Abstract
The melanocortin receptor type 4 (MC4-R) is involved in food intake and represents a potential target for the treatment of some forms of obesity. The fluorescent protein EGFP was fused to the wild-type or mutated coding sequence of the human MC4-R. After transfection in HEK 293, clones stably expressing hMC4-R-EGFP were selected. Wild-type chimeric hMC4-R was well addressed to the cell membrane as demonstrated using confocal microscopy and displayed the same pharmacological characteristics as native hMC4R. NDP-alpha MSH induced a time-dependent internalization of MC4-R that was partially prevented by AgRP. The two mutated chimeric receptors studied here (CTCT-deleted and C271A) showed a high alteration of their response to ligand and were retained inside the cells. In conclusion, we have developed a model of clones stably expressing EGFP-tagged-hMC4-R. This is the only such model available to date and it provides a useful tool to follow the trafficking of MC4-R inside living cells.
Publication types
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Research Support, Non-U.S. Gov't
MeSH terms
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Agouti-Related Protein
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Binding, Competitive
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Cell Line
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Cell Membrane / metabolism
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Cyclic AMP / metabolism
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Cytoplasmic Vesicles / metabolism
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Dose-Response Relationship, Drug
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Genetic Vectors / genetics
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Green Fluorescent Proteins / genetics*
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Green Fluorescent Proteins / metabolism
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Humans
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Microscopy, Fluorescence
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Mutation
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Peptide Fragments / pharmacology
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Polymerase Chain Reaction
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Protein Binding
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Protein Transport / drug effects
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Protein Transport / genetics
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Protein Transport / physiology
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Receptor, Melanocortin, Type 4 / genetics*
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Receptor, Melanocortin, Type 4 / metabolism
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Receptors, Melanocortin / agonists
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Receptors, Melanocortin / antagonists & inhibitors
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Receptors, Melanocortin / metabolism
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Recombinant Fusion Proteins / agonists
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Recombinant Fusion Proteins / antagonists & inhibitors
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Recombinant Fusion Proteins / metabolism
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Transfection
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alpha-MSH / analogs & derivatives*
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alpha-MSH / metabolism
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alpha-MSH / pharmacology
Substances
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Agouti-Related Protein
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Peptide Fragments
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Receptor, Melanocortin, Type 4
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Receptors, Melanocortin
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Recombinant Fusion Proteins
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agouti-related protein-(83-132)
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enhanced green fluorescent protein
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Green Fluorescent Proteins
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alpha-MSH
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MSH, 4-Nle-7-Phe-alpha-
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Cyclic AMP