Stable expression of human melanocortin 3 receptor fused to EGFP in the HEK293 cells.
Among the melanocortins alpha-MSH is known to be involved in feeding behavior. These hormones mediate their effects through G protein-coupled receptors by stimulating adenylate cyclase. In this study, we have developed an in vitro expression model for human melanocortin 3 receptor (hMC3R) tagged at its C terminus with EGFP. The corresponding chimeric cDNA was stably expressed in HEK293 cells. The selected clones expressing the hMC3R-EGFP exhibited cell surface fluorescence and responded to NDP-MSH stimulation by producing cAMP in a dose-dependent manner (EC(50): 0.3 nM). Binding studies revealed a single class of binding sites with a K(D) of 2.24 nM. Moreover, Agouti-related protein was also demonstrated to be an antagonist of the hMC3R-EGFP. Thus, the hMC3R tagged with EGFP stably expressed in HEK293 cells, exhibiting the same characteristics than the wild-type hMC3R, is the only model of expression of this receptor allowing its direct localization inside living cells.
Rached
Mohamed
M
INSERM U418/INRA UMR1245, IFR Laennec, Hôpital Debrousse, 29 rue soeur Bouvier, 69322 05, Lyon Cédex, France.
Buronfosse
Anna
A
Durand
Philippe
P
Begeot
Martine
M
Penhoat
Armelle
A
D001483:Base Sequence; D001665:Binding Sites; D002460:Cell Line; D000242:Cyclic AMP / Q000096:biosynthesis; D018076:DNA, Complementary / Q000235:genetics; D015870:Gene Expression; D049452:Green Fluorescent Proteins; D006801:Humans; D007700:Kinetics; D008164:Luminescent Proteins / Q000235:genetics / Q000378:metabolism; D018613:Microscopy, Confocal; D008856:Microscopy, Fluorescence; D044104:Receptor, Melanocortin, Type 3; D018043:Receptors, Corticotropin / Q000235:genetics* / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D000521:alpha-MSH / Q000031:analogs & derivatives* / Q000494:pharmacology
D018076:DNA, Complementary; D008164:Luminescent Proteins; D044104:Receptor, Melanocortin, Type 3; D018043:Receptors, Corticotropin; D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins; D000521:alpha-MSH; C027756:MSH, 4-Nle-7-Phe-alpha-; D000242:Cyclic AMP