title: The cellular distribution of serotonin transporter is impeded on serotonin-altered vimentin network.
abstract: BACKGROUND
  The C-terminus of the serotonin transporter (SERT) contains binding domains for different proteins and is critical for its functional expression. In endogenous and heterologous expression systems, our proteomic and biochemical analysis demonstrated that an intermediate filament, vimentin, binds to the C-terminus of SERT. It has been reported that 5HT-stimulation of cells leads to disassembly and spatial reorientation of vimentin filaments.
  
  
  RESULTS
  We tested the impact of 5HT-stimulation on vimentin-SERT association and found that 5HT-stimulation accelerates the translocation of SERT from the plasma membrane via enhancing the level of association between phosphovimentin and SERT. Furthermore a progressive truncation of the C-terminus of SERT was performed to map the vimentin-SERT association domain. Deletion of up to 20, but not 14 amino acids arrested the transporters at intracellular locations. Although, truncation of the last 14 amino acids, did not alter 5HT uptake rates of transporter but abolished its association with vimentin. To understand the involvement of 5HT in phosphovimentin-SERT association from the plasma membrane, we further investigated the six amino acids between Delta14 and Delta20, i.e., the SITPET sequence of SERT. While the triple mutation on the possible kinase action sites, S(611), T(613), and T(616) arrested the transporter at intracellular locations, replacing the residues with aspartic acid one at a time altered neither the 5HT uptake rates nor the vimentin association of these mutants. However, replacing the three target sites with alanine, either simultaneously or one at a time, had no significant effect on 5HT uptake rates or the vimentin association with transporter.
  
  
  CONCLUSIONS
  Based on our findings, we propose that phosphate modification of the SITPET sequence differentially, one at a time exposes the vimentin binding domain on the C-terminus of SERT. Conversely, following 5HT stimulation, the association between vimentin-SERT is enhanced which changes the cellular distribution of SERT on an altered vimentin network.
authors.affiliation: Department of Biochemistry and Molecular Biology, College of Medicine, The University of Arkansas for Medical Sciences, Little Rock, Arkansas, United States of America.
authors.forename: Billow A
authors.initials: BA
authors.lastname: Ahmed
authors.forename: Irfan A
authors.initials: IA
authors.lastname: Bukhari
authors.forename: Brandon C
authors.initials: BC
authors.lastname: Jeffus
authors.forename: Justin T
authors.initials: JT
authors.lastname: Harney
authors.forename: Sheeno
authors.initials: S
authors.lastname: Thyparambil
authors.forename: Endrit
authors.initials: E
authors.lastname: Ziu
authors.forename: Mony
authors.initials: M
authors.lastname: Fraer
authors.forename: Nancy J
authors.initials: NJ
authors.lastname: Rusch
authors.forename: Piotr
authors.initials: P
authors.lastname: Zimniak
authors.forename: Vladimir
authors.initials: V
authors.lastname: Lupashin
authors.forename: Dale
authors.initials: D
authors.lastname: Tang
authors.forename: Fusun
authors.initials: F
authors.lastname: Kilic
mesh_terms: D019921:Biotinylation; D001792:Blood Platelets; D015153:Blotting, Western; D002462:Cell Membrane / Q000378:metabolism*; D002478:Cells, Cultured; D002846:Chromatography, Affinity; D005455:Fluorescent Antibody Technique; D006801:Humans; D009154:Mutation / Q000235:genetics; D010446:Peptide Fragments / Q000378:metabolism; D010766:Phosphorylation; D012701:Serotonin / Q000494:pharmacology*; D050486:Serotonin Plasma Membrane Transport Proteins / Q000235:genetics / Q000378:metabolism*; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D014746:Vimentin / Q000235:genetics / Q000378:metabolism*
chemical_list: D010446:Peptide Fragments; C493230:SLC6A4 protein, human; D050486:Serotonin Plasma Membrane Transport Proteins; D014746:Vimentin; D012701:Serotonin