title: MT1 melatonin receptor internalization underlies melatonin-induced morphologic changes in Chinese hamster ovary cells and these processes are dependent on Gi proteins, MEK 1/2 and microtubule modulation.
abstract: Melatonin induces cellular differentiation in numerous cell types. Data show that multiple mechanisms are involved in these processes that are cell-type specific and may be receptor dependent or independent. The focus of this study was to specifically assess the role of human MT1 melatonin receptors in cellular differentiation using an MT1-Chinese hamster ovary (CHO) model; one that reproducibly produces measurable morphologic changes in response to melatonin. Using multiple approaches, we show that melatonin induces MT1-CHO cells to hyperelongate through a MEK 1/2, and ERK 1/2-dependent mechanism that is dependent upon MT1 receptor internalization, Gi protein activation, and clathrin-mediated endocytosis. Using immunoprecipitation analysis, we show that MT1 receptors form complexes with Gi(alpha) 2,3, Gq(alpha), beta-arrestin-2, MEK 1/2, and ERK 1/2 in the presence of melatonin. We also show that MEK and ERK activity that is induced by melatonin is dependent on Gi protein activation, clathrin-mediated endocytosis and is modulated by microtubules. We conclude from these studies that melatonin-induced internalization of human MT1 melatonin receptors in CHO cells is responsible for activating both MEK 1/2 and ERK 1/2 to drive these morphologic changes. These events, as mediated by melatonin, require Gi protein activation and endocytosis mediated through clathrin, to form MT1 receptor complexes with beta-arrestin-2/MEK 1/2 and ERK 1/2. The MT1-CHO model is invaluable to mapping out signaling cascades as mediated through MT1 receptors especially because it separates out MEK/ERK 1/2 activation by MT1 receptors from that of receptor tyrosine kinases.
authors.affiliation: Division of Pharmaceutical Sciences, School of Pharmacy, Duquesne University, Pittsburgh, PA 15282, USA.
authors.forename: C Dominic
authors.initials: CD
authors.lastname: Bondi
authors.forename: Raelene M
authors.initials: RM
authors.lastname: McKeon
authors.forename: Jennifer M
authors.initials: JM
authors.lastname: Bennett
authors.forename: Paul F
authors.initials: PF
authors.lastname: Ignatius
authors.forename: Lena
authors.initials: L
authors.lastname: Brydon
authors.forename: Ralf
authors.initials: R
authors.lastname: Jockers
authors.forename: Melissa A
authors.initials: MA
authors.lastname: Melan
authors.forename: Paula A
authors.initials: PA
authors.lastname: Witt-Enderby
mesh_terms: D000818:Animals; D016466:CHO Cells; D002103:Cadaverine / Q000031:analogs & derivatives / Q000494:pharmacology; D002454:Cell Differentiation / Q000187:drug effects; D002966:Clathrin / Q000502:physiology; D006224:Cricetinae; D003412:Cricetulus; D003703:Demecolcine / Q000494:pharmacology; D048049:Extracellular Signal-Regulated MAP Kinases / Q000502:physiology; D016244:Guanosine 5'-O-(3-Thiotriphosphate) / Q000494:pharmacology; D006801:Humans; D008162:Lumicolchicines / Q000494:pharmacology; D048369:MAP Kinase Kinase 1 / Q000502:physiology*; D048730:MAP Kinase Kinase Kinase 2 / Q000502:physiology*; D008550:Melatonin / Q000494:pharmacology*; D008870:Microtubules / Q000187:drug effects* / Q000502:physiology*; D037342:Pertussis Toxin / Q000494:pharmacology; D044122:Receptor, Melatonin, MT1 / Q000378:metabolism*; D014363:Tryptamines / Q000494:pharmacology
chemical_list: D002966:Clathrin; D008162:Lumicolchicines; D044122:Receptor, Melatonin, MT1; D014363:Tryptamines; C057154:luzindole; D016244:Guanosine 5'-O-(3-Thiotriphosphate); D037342:Pertussis Toxin; D048049:Extracellular Signal-Regulated MAP Kinases; D048730:MAP Kinase Kinase Kinase 2; D048369:MAP Kinase Kinase 1; C008542:monodansylcadaverine; D008550:Melatonin; D002103:Cadaverine; D003703:Demecolcine