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

Biochem Biophys Res Commun. 2005 Aug 5;333(3):671-8. doi: 10.1016/j.bbrc.2005.05.165.

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.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

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

Substances

  • 14-3-3 Proteins
  • Norepinephrine Plasma Membrane Transport Proteins
  • Proteome
  • SLC6A2 protein, human
  • Slc6a2 protein, mouse
  • Symporters
  • Phosphoprotein Phosphatases
  • Protein Phosphatase 2