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The Journal of Neuroscience, August 4, 2004, 24(31):7024-7036; doi:10.1523/JNEUROSCI.1863-04.2004

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Cellular/Molecular
Surface Targeting of the Dopamine Transporter Involves Discrete Epitopes in the Distal C Terminus But Does Not Require Canonical PDZ Domain Interactions

Christian Bjerggaard,¹ Jacob U. Fog,¹ Hanne Hastrup,² Kenneth Madsen,¹ Claus J. Loland,¹ Jonathan A. Javitch,² and Ulrik Gether¹

¹Molecular Neuropharmacology Group, Department of Pharmacology, The Panum Institute, University of Copenhagen, DK-2200 Copenhagen, Denmark, and ²Center for Molecular Recognition and the Departments of Psychiatry and Pharmacology, Columbia University College of Physicians and Surgeons, New York, New York 10032

The human dopamine transporter (hDAT) contains a C-terminal type 2 PDZ (postsynaptic density 95/Discs large/zona occludens 1) domain-binding motif (LKV) known to interact with PDZ domain proteins such as PICK1 (protein interacting with C-kinase 1). As reported previously, we found that, after deletion of this motif, hDAT was retained in the endoplasmic reticulum (ER) of human embryonic kidney (HEK) 293 and Neuro2A cells, suggesting that PDZ domain interactions might be critical for hDAT targeting. Nonetheless, substitution of LKV with SLL, the type 1 PDZ-binding sequence from the ₂-adrenergic receptor, did not disrupt plasma membrane targeting. Moreover, the addition of an alanine to the hDAT C terminus (+Ala), resulting in an LKVA termination sequence, or substitution of LKV with alanines (3xAla_618-620) prevented neither plasma membrane targeting nor targeting into sprouting neurites of differentiated N2A cells. The inability of +Ala and 3xAla_618-620 to bind PDZ domains was confirmed by lack of colocalization with PICK1 in cotransfected HEK293 cells and by the inability of corresponding C-terminal fusion proteins to pull down purified PICK1. Thus, although residues in the hDAT C terminus are indispensable for proper targeting, PDZ domain interactions are not required. By progressive substitutions with ₂-adrenergic receptor sequence, and by triple-alanine substitutions in the hDAT C terminus, we examined the importance of epitopes preceding the LKV motif. Substitution of RHW_615-617 with alanines caused retention of the transporter in the ER despite preserved ability of this mutant to bind PICK1. We propose dual roles of the hDAT C terminus: a role independent of PDZ interactions for ER export and surface targeting, and a not fully clarified role involving PDZ interactions with proteins such as PICK1.

Key words: targeting; trafficking; ER export; neurotransmitter transporters; PDZ domains; oligomerization

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Received Nov 13, 2003; revised June 23, 2004; accepted June 23, 2004.

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