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Multiple molecular determinants in the carboxyl terminus regulate dopamine transporter export from endoplasmic
Manuel Miranda1, Tatiana Sorkina, Tom N Grammatopoulos
1Department of Pharmacology, University of Colorado Health Sciences Center, Denver, Colorado 80262, USA.
The Journal of Biological Chemistry
|May 7, 2004
Summary
The dopamine transporter's (DAT) carboxyl terminus is crucial for its cell surface expression. Specific mutations in this region prevent DAT from reaching the cell surface, impacting dopamine signaling.
Area of Science:
- Neuroscience
- Molecular Biology
- Cell Biology
Background:
- The plasma membrane dopamine transporter (DAT) regulates dopaminergic neurotransmission by dopamine reuptake.
- Cell surface DAT levels are critical for transporter function.
- The carboxyl terminus of DAT plays a role in its trafficking and localization.
Purpose of the Study:
- To investigate the role of the DAT carboxyl terminus in transporter trafficking to the plasma membrane.
- To identify specific residues within the carboxyl terminus essential for ER export and surface expression.
Main Methods:
- Live cell fluorescence microscopy
- Cell surface biotinylation assays
- Systematic deletions and alanine substitutions in the DAT carboxyl terminus
- Studies in porcine aortic endothelial cells, 1RB3AN27 neuronal cells, and primary rat midbrain neurons
Main Results:
- Alanine substitutions at Lys-590 and Asp-600 delayed DAT delivery to the plasma membrane due to ER retention.
- Mutation of Gly-585 to alanine completely blocked DAT ER export and surface expression.
- Mutant DAT proteins (G585A, K590A, D600A) were confined to the cell soma in primary neurons, failing to traffic to dendrites or axons.
Conclusions:
- The carboxyl terminus sequence proximal to the last transmembrane domain is essential for DAT ER export.
- Local conformation and/or intramolecular interactions within the DAT carboxyl terminus regulate transporter trafficking.
- These findings provide insights into the molecular mechanisms governing DAT surface expression and dopaminergic signaling.