Modified receptor internalization upon coexpression of 5-HT1B receptor and 5-HT2B receptors

Serotonin 5-HT2B 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-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 wholecell 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-HT 2B 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, coexpression 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-HT1B receptors (expressed alone) was entirely clathrinin-dependent 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 C epsilon-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.