title: Cocaine and antidepressant-sensitive biogenic amine transporters exist in regulated complexes with protein phosphatase 2A.
abstract: Presynaptic transporter proteins regulate the clearance of extracellular biogenic amines after release and are important targets for multiple psychoactive agents, including amphetamines, cocaine, and antidepressant drugs. Recent studies reveal that dopamine (DA), norepinephrine (NE), and serotonin (5-HT) transporters (DAT, NET, and SERT, respectively) are rapidly regulated by direct or receptor-mediated activation of cellular kinases, particularly protein kinase C (PKC). With SERTs, PKC activation results in activity-dependent transporter phosphorylation and sequestration. Protein phosphatase 1/2A (PP1/PP2A) inhibitors, such as okadaic acid (OA) and calyculin A, also promote SERT phosphorylation and functional downregulation. How kinase, phosphatase, and transporter activities are linked mechanistically is unclear. In the present study, we found that okadaic acid-sensitive phosphatase activity is enriched in SERT immunoprecipitates from human SERT stably transfected cells. Moreover, blots of these immunoprecipitates reveal the presence of PP2A catalytic subunit (PP2Ac), findings replicated using brain preparations. Whole-cell treatments with okadaic acid or calyculin A diminished SERT/PP2Ac associations. Phorbol esters, which trigger SERT phosphorylation, also diminish SERT/PP2Ac associations, effects that can be blocked by PKC antagonists as well as the SERT substrate 5-HT. Similar transporter/PP2Ac complexes were also observed in coimmunoprecipitation studies with NETs and DATs. Our findings provide evidence for the existence of regulated heteromeric assemblies involving biogenic amine transporters and PP2A and suggest that the dynamic stability of these complexes may govern transporter phosphorylation and sequestration.
authors.affiliation: Department of Pharmacology and Center for Molecular Neuroscience, Vanderbilt University School of Medicine, Nashville, Tennessee 37232-6420, USA.
authors.forename: A L
authors.initials: AL
authors.lastname: Bauman
authors.forename: S
authors.initials: S
authors.lastname: Apparsundaram
authors.forename: S
authors.initials: S
authors.lastname: Ramamoorthy
authors.forename: B E
authors.initials: BE
authors.lastname: Wadzinski
authors.forename: R A
authors.initials: RA
authors.lastname: Vaughan
authors.forename: R D
authors.initials: RD
authors.lastname: Blakely
mesh_terms: D000818:Animals; D000928:Antidepressive Agents / Q000494:pharmacology*; D001679:Biogenic Amines / Q000378:metabolism*; D001692:Biological Transport / Q000187:drug effects; D002352:Carrier Proteins / Q000378:metabolism; D002460:Cell Line; D003042:Cocaine / Q000494:pharmacology*; D050483:Dopamine Plasma Membrane Transport Proteins; D006801:Humans; D046911:Macromolecular Substances; D008387:Marine Toxins; D008562:Membrane Glycoproteins / Q000378:metabolism; D026901:Membrane Transport Proteins; D009419:Nerve Tissue Proteins; D050484:Norepinephrine Plasma Membrane Transport Proteins; D019319:Okadaic Acid / Q000494:pharmacology; D010080:Oxazoles / Q000494:pharmacology; D010703:Phorbol Esters / Q000494:pharmacology; D010749:Phosphoprotein Phosphatases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D010766:Phosphorylation / Q000187:drug effects; D011233:Precipitin Tests; D011493:Protein Kinase C / Q000037:antagonists & inhibitors; D054645:Protein Phosphatase 1; D054648:Protein Phosphatase 2; D021381:Protein Transport / Q000187:drug effects; D012701:Serotonin / Q000378:metabolism / Q000494:pharmacology; D050486:Serotonin Plasma Membrane Transport Proteins; D027981:Symporters; D014162:Transfection
chemical_list: D000928:Antidepressive Agents; D001679:Biogenic Amines; D002352:Carrier Proteins; D050483:Dopamine Plasma Membrane Transport Proteins; D046911:Macromolecular Substances; D008387:Marine Toxins; D008562:Membrane Glycoproteins; D026901:Membrane Transport Proteins; D009419:Nerve Tissue Proteins; D050484:Norepinephrine Plasma Membrane Transport Proteins; D010080:Oxazoles; D010703:Phorbol Esters; C493227:SLC6A2 protein, human; C493214:SLC6A3 protein, human; C493230:SLC6A4 protein, human; D050486:Serotonin Plasma Membrane Transport Proteins; D027981:Symporters; D019319:Okadaic Acid; D012701:Serotonin; C059041:calyculin A; D011493:Protein Kinase C; D010749:Phosphoprotein Phosphatases; D054645:Protein Phosphatase 1; D054648:Protein Phosphatase 2; D003042:Cocaine