title: Differential regulation of the serotonin transporter by vesicle-associated membrane protein 2 in cells of neuronal versus non-neuronal origin.
abstract: The serotonin transporter (SERT) is a key regulator of serotonergic signalling as it mediates the re-uptake of synaptic serotonin into nerve terminals, thereby terminating or modulating its signal. It is well-known that SERT regulation is a dynamic process orchestrated by a wide array of proteins and mechanisms. However, molecular details on possible coordinated regulation of SERT activity and 5-HT release are incomplete. Here, we report that vesicle-associated membrane protein 2 (VAMP2), a SNARE protein that mediates vesicle fusion with the plasma membrane, interacts with SERT. This was documented in vitro, through GST pull-down assays, by co-immunoprecipitation experiments on heterologous cells and rat hippocampal synaptosomes, and with FRET analysis in live transfected HEK-293 MSR cells. The related isoforms VAMP1 and VAMP3 also physically interact with SERT. However, comparison of the three VAMP isoforms shows that only VAMP2 possesses a functionally distinct role in relation to SERT. VAMP2 influences 5-HT uptake, cell surface expression and the delivery rate of SERT to the plasma membrane differentially in HEK-293 MSR and PC12 cells. Moreover, siRNA-mediated knock-down of endogenous VAMP2 reduces 5-HT uptake in CAD cells stably expressing low levels of heterologous SERT. Deletion and mutant analysis suggest a role for the isoform specific C-terminal domain of VAMP2 in regulating SERT function. Our data identify a novel interaction between SERT and a synaptic vesicle protein and support a link between 5-HT release and re-uptake.
authors.affiliation: Translational Neuropsychiatry Unit, Department of Clinical Medicine, Aarhus University Hospital, Risskov, Denmark.
authors.forename: Heidi Kaastrup
authors.initials: HK
authors.lastname: Müller
authors.affiliation: Translational Neuropsychiatry Unit, Department of Clinical Medicine, Aarhus University Hospital, Risskov, Denmark.
authors.forename: Marie
authors.initials: M
authors.lastname: Kragballe
authors.affiliation: Stereology and Electron Microscopy Laboratory, Centre for Stochastic Geometry and Advanced Bioimaging, Department of Clinical Medicine, University of Aarhus, Aarhus, Denmark.
authors.forename: Anja Winther
authors.initials: AW
authors.lastname: Fjorback
authors.affiliation: Translational Neuropsychiatry Unit, Department of Clinical Medicine, Aarhus University Hospital, Risskov, Denmark.
authors.forename: Ove
authors.initials: O
authors.lastname: Wiborg
mesh_terms: D000818:Animals; D055785:Gene Knockdown Techniques; D057809:HEK293 Cells; D006624:Hippocampus / Q000166:cytology; D006801:Humans; D008297:Male; D009474:Neurons / Q000166:cytology / Q000378:metabolism*; D021381:Protein Transport; D034741:RNA, Small Interfering / Q000235:genetics; D051381:Rats; D017207:Rats, Sprague-Dawley; D012701:Serotonin / Q000378:metabolism; D050486:Serotonin Plasma Membrane Transport Proteins / Q000378:metabolism*; D013569:Synapses / Q000378:metabolism; D050988:Vesicle-Associated Membrane Protein 2 / Q000172:deficiency / Q000235:genetics / Q000378:metabolism*
chemical_list: D034741:RNA, Small Interfering; D050486:Serotonin Plasma Membrane Transport Proteins; D050988:Vesicle-Associated Membrane Protein 2; D012701:Serotonin