Ligand dependency of 5-hydroxytryptamine 2C receptor internalization

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 (Array-scan) 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 similar to50% 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.