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J. Biol. Chem., Vol. 278, Issue 30, 28274-28283, July 25, 2003

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

Tatiana Sorkina, Suzanne Doolen, Emilia Galperin, Nancy R. Zahniser and Alexander Sorkin 

From the Department of Pharmacology and Neuroscience Program, University of Colorado Health Sciences Center, Denver, Colorado 80262

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.

Received for publication, October 18, 2002 , and in revised form, May 5, 2003.

^* This work was supported by Grants DA14204 (to A. S. and N. R. Z.) and DA1505 from National Institute on Drug Abuse (to N. R. Z.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

To whom correspondence should be addressed: Dept. of Pharmacology, University of Colorado Health Science Center, 4200 E. Ninth Ave., Denver, CO 80262. Tel.: 303-315-7252; Fax: 303-315-7097; E-mail: alexander.sorkin{at}uchsc.edu./.

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