Stable expression of human melanocortin 3 receptor fused to EGFP in the HEK293 cells

详细信息    查看全文

• 作者：Rached ; Mohamed ; Buronfosse ; Anna ; Durand ; Philippe ; Begeot ; Martine ; Penhoat ; Armelle
• 关键词：Melanocortin 3 receptors ; Enhanced green fluorescent protein ; Chimeric protein ; Receptor binding ; Confocal microscopy ; cAMP
• 刊名：Biochemical and Biophysical Research Communications
• 出版年：2003
• 出版时间：June 20, 2003
• 年：2003
• 卷：306
• 期：1
• 页码：208-212
• 全文大小：142 K

文摘

Among the melanocortins α-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 (EC₅₀: 0.3nM). Binding studies revealed a single class of binding sites with a K_D of 2.24nM. 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.