title: Substrate-induced regulation of gamma-aminobutyric acid transporter trafficking requires tyrosine phosphorylation.
abstract: Neurotransmitter transporters regulate synaptic transmitter levels and are themselves functionally regulated by a number of different signal transduction cascades. A common theme in transporter regulation is redistribution of transporter protein between intracellular stores and the plasma membrane. The triggers and mechanisms underlying this regulation are important in the control of extracellular transmitter concentrations and hence synaptic signaling. Previously, we demonstrated that the gamma-aminobutyric acid transporter GAT1 is regulated by direct tyrosine phosphorylation, resulting in an up-regulation of transporter expression on the plasma membrane. In the present report, we show that two tyrosine residues on GAT1 contribute to the phosphorylation and transporter redistribution. Tyrosine phosphorylation is concomitant with a decrease in the rate of transporter internalization from the plasma membrane. A decrease in GAT internalization rates also occurs in the presence of GAT1 substrates, suggesting the hypothesis that tyrosine phosphorylation is required for the substrate-induced up-regulation of GAT1 surface expression. In support of this hypothesis, incubation of GAT1-expressing cells with transporter ligands alters the amount of GAT1 tyrosine phosphorylation, and substrate-induced surface expression is unchanged in a GAT1 mutant lacking tyrosine phosphorylation sites. These data suggest a model in which substrates permit the phosphorylation of GAT1 on tyrosine residues and that the phosphorylated state of the transporter is refractory for internalization.
authors.affiliation: Department of Neurobiology, University of Alabama at Birmingham, Birmingham, Alabama 35294-0021, USA.
authors.forename: T L
authors.initials: TL
authors.lastname: Whitworth
authors.forename: M W
authors.initials: MW
authors.lastname: Quick
mesh_terms: D000818:Animals; D001665:Binding Sites; D019921:Biotinylation; D016466:CHO Cells; D002352:Carrier Proteins / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism*; D002462:Cell Membrane / Q000378:metabolism; D006224:Cricetinae; D050485:GABA Plasma Membrane Transport Proteins; D019833:Genistein / Q000494:pharmacology; D015151:Immunoblotting; D008024:Ligands; D008565:Membrane Proteins / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism*; D026901:Membrane Transport Proteins; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D009557:Nipecotic Acids / Q000494:pharmacology; D027361:Organic Anion Transporters; D010766:Phosphorylation; D011233:Precipitin Tests; D011485:Protein Binding; D011505:Protein-Tyrosine Kinases / Q000378:metabolism; D015398:Signal Transduction; D014162:Transfection; D014443:Tyrosine / Q000737:chemistry* / Q000378:metabolism*; D015854:Up-Regulation
chemical_list: D002352:Carrier Proteins; D050485:GABA Plasma Membrane Transport Proteins; D008024:Ligands; D008565:Membrane Proteins; D026901:Membrane Transport Proteins; D009557:Nipecotic Acids; D027361:Organic Anion Transporters; C030278:nipecotic acid; D014443:Tyrosine; D019833:Genistein; D011505:Protein-Tyrosine Kinases