title: Role of C-terminal region in the functional regulation of rat serotonin transporter (SERT).
abstract: Previously, we revealed that the state of the actin cytoskeleton affects the uptake activity of the serotonin transporter (SERT). Recently, it was reported that the C-terminus of SERT interacts with MacMARCKS, a substrate of PKC that can bind to the actin cytoskeleton. To elucidate the importance of the C-terminal region in the regulation of SERT activity and the interaction with the actin cytoskeleton, we examined whether the overexpression of the C-terminus affects the transport activity of SERT. To this end, we overexpressed a GFP-fused 30-amino acid construct of the SERT C-terminus (GFP-SERT-CT) in HEK293 cells stably expressing FLAG-tagged SERT (FL-SERT-HEK293 cells). The SERT uptake activity and transporter current were attenuated in GFP-SERT-CT-expressing FL-SERT-HEK293 cells, as compared with GFP-expressing FL-SERT-HEK293 cells. Eadie-Hofstee analysis revealed that GFP-SERT-CT overexpression attenuated the SERT uptake activity by reducing the Vmax, but not changing the Km, which was consistent with the results of experiments on the cell-surface expression of SET using biotinylation/immunoblot analysis. Immunocytochemical analysis demonstrated that GFP-SERT-CT was co-localized with FLAG-SERT and cortical actin at the plasma membrane. In addition, the SERT C-terminus did not affect dopamine transporter activity. These findings showed the significance of the C-terminal region to the functional regulation of SERT, suggesting that GFP-SERT-CT acts as a molecular decoy to disrupt the interaction between SERT and the actin cytoskeleton.
authors.affiliation: Department of Ophthalmology and Visual sciences, Graduate School of Biomedical Sciences, Hiroshima University, 1-2-3 Kasumi Minami-ku, Hiroshima 734-8551, Japan.
authors.forename: Hideki
authors.initials: H
authors.lastname: Mochizuki
authors.forename: Taku
authors.initials: T
authors.lastname: Amano
authors.forename: Takahiro
authors.initials: T
authors.lastname: Seki
authors.forename: Hiroaki
authors.initials: H
authors.lastname: Matsubayashi
authors.forename: Chieko
authors.initials: C
authors.lastname: Mitsuhata
authors.forename: Katsuya
authors.initials: K
authors.lastname: Morita
authors.forename: Shigeo
authors.initials: S
authors.lastname: Kitayama
authors.forename: Toshihiro
authors.initials: T
authors.lastname: Dohi
authors.forename: Hiromu K
authors.initials: HK
authors.lastname: Mishima
authors.forename: Norio
authors.initials: N
authors.lastname: Sakai
mesh_terms: D000818:Animals; D015153:Blotting, Western; D002460:Cell Line; D050483:Dopamine Plasma Membrane Transport Proteins; D004594:Electrophysiology; D049452:Green Fluorescent Proteins / Q000378:metabolism; D006801:Humans; D007150:Immunohistochemistry; D007700:Kinetics; D008562:Membrane Glycoproteins / Q000378:metabolism*; D008565:Membrane Proteins / Q000096:biosynthesis; D026901:Membrane Transport Proteins / Q000378:metabolism*; D009419:Nerve Tissue Proteins / Q000378:metabolism*; D051381:Rats; D050486:Serotonin Plasma Membrane Transport Proteins
chemical_list: D050483:Dopamine Plasma Membrane Transport Proteins; D008562:Membrane Glycoproteins; D008565:Membrane Proteins; D026901:Membrane Transport Proteins; D009419:Nerve Tissue Proteins; C493230:SLC6A4 protein, human; D050486:Serotonin Plasma Membrane Transport Proteins; C493232:Slc6a4 protein, rat; D049452:Green Fluorescent Proteins