title: Proteomic analysis of human norepinephrine transporter complexes reveals associations with protein phosphatase 2A anchoring subunit and 14-3-3 proteins.
abstract: The norepinephrine transporter (NET) terminates noradrenergic signals by clearing released NE at synapses. NET regulation by receptors and intracellular signaling pathways is supported by a growing list of associated proteins including syntaxin1A, protein phosphatase 2A (PP2A) catalytic subunit (PP2A-C), PICK1, and Hic-5. In the present study, we sought evidence for additional partnerships by mass spectrometry-based analysis of proteins co-immunoprecipitated with human NET (hNET) stably expressed in a mouse noradrenergic neuroblastoma cell line. Our initial proteomic analyses reveal multiple peptides derived from hNET, peptides arising from the mouse PP2A anchoring subunit (PP2A-Ar) and peptides derived from 14-3-3 proteins. We verified physical association of NET with PP2A-Ar via co-immunoprecipitation studies using mouse vas deferens extracts and with 14-3-3 via a fusion pull-down approach, implicating specifically the hNET NH2-terminus for interactions. The transporter complexes described likely support mechanisms regulating transporter activity, localization, and trafficking.
authors.affiliation: Department of Pharmacology, Vanderbilt University School of Medicine, Nashville, TN 37232-8548, USA.
authors.forename: Uhna
authors.initials: U
authors.lastname: Sung
authors.forename: Jennifer L
authors.initials: JL
authors.lastname: Jennings
authors.forename: Andrew J
authors.initials: AJ
authors.lastname: Link
authors.forename: Randy D
authors.initials: RD
authors.lastname: Blakely
mesh_terms: D048948:14-3-3 Proteins / Q000378:metabolism*; D000595:Amino Acid Sequence; D000818:Animals; D020134:Catalytic Domain; D045744:Cell Line, Tumor; D006801:Humans; D047468:Immunoprecipitation; D013058:Mass Spectrometry; D051379:Mice; D008969:Molecular Sequence Data; D050484:Norepinephrine Plasma Membrane Transport Proteins; D010749:Phosphoprotein Phosphatases / Q000378:metabolism*; D011485:Protein Binding; D054648:Protein Phosphatase 2; D020543:Proteome; D027981:Symporters / Q000737:chemistry / Q000378:metabolism*
chemical_list: D048948:14-3-3 Proteins; D050484:Norepinephrine Plasma Membrane Transport Proteins; D020543:Proteome; C493227:SLC6A2 protein, human; C493228:Slc6a2 protein, mouse; D027981:Symporters; D010749:Phosphoprotein Phosphatases; D054648:Protein Phosphatase 2