Insertion of tetracysteine motifs into dopamine transporter extracellular domains
Student Authors
Deanna M. NavaroliUMass Chan Affiliations
Melikian LabGraduate School of Biomedical Sciences
Department of Psychiatry
Brudnick Neuropsychiatric Research Institute
Document Type
Journal ArticlePublication Date
2010-02-09Keywords
Dopamine Plasma Membrane Transport Proteins; Extracellular Space; Endocytosis; Amino Acid Motifs; Cysteine; ArsenicalsLife Sciences
Medicine and Health Sciences
Neuroscience and Neurobiology
Metadata
Show full item recordAbstract
The neuronal dopamine transporter (DAT) is a major determinant of extracellular dopamine (DA) levels and is the primary target for a variety of addictive and therapeutic psychoactive drugs. DAT is acutely regulated by protein kinase C (PKC) activation and amphetamine exposure, both of which modulate DAT surface expression by endocytic trafficking. In order to use live imaging approaches to study DAT endocytosis, methods are needed to exclusively label the DAT surface pool. The use of membrane impermeant, sulfonated biarsenic dyes holds potential as one such approach, and requires introduction of an extracellular tetracysteine motif (tetraCys; CCPGCC) to facilitate dye binding. In the current study, we took advantage of intrinsic proline-glycine (Pro-Gly) dipeptides encoded in predicted DAT extracellular domains to introduce tetraCys motifs into DAT extracellular loops 2, 3, and 4. [(3)H]DA uptake studies, surface biotinylation and fluorescence microscopy in PC12 cells indicate that tetraCys insertion into the DAT second extracellular loop results in a functional transporter that maintains PKC-mediated downregulation. Introduction of tetraCys into extracellular loops 3 and 4 yielded DATs with severely compromised function that failed to mature and traffic to the cell surface. This is the first demonstration of successful introduction of a tetracysteine motif into a DAT extracellular domain, and may hold promise for use of biarsenic dyes in live DAT imaging studies.Source
Navaroli DM, Melikian HE (2010) Insertion of Tetracysteine Motifs into Dopamine Transporter Extracellular Domains. PLoS ONE 5(2): e9113. Link to article on publisher's website
DOI
10.1371/journal.pone.0009113Permanent Link to this Item
http://hdl.handle.net/20.500.14038/33092PubMed ID
20161733Related Resources
Link to article in PubMedRights
Copyright: 2010 Navaroli, Melikian. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.ae974a485f413a2113503eed53cd6c53
10.1371/journal.pone.0009113