Amy E. Moritz

Date of Award

9-7-2012

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Department

Biology

First Advisor

Roxanne Vaughan

Abstract

The dopamine transporter (DAT) is an integral membrane phosphoprotein found on dopaminergic neurons that mediates the presynaptic reuptake of dopamine and is a site of action for psychostimulants. Numerous DAT functions including efflux, uptake activity, and surface expression are regulated by protein kinase C (PKC) through unknown mechanisms. Basal and PKC stimulated phosphorylation of DAT occurs on multiple serines in the distal N-terminus, but the exact sites modified and the kinases involved are unknown. To address these issues, we examined in vitro phosphorylation of a recombinant DAT N-terminal tail peptide (NDAT). We found that NDAT is phosphorylated at multiple sites (S4, S7, and S13) by PKC, and at single distinct sites by Protein Kinase A (S7) and Calcium-Calmodulin Dependent Protein Kinase II (S13), suggesting these kinases may act differentially on DAT in vivo. Using multiple approaches, we identified S7 as a phosphorylation site in rat striatal and heterologously expressed DAT, and also found that PKC-induced DAT phosphorylation occurs on the same sites utilized basally. Unexpectedly, we found that mutation of S7 and nearby residues strongly reduced the binding affinity of DAT for a cocaine analog, and that S7 mutation alters zinc modulation of cocaine analog binding, while conditions promoting DAT phosphorylation caused increased cocaine analog affinity. These results suggest that the N-terminus impacts cocaine analog binding in a manner that may be regulated by S7 phosphorylation. DAT is also S-palmitoylated on Cys580 in the C-terminus. Inhibition of DAT palmitoylation leads to reduced dopamine uptake and increased DAT degradation, while PKC activation leads to these same processes. Results also indicated that depalmitoylated DATs are more sensitive to PKC-induced down-regulation than palmitoylated DATs, suggesting that palmitoylation and kinase pathways can interact to regulate DAT. We have now identified a reciprocal relationship between these post-translational modifications, as S7A DAT shows decreased phosphorylation and increased palmitoylation, while C580A DAT shows decreased palmitoylation and increased phosphorylation. In addition DAT palmitoylation is decreased by PKC activation and increased by PKC inhibition, further supporting reciprocal regulation. To explore the functional relationship between these modifications, we examined regulation of transport by PKC in S7A and C580A DATs. We found that S7A DAT has decreased PKC-induced down-regulation, while C580A DAT has increased PKC-induced down-regulation, indicating that DAT phosphorylation is essential for down-regulation. The reciprocal nature of DAT phosphorylation and palmitoylation suggests the potential for functionally distinct DAT populations to differentially regulate dopaminergic neurotransmission.

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