title: Involvement of threonine 258 and serine 259 motif in amphetamine-induced norepinephrine transporter endocytosis.
abstract: D-amphetamine (AMPH) down-regulates the norepinephrine transporter (NET), although the exact trafficking pathways altered and motifs involved are not known. Therefore, we examined the cellular and molecular mechanisms involved in AMPH-induced NET regulation in human placental trophoblast cells expressing the wild-type (WT)-hNET and the hNET double mutant (DM)-bearing protein kinase C (PKC)-resistant T258A + S259A motif. NET function and surface expression were significantly reduced in cells expressing WT-hNET but not in cells expressing hNET-DM following AMPH treatment. AMPH inhibited plasma membrane recycling of both WT-hNET and hNET-DM. In contrast, AMPH stimulated endocytosis of WT-hNET, and did not affect hNET-DM endocytosis. Although PKC or calcium/calmodulin- dependent kinase-II (CaMKII) inhibition or depletion of calcium failed to block AMPH-mediated down-regulation of WT-hNET, NET-specific blocker desipramine completely prevented AMPH-induced down-regulation. Furthermore, AMPH treatment had no effect on phospho-CaMKII immunoreactivity. The inhibitory potency of AMPH was highest on hNET-DM, intermediary on T258A and S259A single mutants and lowest on WT-hNET. Single mutants exhibited partial resistance to AMPH-mediated down-regulation. AMPH accumulation was similar in cells expressing WT-hNET or hNET-DM. The results demonstrate that reduced plasma membrane insertion and enhanced endocytosis account for AMPH-mediated NET down-regulation, and provide the first evidence that T258/S259 motif is involved only in AMPH-induced NET endocytosis that is desipramine-sensitive, but PKC and CaMKII independent.
authors.affiliation: Department of Neurosciences, Division of Neuroscience Research, Medical University of South Carolina, Charleston, South Carolina 29425, USA.
authors.forename: Balasubramaniam
authors.initials: B
authors.lastname: Annamalai
authors.forename: Padmanabhan
authors.initials: P
authors.lastname: Mannangatti
authors.forename: Obulakshmi
authors.initials: O
authors.lastname: Arapulisamy
authors.forename: Sammanda
authors.initials: S
authors.lastname: Ramamoorthy
authors.forename: Lankupalle D
authors.initials: LD
authors.lastname: Jayanthi
mesh_terms: D018759:Adrenergic Uptake Inhibitors / Q000494:pharmacology; D000661:Amphetamine / Q000494:pharmacology*; D019921:Biotinylation; D002118:Calcium / Q000502:physiology; D054732:Calcium-Calmodulin-Dependent Protein Kinase Type 2 / Q000037:antagonists & inhibitors; D002462:Cell Membrane / Q000187:drug effects; D000697:Central Nervous System Stimulants / Q000494:pharmacology*; D003891:Desipramine / Q000494:pharmacology; D015536:Down-Regulation / Q000187:drug effects; D004705:Endocytosis / Q000187:drug effects* / Q000235:genetics*; D005260:Female; D006801:Humans; D009154:Mutation / Q000502:physiology; D050484:Norepinephrine Plasma Membrane Transport Proteins / Q000037:antagonists & inhibitors / Q000235:genetics* / Q000502:physiology*; D010920:Placenta / Q000166:cytology; D011247:Pregnancy; D011493:Protein Kinase C / Q000037:antagonists & inhibitors / Q000378:metabolism; D047428:Protein Kinase Inhibitors / Q000494:pharmacology; D012694:Serine / Q000235:genetics*; D015398:Signal Transduction / Q000187:drug effects; D013912:Threonine / Q000235:genetics*; D014327:Trophoblasts / Q000187:drug effects
chemical_list: D018759:Adrenergic Uptake Inhibitors; D000697:Central Nervous System Stimulants; D050484:Norepinephrine Plasma Membrane Transport Proteins; D047428:Protein Kinase Inhibitors; D013912:Threonine; D012694:Serine; D000661:Amphetamine; D011493:Protein Kinase C; D054732:Calcium-Calmodulin-Dependent Protein Kinase Type 2; D002118:Calcium; D003891:Desipramine