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 (Kd = 1.3 nM; binding capacity, 3.6 pmol/mg protein) is used to assess the ability of the MCH receptor to couple to Gi, Go, and Gq 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, MCH 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 PTX sensitive and therefore mediated through a Gi/Go protein. A distinct G protein-coupled (probably Gq type) mediates the PTX-insensitive component. To distinguish Gi vs. Go coupling, MCH-stimulated mitogen-activated protein (MAP) kinase activity was examined. Gi and Go use separate signaling pathways to mediate MAP kinase activation in CHOcells. Protein kinase C (PKC) activity is essential in the Go-dependent MAP kinase signaling pathway, but is not required in the GC-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 Gi- and Go-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.
Hawes
B E
BE
Central Nervous System/Cardiovascular Department, Schering-Plough Corp. Research Institute, Kenilworth, New Jersey 07033, USA. brian.hawes@spcorp.com
Kil
E
E
Green
B
B
O'Neill
K
K
Fried
S
S
Graziano
M P
MP
D000262:Adenylyl Cyclases / Q000378:metabolism; D000818:Animals; D016466:CHO Cells; D002462:Cell Membrane / Q000378:metabolism; D005576:Colforsin / Q000494:pharmacology; D006224:Cricetinae; D000242:Cyclic AMP / Q000096:biosynthesis; D019206:GTP-Binding Protein alpha Subunits, Gi-Go / Q000502:physiology; D019204:GTP-Binding Proteins / Q000502:physiology*; D015870:Gene Expression; D020962:Heterotrimeric GTP-Binding Proteins / Q000502:physiology; D006801:Humans; D007028:Hypothalamic Hormones / Q000378:metabolism / Q000494:pharmacology; D007295:Inositol Phosphates / Q000096:biosynthesis; D008246:Lysophospholipids / Q000494:pharmacology; D008543:Melanins / Q000378:metabolism / Q000494:pharmacology; D020928:Mitogen-Activated Protein Kinases / Q000378:metabolism; D037342:Pertussis Toxin; D010907:Pituitary Hormones / Q000378:metabolism / Q000494:pharmacology; D011493:Protein Kinase C / Q000037:antagonists & inhibitors; D011979:Receptors, Pituitary Hormone / Q000235:genetics / Q000502:physiology*; D015398:Signal Transduction; D014162:Transfection; D010566:Virulence Factors, Bordetella / Q000494:pharmacology
D007028:Hypothalamic Hormones; D007295:Inositol Phosphates; D008246:Lysophospholipids; D008543:Melanins; D010907:Pituitary Hormones; D011979:Receptors, Pituitary Hormone; D010566:Virulence Factors, Bordetella; C092274:melanin-concentrating hormone receptor; D005576:Colforsin; C019463:melanin-concentrating hormone; D000242:Cyclic AMP; D037342:Pertussis Toxin; D011493:Protein Kinase C; D020928:Mitogen-Activated Protein Kinases; D019204:GTP-Binding Proteins; D019206:GTP-Binding Protein alpha Subunits, Gi-Go; D020962:Heterotrimeric GTP-Binding Proteins; D000262:Adenylyl Cyclases