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Received for publication February 24, 2004.
Revised April 21, 2004.
Accepted for publication April 26, 2004.
Agonist induced internalization of G-protein coupled receptors (GPCRs) is a well-characterized phenomenon believed to contribute to receptor desensitization. The 5-HT2C 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 1-(m-chlorophenyl)piperazine (mCPP) and (+)-1-(2,5-Dimethoxy-4-iodophenyl)-2-amino-propane (DOI), inverse agonists SB-206553 (N-3-pyridinyl-3,5-dihydro-5-methyl-benzo(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 GFP tagged 5-HT2C receptor (VSV isoform) expressed in transiently transfected HEK cells. We detected internalization with an automated, cell based fluorescence-imaging system (Cellomics' Arrayscan) and monitored function with intracellular Ca2+ measurements (FLIPR). The 5-HT2C-GFP construct exhibited appropriate pharmacology and we observed that while 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-HT2C receptor is internalized by incubation with agonists and partial agonists, but not with inverse agonists or neutral antagonists.
Key words:
5-HT, Arrayscan, GFP, GPCR, internalization, receptor
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