Proteomic analysis of human norepinephrine transporter complexes reveals associations with protein phosphatase 2A anchoring subunit and 14-3-3 proteins.

Sung U, Jennings JL, Link AJ, Blakely RD
Biochem Biophys Res Commun. 2005 333 (3): 671-8

PMID: 15963952 · DOI:10.1016/j.bbrc.2005.05.165

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.

MeSH Terms (16)

14-3-3 Proteins Amino Acid Sequence Animals Catalytic Domain Cell Line, Tumor Humans Immunoprecipitation Mass Spectrometry Mice Molecular Sequence Data Norepinephrine Plasma Membrane Transport Proteins Phosphoprotein Phosphatases Protein Binding Protein Phosphatase 2 Proteome Symporters

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