Oligomerization of Dopamine Transporters Visualized in Living Cells by Fluorescence Resonance Energy Transfer Microscopy

Abstract

Plasma membrane transporters belonging to the family of Na ϩ /Cl Ϫ -dependent neurotransmitter transporters play an important role in terminating the activity of the monoamine neurotransmitters and of ␥-aminobutyric acid (see Ref. 1). Thus, reuptake of dopamine (DA) 1 from the synaptic cleft by the dopamine transporter (DAT) serves as the major mechanism for terminating dopaminergic neurotransmission in the brain (2). Because the efficiency of DA removal depends on the number of DAT molecules expressed at the plasma membrane, trafficking processes that control transporter distribution in the cell represent a potentially important mechanism by which neurotransmission could be regulated. Newly synthesized DAT acquires glycosylation in the endoplasmic reticulum (ER) and Golgi complex and is then trafficked to the plasma membrane. Typically, a large pool of mature DAT molecules are found at the cell surface of dopaminergic neurons and when DAT is heterologously expressed in tissue culture cells. However, DAT localization can be altered rapidly. Acute exposure of cells to either phorbol esters or substrates reduces the number of plasma membrane DATs and thus DAT function, and this reduction is due to acceleration of DAT endocytosis through a dynamin-dependent mechanism (3-6). Recently, it has been shown that DAT can interact with the anchoring protein PICK 1 and the adaptor protein 8). However, in general, the molecular mechanisms controlling DAT trafficking are not yet well understood.The DAT molecule is predicted to have 12 membrane-spanning sequences with both amino and carboxyl termini oriented intracellularly. The specific function of DAT transmembrane (TM) motifs and termini are not well established. TM domains may play a role in intra-and intermolecular interactions. Many membrane receptors and other integral membrane proteins require dimerization or higher oligomerization for their activity. Several lines of evidence have suggested that monoamine transporters are also dimers or oligomers. Results with dominant negative forms of the serotonin transporter (SERT) and norepinephrine transporter were consistent with this idea (9, 10). Oligomerization of SERT was demonstrated directly by co-immunoprecipitation (11). The results of fluorescence resonance energy transfer (FRET) studies further confirmed that SERT and a ␥-aminobutyric acid transporter (GAT-1) are homo-oligomers (12, 13). Early radiation inactivation studies also suggested that DAT exists as a dimer or oligomer (14,15). Recently, the potential for DAT to exist as dimer or higherorder oligomer in the plasma membrane has been demonstrated using chemical cross-linking (16). More recently, Torres and co-workers (17) reported detection of DAT oligomerization by co-immunoprecipitation. In general, the mechanisms and functional roles of oligomerization of monoamine transporters remain to be defined. However, it has been proposed that SERT oligomerization may be important for its transport activity (9). Oligomerization may be also required for proper traffic...

Dopamine transporter endocytic determinants: Carboxy terminal residues critical for basal and PKC-stimulated internalization

“…All members of the family share a similar predicted topology of twelve transmembrane domains, cytoplasmic amino-and carboxy-terminals, a large second extracellular loop with multiple N-linked glycosylation sites, and cytoplasmic phosphorylation sites. Recent crystallographic studies focusing on LeuT Aa , a bacterial SLC6 transporter homolog, support the predicted DAT topology and reveal a dimeric transporter assembly (Yamashita et al, 2005), consistent with FRET and co-immunoprecipitation studies that suggest SLC6 transporter homooligomerization (Schmid et al, 2001;Scholze et al, 2002;Sorkina et al, 2003;Torres et al, 2003b).…”

Dopamine Transporter Activity Is Modulated by α-Synuclein

“…The plasmid cDNAs for YFP-DAT and YFP-HA-DAT were obtained from Dr. Alexander Sorkin, University of Pittsburgh (26). The plasmid cDNA for ␣-synuclein was obtained from Dr. Ted Dawson, Johns Hopkins University (27).…”

Enhanced Ubiquitylation and Accelerated Degradation of the Dopamine Transporter Mediated by Protein Kinase C

“…All constructs and point mutations were verified by automatic dideoxynucleotide sequencing. The plasmid CFP-DAT was described previously (6). YFP-Hrs (hepatocyte growth factor receptor substrate) was provided by Dr. H. Stenmark (Radium Institute, Oslo, Norway).…”