Received for publication November 14, 2006.
Revised February 17, 2007.
Accepted for publication February 26, 2007.

Modified Receptor Internalization upon Co-expression of 5-HT_1B Receptor and 5-HT_2B Receptors

Abstract

Serotonin 5-HT_2B receptors are often co-expressed with 5-HT_1B receptors, and crosstalk between the two receptors has been reported in various cell types. However, many mechanistic details underlying 5-HT_1B and 5-HT_2B receptors crosstalk have not been elucidated. We hypothesized that 5-HT_2B and 5-HT_1B receptors affect each other's signaling by modulating each other's 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 co-expression in LMTK^- murine fibroblasts. Time-lapse confocal microscopy and whole-cell radioligand binding analyses revealed that 5-HT_2B and 5-HT_1B receptors when expressed alone displayed distinct half-lives. Upon co-expression, serotonin-induced internalization of 5-HT_2B receptors was accelerated five-fold, and 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 co-expression was likely not due to direct interaction as only low levels of co-localization were observed. Antibody knock-down experiments revealed that internalization of 5-HT_1B receptors (expressed alone) was entirely clathrin-independent and Caveolin1-dependent, while that of 5-HT_2B receptors (expressed alone) was Caveolin1-independent and clathrin-dependent. Upon co-expression, 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 Cepsilon-dependent fashion. In conclusion, these data demonstrate that co-expression of 5-HT_1B and 5-HT_2B receptors influences the internalization pathways and kinetics of both receptors.

Key words: Serotonin, Gi family, Gq/11 family, Protein Kinase C, G protein regulation, Receptor synthesis/trafficking, Desensitization/uncoupling, GRKs, barrestins, Fluorescence techniques, Receptor binding studies