PHOSPHORYLATION AND REGULATION OF DOPAMINE TRANSPORTER

多巴胺转运体（DAT）是清除突触空间细胞外多巴胺的主要机制。因此，它在终止突触传递和调节可用于结合突触前和突触后多巴胺受体的多巴胺浓度方面发挥关键作用。最近发现，蛋白激酶C的激活导致dat的磷酸化，并伴随多巴胺转运的减少，这表明dat通过磷酸化进行功能调节。这将为神经元提供一种精细的时间和空间控制细胞外多巴胺浓度的机制，以及随后的下游多巴胺能神经活动。因此，DAT的这种特性有可能深刻影响正常的多巴胺能神经生理，也可能与可卡因或其他药物滥用和多巴胺能神经变性的机制有关。本研究旨在全面表征DAT磷酸化特性，明确DAT磷酸化与功能调控之间的关系。为实现这些目标而设计的具体目标是：1。确定DATs上pkc刺激磷酸化的位点。2. 构建磷酸化位点改变为非磷酸盐受体的突变体，并检查这些突变的功能后果。3. 通过鉴定作用于该蛋白的特定激酶和磷酸酶，彻底表征DAT的磷酸化和去磷酸化特性。4. 检测多巴胺转运、拮抗剂结合和表面表达的相关变化。5. 确定体内控制DAT磷酸化的内源性途径。

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.