Abstract
Serotonin 5-HT2B receptors are often coexpressed with 5-HT1B receptors, and cross-talk between the two receptors has been reported in various cell types. However, many mechanistic details underlying 5-HT1B and 5-HT2B receptor cross-talk have not been elucidated. We hypothesized that 5-HT2B and 5-HT1B receptors each affect the others' signaling by modulating the others' trafficking. We thus examined the agonist stimulated internalization kinetics of fluorescent protein-tagged 5-HT2B and 5-HT1B receptors when expressed alone and upon coexpression in LMTK– murine fibroblasts. Time-lapse confocal microscopy and whole-cell radioligand binding analyses revealed that, when expressed alone, 5-HT2B and 5-HT1B receptors displayed distinct half-lives. Upon coexpression, serotonin-induced internalization of 5-HT2B receptors was accelerated 5-fold and was insensitive to a 5-HT2B receptor antagonist. In this context, 5-HT2B receptors did internalize in response to a 5-HT1B receptor agonist. In contrast, co-expression did not render 5-HT1B receptor internalization sensitive to a 5-HT2B receptor agonist. The altered internalization kinetics of both receptors upon coexpression was probably not due to direct interaction because only low levels of colocalization were observed. Antibody knockdown experiments revealed that internalization of 5-HT1B receptors (expressed alone) was entirely clathrin-independent and Caveolin1-dependent, whereas that of 5-HT2B receptors (expressed alone) was Caveolin1-independent and clathrin-dependent. Upon coexpression, serotonin-induced 5-HT2B receptor internalization became partially Caveolin1-dependent, and serotonin-induced 5-HT1B receptor internalization became entirely Caveolin1-independent in a protein kinase Cϵ-dependent fashion. In conclusion, these data demonstrate that coexpression of 5-HT1B and 5-HT2B receptors influences the internalization pathways and kinetics of both receptors.
Footnotes
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This work has been supported by funds from the Centre National de la Recherche Scientifique, the Institut National de la Santé et de la Recherche Médicale, the Université Pierre et Marie Curie, the Université Louis Pasteur, and by grants from the Fondation de France, the Fondation pour la Recherche Médicale, the Association pour la Recherche contre le Cancer, the french ministry of research (ACI and ANR) and the European community. L.M.'s team is an “Equipe FRM.”
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*A.J. and M.D. contributed equally to this work
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Article, publication date, and citation information can be found at http://molpharm.aspetjournals.org.
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doi:10.1124/mol.106.032656.
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ABBREVIATIONS: 5-HT, 5-hydroxytryptamine; GPCR, G protein-coupled receptor; GRK, GPCR kinase; PKA, protein kinase A; PDZ, postsynaptic density 95/disc-large/zona occludens (PSD-95, Dlg, ZO-1); CFP, cyan fluorescent protein; YFP, yellow fluorescent protein; GFP, green fluorescent protein; RS, RS127445; RS-127445, 2-amino-4-(4-fluoronaphth-1-yl)-6-isopropylpyrimidine; CP, CP93129; CP93129, 1,4-dihydro-3-(1,2,5,6-tetrahydropyrid-4-yl)pyrrolo{3,2-b}pyridin-5-one dihydrochloride; BW, BW723C86; BW723C86, 1-{5-(2-thienylmethoxy)-1H-3-indolyl}propan-2-amine hydrochloride; H-89, N-{2-((p-bromocinnamyl)amino)ethyl}-5-isoquinolinesulfonamide dihydrochloride; Gö 6850-Bisindolylmaleimide I, 2-{1-(3-dimethylaminopropyl)-1H-indol-3-yl}-3-(1H-indol-3-yl)-maleimide; Gö 6976, 12-(2-cyanoethyl)-6,7,12,13-tetrahydro-13-methyl-5-oxo-5H-indolo(2,3-a)pyrrolo(3,4-c)-carbazole; [125I]DOI, [125I]1-(2,5-dimethoxy-4-iodophenyl)-2-aminopropane hydrochloride; [125I]GTI, [125I]serotonin-5-O-carboxymethyl-glycil-iodo-tyrosamine; PKC, protein kinase C; ROI, regions of interest; FRET, fluorescent resonance energy transfer; PA, photoactivatable; Cav-1, caveolin-1.
- Received November 14, 2006.
- Accepted February 26, 2007.
- The American Society for Pharmacology and Experimental Therapeutics
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