Oligomerization of dopamine transporters visualized in living cells by fluorescence resonance energy transfer microscopy.
To examine the oligomeric state and trafficking of the dopamine transporter (DAT) in different compartments of living cells, human DAT was fused to yellow (YFP) or cyan fluorescent protein (CFP). YFP-DAT and CFP-DAT were transiently and stably expressed in porcine aortic endothelial (PAE) cells, human embryonic kidney (HEK) 293 cells, and an immortalized dopaminergic cell line 1RB3AN27. Fluorescence microscopic imaging of cells co-expressing YFP-DAT and CFP-DAT revealed fluorescence resonance energy transfer (FRET) between CFP and YFP, which is consistent with an intermolecular interaction of DAT fusion proteins. FRET signals were detected between CFP- and YFP-DAT located at the plasma membrane and in intracellular membrane compartments. Phorbol esters or amphetamine induced the endocytosis of YFP/CFP-DAT to early and recycling endosomes, identified by Rab5, Rab11, Hrs and EEA.1 proteins. Interestingly, however, DAT was mainly excluded from Rab5- and Hrs-containing microdomains within the endosomes. The strongest FRET signals were measured in endosomes, indicative of efficient oligomerization of internalized DAT. The intermolecular DAT interactions were confirmed by co-immunoprecipitation. A DAT mutant that was retained in the endoplasmic reticulum (ER) after biosynthesis was used to show that DAT is oligomeric in the ER. Moreover, co-expression of an ER-retained DAT mutant and wild-type DAT resulted in the retention of wild-type DAT in the ER. These data suggest that DAT oligomers are formed in the ER and then are constitutively maintained both at the cell surface and during trafficking between the plasma membrane and endosomes.
Department of Pharmacology and Neuroscience Program, University of Colorado Health Sciences Center, Denver, Colorado 80262, USA.
Tatiana
T
Sorkina
Suzanne
S
Doolen
Emilia
E
Galperin
Nancy R
NR
Zahniser
Alexander
A
Sorkin
D000662:Amphetamines / Q000494:pharmacology; D000818:Animals; D001426:Bacterial Proteins / Q000378:metabolism; D019921:Biotinylation; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism; D004298:Dopamine / Q000378:metabolism*; D050483:Dopamine Plasma Membrane Transport Proteins; D004705:Endocytosis; D056827:Endosomal Sorting Complexes Required for Transport; D011992:Endosomes / Q000378:metabolism; D031541:Fluorescence Resonance Energy Transfer / Q000379:methods*; D049452:Green Fluorescent Proteins; D006801:Humans; D008164:Luminescent Proteins / Q000378:metabolism; D008562:Membrane Glycoproteins; D008565:Membrane Proteins / Q000378:metabolism; D026901:Membrane Transport Proteins / Q000737:chemistry* / Q000502:physiology*; D008856:Microscopy, Fluorescence; D009154:Mutation; D009419:Nerve Tissue Proteins; D010750:Phosphoproteins / Q000378:metabolism; D010957:Plasmids / Q000378:metabolism; D011233:Precipitin Tests; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D013552:Swine; D033921:Vesicular Transport Proteins; D020691:rab GTP-Binding Proteins / Q000378:metabolism; D020696:rab5 GTP-Binding Proteins / Q000378:metabolism; D000099193:rab11 GTP-Binding Proteins
D000662:Amphetamines; D001426:Bacterial Proteins; D004298:Dopamine; D050483:Dopamine Plasma Membrane Transport Proteins; D056827:Endosomal Sorting Complexes Required for Transport; D049452:Green Fluorescent Proteins; D008164:Luminescent Proteins; D008562:Membrane Glycoproteins; D008565:Membrane Proteins; D026901:Membrane Transport Proteins; D009419:Nerve Tissue Proteins; D010750:Phosphoproteins; D011993:Recombinant Fusion Proteins; D033921:Vesicular Transport Proteins; D020691:rab GTP-Binding Proteins; D020696:rab5 GTP-Binding Proteins; D000099193:rab11 GTP-Binding Proteins; C493214:SLC6A3 protein, human; C093846:early endosome antigen 1; C096164:hepatocyte growth factor-regulated tyrosine kinase substrate; C054562:yellow fluorescent protein, Bacteria