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Modified Receptor Internalization upon Coexpression of 5-HT_1B Receptor and 5-HT_2B Receptors

Institut National de la Santé et de la Recherche Médicale, U839, Paris, France (V.S., L.M.); Université Pierre et Marie Curie-Paris 6, Institut du Fer à Moulin, Unité Mixte de Recherche S0839, Paris, France (V.S., L.M.); Centre National de la Recherche Scientifique UMR7104, Illkirch, France (A.J., M.D., S.G., L.M.); Assistance Publique-Hopitaux de Paris, Hôpital Lariboisière, Service de Biochimie, Paris, France (J.C., B.S., P.M., J.M.L.); EA3621, IFR139, Paris, France (J.C., B.S., P.M., J.M.L.)

Serotonin 5-HT_2B receptors are often coexpressed with 5-HT_1B receptors, and cross-talk between the two receptors has been reported in various cell types. However, many mechanistic details underlying 5-HT_1B and 5-HT_2B receptor cross-talk have not been elucidated. We hypothesized that 5-HT_2B and 5-HT_1B receptors each affect the others' signaling by modulating the others' trafficking. We thus examined the agonist stimulated internalization kinetics of fluorescent protein-tagged 5-HT_2B and 5-HT_1B 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-HT_2B and 5-HT_1B receptors displayed distinct half-lives. Upon coexpression, serotonin-induced internalization of 5-HT_2B receptors was accelerated 5-fold and was insensitive to a 5-HT_2B receptor antagonist. In this context, 5-HT_2B receptors did internalize in response to a 5-HT_1B receptor agonist. In contrast, co-expression did not render 5-HT_1B receptor internalization sensitive to a 5-HT_2B 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-HT_1B receptors (expressed alone) was entirely clathrin-independent and Caveolin1-dependent, whereas that of 5-HT_2B receptors (expressed alone) was Caveolin1-independent and clathrin-dependent. Upon coexpression, serotonin-induced 5-HT_2B receptor internalization became partially Caveolin1-dependent, and serotonin-induced 5-HT_1B receptor internalization became entirely Caveolin1-independent in a protein kinase C-dependent fashion. In conclusion, these data demonstrate that coexpression of 5-HT_1B and 5-HT_2B receptors influences the internalization pathways and kinetics of both receptors.

Address correspondence to: Luc Maroteaux, INSERM, U616-U839, Hôpital Pitié-Salpetrière, Bat Pédiatrie, 47 Bd de l'Hôpital 75013 Paris, France. E-mail: luc.maroteaux{at}chups.jussieu.fr