Oligomerization and trafficking of the human dopamine transporter. Mutational analysis identifies critical domains important for the functional expression of the transporter

J Biol Chem. 2003 Jan 24;278(4):2731-9. doi: 10.1074/jbc.M201926200. Epub 2002 Nov 11.

Abstract

The dopamine transporter (DAT) is a presynaptic plasma membrane protein responsible for the termination of dopaminergic neurotransmission in the central nervous system. While most studies have focused on structure/function analysis, much less information is available regarding the assembly and the trafficking of this protein. To address this problem, we performed a mutational analysis of the DAT protein, combined with biochemical, immunological, and functional approaches. In mammalian cells co-expressing differentially tagged DAT molecules, HA-tagged DAT co-purified with 6His-tagged DAT demonstrating a physical interaction between transporter proteins. Evidence for the functional oligomerization of DAT was obtained using dominant-negative mutants of DAT. Two loss-of-function mutant transporters (Y335A and D79G) that were targeted to the cell surface inhibited wild-type DAT uptake activity without affecting the membrane targeting of the wild-type transporter. Moreover, non-functional amino and carboxyl termini-truncated mutants of DAT inhibited wild-type DAT function by interfering with the normal processing of the wild-type transporter to the cell membrane. Mutations in the leucine repeat of the second transmembrane domain of the transporter could eliminate the dominant-negative effect of all these mutants. In addition, a small fragment comprising the first two transmembrane domains of DAT inhibited wild-type transporter function but not when the leucine repeat motif was mutated. Taken together, our results suggest that the assembly of DAT monomers plays a critical role in the expression and function of the transporter.

Publication types

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

MeSH terms

  • Blotting, Western
  • Cell Line
  • DNA Mutational Analysis*
  • DNA, Complementary / metabolism
  • Dopamine Plasma Membrane Transport Proteins
  • Dose-Response Relationship, Drug
  • Genes, Dominant
  • Glycosylation
  • Humans
  • Immunohistochemistry
  • Leucine / chemistry
  • Membrane Glycoproteins*
  • Membrane Transport Proteins / chemistry*
  • Membrane Transport Proteins / genetics
  • Membrane Transport Proteins / metabolism*
  • Microscopy, Confocal
  • Mutagenesis, Site-Directed
  • Mutation
  • Nerve Tissue Proteins*
  • Protein Binding
  • Protein Structure, Tertiary
  • Protein Transport
  • Transfection

Substances

  • DNA, Complementary
  • Dopamine Plasma Membrane Transport Proteins
  • Membrane Glycoproteins
  • Membrane Transport Proteins
  • Nerve Tissue Proteins
  • SLC6A3 protein, human
  • Leucine