Expression of Functional Melanocortin-4 Receptor in the Hypothalamic GT1-1 Cell Line

Mutations in the melanocortin-4 receptor (MC4-R) cause obesity in both mice and humans, and the receptor is presumed to have an important role in the regulation of energy homeostasis. The MC4-R is expressed in discrete sets of neurons in the central nervous system, and thus it has been technically difficult to study the regulation of expression and the signaling mechanisms of this receptor. We report here a neuronal cell line that exhibits endogenous functional expression for the MC4-R. Initially, RT-PCR analysis showed the presence of MC4-R RNA in the hypothalamic GT1-1 and GT1-7 cells. In addition, GT1-7 cells expressed melanocortin-3 receptor while the GT1-1 subclone specifically expressed predominantly the MC4-R RNA. High-affinity binding sites were demonstrated in the GT1-1 and GT1-7 cells for NDP-α melanocyte-stimulating hormone (MSH; K i = 1.1 × 10 –10 and 1.8 × 10 –10 M) and agouti-related protein (AGRP; K i = 1.548 × 10 –9 and 1.663 –9 M). α-MSH-stimulated cAMP production in GT1-1 cells with an EC 50 of 2.2 × 10 –8 M, and cAMP production was inhibited in the presence of AGRP, an endogenous antagonist of the MC4-R. Stimulation of gonadotropin-releasing hormone (GnRH) secretion was achieved with 1 nM to 1µM concentrations of NDP-α-MSH while no GnRH secretion was observed when the GT1-1 cells were treated with AGRP. The data presented here show that GT1-1 cells specifically express a functional MC4-R that couples to GnRH release.