FIGURE 4.

Hypothalamic GT1-7 cells exclusively express the MC4R subtype. A, the presence of MC3R or MC4R transcripts in GT1-7 cells was detected by RTQ-PCR and is given relative to the expression of β-actin. As a control, cDNA from HEK-293 cells was used. B, cAMP accumulation in GT1-7 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 in percentage of basal cAMP production. Cells were stimulated with increasing concentrations of α-MSH, γ-MSH, or AGRP for 30 min at 37 °C. Results are expressed as the mean ± S.E. of three independent experiments carried out in triplicate. C, cell surface expression of the MC4R in HEK293 or GT1-7 cells was monitored by performing an enzyme-linked immunosorbent assay with intact cells using an anti-MC4R-peptide antibody raised against an N-terminal peptide of the murine MC4R.