C-terminal di-isoleucine motif in MC4R cell surface expression and function.
Main Methods:
- Oligonucleotide-directed mutagenesis was used to create single (I316T, I317T) and double (I316T,I317T) MC4R mutants.
- Receptor function was assessed by measuring cAMP accumulation in response to NDP-alpha-MSH or forskolin.
- Cell surface expression was quantified using radioligand binding, immunocytochemistry, and flow cytometry.
Main Results:
- Single MC4R mutants (I316T, I317T) showed a 40% reduction in maximal cAMP generation compared to wild-type.
- The double mutant (I316T,I317T) further decreased function to 40% of control levels.
- Reduced receptor function correlated with decreased cell surface expression, while transcript levels remained unaffected.
Conclusions:
- The C-terminal di-isoleucine motif is critical for MC4R cell surface expression.
- Mutations affecting residues 316 or 317 of MC4R are predicted to cause MC4R hypofunction, potentially leading to obesity.