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Research ArticleCELLULAR AND MOLECULAR

Ligand Dependency of 5-Hydroxytryptamine 2C Receptor Internalization

Brian D. Schlag, Zhuangwei Lou, Myles Fennell and John Dunlop
Journal of Pharmacology and Experimental Therapeutics September 2004, 310 (3) 865-870; DOI: https://doi.org/10.1124/jpet.104.067306
Brian D. Schlag
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Zhuangwei Lou
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Myles Fennell
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John Dunlop
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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
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Journal of Pharmacology and Experimental Therapeutics: 310 (3)
Journal of Pharmacology and Experimental Therapeutics
Vol. 310, Issue 3
1 Sep 2004
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Research ArticleCELLULAR AND MOLECULAR

Ligand Dependency of 5-Hydroxytryptamine 2C Receptor Internalization

Brian D. Schlag, Zhuangwei Lou, Myles Fennell and John Dunlop
Journal of Pharmacology and Experimental Therapeutics September 1, 2004, 310 (3) 865-870; DOI: https://doi.org/10.1124/jpet.104.067306

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Research ArticleCELLULAR AND MOLECULAR

Ligand Dependency of 5-Hydroxytryptamine 2C Receptor Internalization

Brian D. Schlag, Zhuangwei Lou, Myles Fennell and John Dunlop
Journal of Pharmacology and Experimental Therapeutics September 1, 2004, 310 (3) 865-870; DOI: https://doi.org/10.1124/jpet.104.067306
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