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-HT_2C 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-HT_2C 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-HT_2C-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 ∼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-HT_2C receptor is internalized by incubation with agonists and partial agonists but not with inverse agonists or neutral antagonists.