FIGURE 1.

Stable expression of the mouse MC4R and the MC4R-D90N mutant in HEK293 cells. A, cell surface expression in HEK293 cells stably expressing the MC4R wild type or the D90N mutant fused on the N terminus with the Xpress epitope was monitored by the enzyme-linked immunosorbent assay technique with intact cells. B, HEK293-Ex-MC4R or HEK293-Ex-MC4R-D90N cells were transiently transfected with a reporter gene construct harboring the firefly luciferase gene under the control of the CRE promoter. Thirty-six hours after transfection (including 12 h of cell starvation), cells were stimulated with increasing concentrations of α-MSH. After lysis of the cells, firefly luciferase activity was determined. Dose-response curves were drawn from data obtained in three independent experiments carried out in triplicate. C, cAMP accumulation in HEK293-Ex-MC4R or HEK293-Ex-MC4R-D90N cells was assessed after labeling of the cells with [³H]adenine followed by the purification of [³H]cAMP and [³H]ATP by sequential chromatography and expressed as the ratio of [³H]cAMP/([³H]cAMP + [³H]ATP). Cells were stimulated or not with 1 μm α-MSH for 30 min at 37 °C. As a control, cells were also stimulated with the MC4R-independent direct adenylyl cyclase activator forskolin (FSK, 20 μm). Results are expressed as the mean ± S.E. of five independent experiments performed in triplicate.