The melanin-concentrating hormone receptor couples to multiple G proteins to activate diverse intracellular signaling pathways

The receptor for melanin-concentrating hormone (MCH) was recently identified as the orphan G protein-coupled receptor SLC-1. In this study, a CHO cell line expressing the MCH receptor(K-d = 1.3 nM; binding capacity, 3.6 pmol/mg protein) is used to assess the ability of the MCH receptor to couple to G(i), G(o), and G(q) proteins. The results demonstrate that MCH inhibits forskolin-stimulated cAMP production in a pertussis toxin- (PTX)-sensitive manner in CHO-MCHR cells (EC50 = 100 pM), indicating that the MCH receptor couples to one or more members of the Gi subfamily of G proteins. in addition, CH stimulates increases in phosphoinositide metabolism (EC50 = 50 nM) and in intracellular free Ca2+ levels (EC50 = 10 nM). MCH-stimulated inositol phosphate production and increases in intracellular free Ca2+ are partially inhibited (60% and 40%, respectively) by PTX pretreatment, demonstrating that there are at least two components of each of these signaling pathways. One component is PIX sensitive and therefore mediated through a G(i)/G(o) protein. A distinct G protein-coupled (probably G(q) type) mediates the PTX-insensitive component. To distinguish G(i) vs. G(o) coupling, MCH-stimulated mitogen-activated protein (MAP) kinase activity was examined. G(i) and G(o) use separate signaling pathways to mediate MAP kinase activation in CHO cells. Protein kinase C (PKC) activity is essential in the G(o)-dependent MAP kinase signaling pathway, but is not required in the G(i)-dependent MAP kinase signaling pathway. MCH stimulated MAP kinase activity is decreased (50%), but not abolished, by inhibition of PKC activity or depletion of cellular PKC, indicating that MCH-stimulated MAP kinase activity is mediated through both G(i)- and G(o)-dependent signaling mechanisms. The results of this study are the first to clearly demonstrate that the MCH receptor couples to multiple G proteins to mediate several diverse intracellular signaling pathways.