title: Serotonin (5-HT) transport in human platelets is modulated by Src-catalysed Tyr-phosphorylation of the plasma membrane transporter SERT.
abstract: OBJECTIVE
  platelets possess tightly regulated systems for serotonin (5-HT) transport. This study analysed whether the 5-HT transport mediated by the plasma-membrane transporter SERT is regulated by its Tyr-phosphorylation.
  
  
  METHODS
  5-HT transport was determined by filtration techniques, while immunoblotting procedures were adopted for detecting the Tyr-phosphorylation of SERT in human platelet fractions.
  
  
  RESULTS
  5-HT accumulation in platelets pre-treated with reserpine, which prevents the neurotransmitter transport into the dense granules, decreased upon cellular exposure to PP2 and SU6656, two structurally unrelated inhibitors of Src-kinases. By contrast, the protein Tyr-phosphatase inhibitor pervanadate increased the 5-HT accumulation. Anti-SERT immunostaining of the platelet fractions showed a major band displaying an apparent molecular mass of 50 kappaDa, indicating that, during the analytical procedure, SERT underwent proteolysis, which was counteracted by addition of 4 M urea in the cellular disrupting medium. The Tyr-phosphorylation degree of SERT immunoprecipitated from membrane extracts decreased by platelet treatment with SU6656 or PP2, and enhanced upon pervanadate treatment. The anti-SERT immunoprecipitates displayed anti-Src immunostaining and in vitro kinase activity towards a Src-specific peptide-substrate. Platelet treatment with PP2 or SU6656 also caused a decrease in the imipramine binding to platelets. It was concluded that the Src-mediated SERT Tyr-phosphorylation regulates the 5-HT transport by affecting the neurotransmitter binding sites.
authors.affiliation: Department of Biological Chemistry, University of Padova, Padova, Italy.
authors.forename: Alessandro
authors.initials: A
authors.lastname: Zarpellon
authors.forename: Arianna
authors.initials: A
authors.lastname: Donella-Deana
authors.forename: Alessandra
authors.initials: A
authors.lastname: Folda
authors.forename: Loris
authors.initials: L
authors.lastname: Turetta
authors.forename: Martina
authors.initials: M
authors.lastname: Pavanetto
authors.forename: Renzo
authors.initials: R
authors.lastname: Deana
mesh_terms: D001692:Biological Transport / Q000187:drug effects; D001792:Blood Platelets / Q000187:drug effects / Q000201:enzymology*; D002384:Catalysis / Q000187:drug effects; D002462:Cell Membrane / Q000187:drug effects / Q000201:enzymology; D006801:Humans; D008985:Monensin / Q000494:pharmacology; D019000:Phosphotyrosine / Q000378:metabolism*; D047428:Protein Kinase Inhibitors / Q000494:pharmacology; D016392:Proto-Oncogene Proteins pp60(c-src) / Q000037:antagonists & inhibitors / Q000378:metabolism*; D012110:Reserpine / Q000494:pharmacology; D012701:Serotonin / Q000378:metabolism*; D050486:Serotonin Plasma Membrane Transport Proteins / Q000378:metabolism*; D013347:Subcellular Fractions / Q000187:drug effects
chemical_list: D047428:Protein Kinase Inhibitors; D050486:Serotonin Plasma Membrane Transport Proteins; D019000:Phosphotyrosine; D012701:Serotonin; D012110:Reserpine; D008985:Monensin; D016392:Proto-Oncogene Proteins pp60(c-src)