The dopamine transporter proteome
Torres GE.

Dopamine (DA) uptake through the neuronal plasma membrane DA transporter (DAT) is essential for the maintenance of normal DA homeostasis in the brain. The DAT-mediated re-uptake system limits not only the intensity but also the duration of DA actions at presynaptic and postsynaptic receptors. This protein is the primary target for cocaine and amphetamine, both highly addictive and major substances of abuse worldwide. DAT is also the molecular target for therapeutic agents used in the treatment of mental disorders, such as attention deficit hyperactivity disorder and depression. Given the role played by the DAT in regulation of DA neurotransmission and its contribution to the abuse potential of psychostimulants, it becomes not only important but also necessary to understand the functional regulation of this protein. To investigate the cellular and molecular mechanisms associated with DAT function and regulation, our laboratory and others have embarked on a systematic search for DAT protein-protein interactions. Recently, a growing number of proteins have been shown to interact with DAT. These novel interactions might be important in the assembly, targeting, trafficking and/or regulation of transporter function. In this review, I summarize the main findings obtained from the characterization of DAT-interacting proteins and discuss the functional implications of these novel interactions. Based on these new data, I propose to use the term DAT proteome to explain how interacting proteins regulate DAT function. These novel interactions might help define new mechanisms associated with the function of the transporter.

The identification of DAT-interacting proteins is rapidly facilitating the characterization of cellular processes that regulate the synthesis, assembly, targeting, trafficking and functional properties of the transporter. The proteins identified so far probably represent the 'tip of the iceberg' in terms of the entire complement of proteins that interact with this transporter. A major goal in future years will be to identify all proteins that directly or indirectly form part of the DAT proteome. New methods, such as mass spectrometry coupled with better purification techniques, should be employed in this effort. Once these proteins are identified, we will need to understand the temporal and spatial distribution of these interactions. Some proteins will be required for transporter synthesis and assembly, some for targeting to distinct subcellular microdomains, and some for trafficking and recycling. It is also conceivable that unexpected functions associated with the transporter will be discovered through the identification of novel protein-protein interactions. Disruption of specific protein-protein interactions with competition peptides, mutants lacking binding sites or small interfering RNA-mediated suppression of interacting proteins will be crucial for examining the physiological significance of these novel interactions.

Finally, given the role of the DAT in the regulation of DA homeostasis, it will be important to investigate to what extent disruption of these novel protein-protein interactions is associated with dysfunction of the DA system. These novel interacting proteins might represent important new targets for the treatment of mental diseases.

[J Neurochem. 2006] - open access
Volume 97 Page 3 - April 2006

Defining the dopamine transporter proteome by convergent biochemical and in silico analyses
Maiya R, Ponomarev I, Linse KD, Harris RA, Mayfield RD.; Institute for Cellular and Molecular Biology, Waggoner Center for Alcohol and Addiction Research and Section of Neurobiology, University of Texas at Austin, Austin, TX, USA.

Monoamine transporters play a key role in neuronal signaling by mediating reuptake of neurotransmitters from the synapse. The function of the dopamine transporter (DAT), an important member of this family of transporters, is regulated by multiple signaling mechanisms, which result in altered cell surface trafficking of DAT. Protein-protein interactions are likely critical for this mode of transporter regulation. In this study, we identified proteins associated with DAT by immunoprecipitation (IP) followed by mass spectrometry. We identified 20 proteins with diverse cellular functions that can be classified as trafficking proteins, cytoskeletal proteins, ion channels and extracellular matrix-associated proteins. DAT was found to associate with the voltage-gated potassium channel Kv2.1 and synapsin Ib, a protein involved in regulating neurotransmitter release. An in silico analysis provided evidence for common transcriptional regulation of the DAT proteome genes. In summary, this study identified a network of proteins that are primary candidates for functional regulation of the DAT, an important player in mechanisms of mental disorders and drug addiction.