Characterization of cell lines stably expressing human normal or mutated EGFP-tagged MC4R.
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.
Blondet
Antonine
A
INSERM U418-INRA UMR 1245 and IFR 62, Hôpital Debrousse and Claude Bernard University, Lyon, France.
Doghman
Mabrouka
M
Rached
Mohamed
M
Durand
Philippe
P
Bégeot
Martine
M
Naville
Danielle
D
D054369:Agouti-Related Protein; D001667:Binding, Competitive; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism; D000242:Cyclic AMP / Q000378:metabolism; D022162:Cytoplasmic Vesicles / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D005822:Genetic Vectors / Q000235:genetics; D049452:Green Fluorescent Proteins / Q000235:genetics* / Q000378:metabolism; D006801:Humans; D008856:Microscopy, Fluorescence; D009154:Mutation; D010446:Peptide Fragments / Q000494:pharmacology; D016133:Polymerase Chain Reaction; D011485:Protein Binding; D021381:Protein Transport / Q000187:drug effects / Q000235:genetics / Q000502:physiology; D044105:Receptor, Melanocortin, Type 4 / Q000235:genetics* / Q000378:metabolism; D044101:Receptors, Melanocortin / Q000819:agonists / Q000037:antagonists & inhibitors / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000819:agonists / Q000037:antagonists & inhibitors / Q000378:metabolism; D014162:Transfection; D000521:alpha-MSH / Q000031:analogs & derivatives* / Q000378:metabolism / Q000494:pharmacology
D054369:Agouti-Related Protein; D010446:Peptide Fragments; D044105:Receptor, Melanocortin, Type 4; D044101:Receptors, Melanocortin; D011993:Recombinant Fusion Proteins; C411703:agouti-related protein-(83-132); C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins; D000521:alpha-MSH; C027756:MSH, 4-Nle-7-Phe-alpha-; D000242:Cyclic AMP