title: Ligand dependency of 5-hydroxytryptamine 2C receptor internalization.
abstract: Agonist-induced internalization of G protein-coupled receptors (GPCRs) is a well characterized phenomenon believed to contribute to receptor desensitization. The 5-hydroxytryptamine (5-HT)2C subtype of serotonin receptor is a GPCR that we have shown to internalize upon agonist incubation. In this study, we have examined the effects of 5-HT2C receptor agonists serotonin, Ro 60-0175 [(S)-2-(6-chloro-5-fluoroindol-1-yl)-1-methylethylamine], and WAY-161503 [(4aR)-8,9-dichloro-2,3,4,4a-tetrahydro-1H-pyrazino[1,2-a]quinoxalin-5(6H)-one]; partial agonists mCPP [1-(m-chlorophenyl)piperazine] and DOI [(+)-1-(2,5-dimethoxy-4-iodophenyl)-2-amino-propane]; inverse agonists SB-206553 [N-3-pyridinyl-3,5-dihydro-5-methylbenzo(1,2-b:4,5-b')dipyrrole-1(2H)carboxamide] and mianserin; and neutral antagonists SB-242084 [6-chloro-5-methyl-1-[[2-[(2-methyl-3-pyridyl)oxy]-5-pyridyl]carbamoyl]-indoline] and 5-methoxygramine on the internalization of a C-terminal green fluorescent protein (GFP)-tagged 5-HT2C receptor (VSV isoform) expressed in transiently transfected human embryonic kidney cells. We detected internalization with an automated, cell-based fluorescence-imaging system (Arrayscan) and monitored function with intracellular Ca2+ measurements (flourometric imaging plate reader). The 5-HT2C-GFP construct exhibited appropriate pharmacology, and we observed that although all three agonists resulted in similar magnitudes of dose-dependent internalization, the partial agonists resulted in approximately 50% less internalization, and the inverse agonists and neutral antagonists failed to induce internalization. These results were confirmed by confocal microscopy. They demonstrate that the 5-HT2C receptor is internalized by incubation with agonists and partial agonists but not with inverse agonists or neutral antagonists.
authors.affiliation: Neuroscience Discovery Research, Wyeth Research, CN 8000, Princeton, NJ 08543, USA.
authors.forename: Brian D
authors.initials: BD
authors.lastname: Schlag
authors.forename: Zhuangwei
authors.initials: Z
authors.lastname: Lou
authors.forename: Myles
authors.initials: M
authors.lastname: Fennell
authors.forename: John
authors.initials: J
authors.lastname: Dunlop
mesh_terms: D002118:Calcium / Q000378:metabolism*; D002478:Cells, Cultured; D004705:Endocytosis / Q000502:physiology; D049452:Green Fluorescent Proteins; D006801:Humans; D008024:Ligands; D008164:Luminescent Proteins / Q000235:genetics / Q000378:metabolism; D044405:Receptor, Serotonin, 5-HT2C / Q000378:metabolism*; D012702:Serotonin Antagonists / Q000378:metabolism*; D017366:Serotonin Receptor Agonists / Q000378:metabolism*
chemical_list: D008024:Ligands; D008164:Luminescent Proteins; D044405:Receptor, Serotonin, 5-HT2C; D012702:Serotonin Antagonists; D017366:Serotonin Receptor Agonists; D049452:Green Fluorescent Proteins; D002118:Calcium