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 ∼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.
Footnotes
-
These results have been presented in poster format (abstract 643.7) at the Experimental Biology Meeting (San Diego, CA), April 11–15, 2003.
-
Article, publication date, and citation information can be found at http://jpet.aspetjournals.org.
-
doi:10.1124/jpet.104.067306.
-
ABBREVIATIONS: GPCR, G protein-coupled receptor; 5-HT, 5-hydroxytryptamine (serotonin); GFP, green fluorescent protein; FLIPR, fluorometric imaging plate reader; ERC, endocytic recycling compartment; HEK, human embryonic kidney; Ro 60-0175, (S)-2-(6-chloro-5-fluoroindol-1-yl)-1-methylethylamine; WAY-161503, (4aR)-8,9-dichloro-2,3,4,4a-tetrahydro-1H-pyrazino[1,2-a]quinoxalin-5(6H)-one; mCPP, 1-(m-chlorophenyl)piperazine; DOI, (+)-1-(2,5-dimethoxy-4-iodophenyl)-2-amino-propane; SB-206553, N-3-pyridinyl-3,5-dihydro-5-methyl-benzo(1,2-b:4,5-b′)dipyrrole-1(2H)carboxamide; SB-242084, 6-chloro-5-methyl-1-[[2-[(2-methyl-3-pyridyl)oxy]-5-pyridyl]carbamoyl]-indoline.
- Received February 19, 2004.
- Accepted April 26, 2004.
- The American Society for Pharmacology and Experimental Therapeutics
JPET articles become freely available 12 months after publication, and remain freely available for 5 years.Non-open access articles that fall outside this five year window are available only to institutional subscribers and current ASPET members, or through the article purchase feature at the bottom of the page.
|