The dopamine transporter (DAT) is the primary mechanism which clears extracellular dopamine from the synaptic space. As such, it performs a key role in terminating synaptic transmission and in regulating the concentration of dopamine available for binding to pre- and post-synaptic dopamine receptors. It has recently been discovered that activation of protein kinase C leads to phosphorylation of DATs and to concomitant reductions in dopamine transport, suggesting that DATs undergo functional regulation by phosphorylation. This would provide the neuron with a mechanism for fine temporal and spatial control of extracellular dopamine concentrations, and subsequent downstream dopaminergic neural activity. This property of DAT therefore has the potential to profoundly influence normal dopaminergic neurophysiology, and may also be related to mechanisms of abuse of cocaine or other drugs and dopaminergic neurodegeneration. This study proposes to thoroughly characterize DAT phosphorylation properties and define the relationship between DAT phosphorylation and functional regulation.
The specific aims designed to achieve these goals are: 1. Identify sites of PKC-stimulated phosphorylation on DATs. 2. Construct mutants with phosphorylation sites changed to non-phosphate acceptors, and examine the functional consequences of these mutations. 3. Thoroughly characterize phosphorylation and dephosphorylation properties of DAT by identifying the specific kinases and phosphatases which act on the protein. 4. Test for related changes in transport of dopamine, binding of antagonists, and surface expression. 5. Identify the endogenous pathways responsible for in vivo control of DAT phosphorylation.

Agency
National Institute of Health (NIH)
Institute
National Institute on Drug Abuse (NIDA)
Type
Research Project (R01)
Project #
1R01DA013147-01
Application #
6081243
Study Section
Molecular, Cellular and Developmental Neurosciences 2 (MDCN)
Program Officer
Pilotte, Nancy S
Project Start
1999-07-01
Project End
2002-04-30
Budget Start
1999-07-01
Budget End
2000-04-30
Support Year
1
Fiscal Year
1999
Total Cost
Indirect Cost
Name
University of North Dakota
Department
Biochemistry
Type
Schools of Medicine
DUNS #
102280781
City
Grand Forks
State
ND
Country
United States
Zip Code
58202
Hovde, Moriah J; Larson, Garret H; Vaughan, Roxanne A et al. (2018) Model systems for analysis of dopamine transporter function and regulation. Neurochem Int :
Foster, James D; Vaughan, Roxanne A (2017) Phosphorylation mechanisms in dopamine transporter regulation. J Chem Neuroanat 83-84:10-18
Challasivakanaka, Sathya; Zhen, Juan; Smith, Margaret E et al. (2017) Dopamine transporter phosphorylation site threonine 53 is stimulated by amphetamines and regulates dopamine transport, efflux, and cocaine analog binding. J Biol Chem 292:19066-19075
Rastedt, Danielle E; Vaughan, Roxanne A; Foster, James D (2017) Palmitoylation mechanisms in dopamine transporter regulation. J Chem Neuroanat 83-84:3-9
Moritz, Amy E; Rastedt, Danielle E; Stanislowski, Daniel J et al. (2015) Reciprocal Phosphorylation and Palmitoylation Control Dopamine Transporter Kinetics. J Biol Chem 290:29095-105
Gaffaney, Jon D; Shetty, Madhur; Felts, Bruce et al. (2014) Antagonist-induced conformational changes in dopamine transporter extracellular loop two involve residues in a potential salt bridge. Neurochem Int 73:16-26
Moritz, Amy E; Foster, James D; Gorentla, Balachandra K et al. (2013) Phosphorylation of dopamine transporter serine 7 modulates cocaine analog binding. J Biol Chem 288:20-32
Vaughan, Roxanne A; Foster, James D (2013) Mechanisms of dopamine transporter regulation in normal and disease states. Trends Pharmacol Sci 34:489-96
Foster, James D; Yang, Jae-Won; Moritz, Amy E et al. (2012) Dopamine transporter phosphorylation site threonine 53 regulates substrate reuptake and amphetamine-stimulated efflux. J Biol Chem 287:29702-12
Foster, James D; Vaughan, Roxanne A (2011) Palmitoylation controls dopamine transporter kinetics, degradation, and protein kinase C-dependent regulation. J Biol Chem 286:5175-86

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