Monoamine reuptake is a definitive step in the termination of neural transmission and is mediated by a family of Na+-/C1-dependent plasma membrane transporters. Monoamine transporters are the molecular targets for the psychostimulants cocaine, amphetamines, and MDMA ("ecstasy"), which bind to and inhibit transporters, thereby increasing extracellular monoamine concentrations and enhancing post-synaptic responses. As the major psychostimulant targets, transporter presentation at the cell surface is paramount to psychostimulant binding and action. Mounting evidence from a number of investigators demonstrates that activation of cellular signaling pathways dynamically modulates monoamine transporter cell surface presentation. Moreover, data from our laboratory demonstrates that transporters dynamically traffic under steady state conditions. Transporter regulation and trafficking correlate with changes in transporter phosphorylation states. However, the relationship between transporter trafficking and phosphorylation is not well defined. The major goals of this project are to understand the role of phosphorylation in transporter function and trafficking. We will pursue this area of investigation by testing the following hypotheses: (1) transporters are phosphorylated and dephosphorylated as part of their constitutive trafficking process, and (2) protein kinase C (PKC) activation blocks basal transporter dephosphorylation, thereby perturbing endocytic transporter trafficking. The proposed studies will focus on a representative monoamine transporter, the dopamine transporter (DAT). We will test our hypotheses by directly analyzing basal and regulated transporter phosphorylation and trafficking in cell lines. Intrinsic domains mediating DAT phosphorylation will be identified using molecular truncation and mutagenesis approaches. It is expected that these approaches will provide a clearer picture of how transporter trafficking and phosphorylation are coupled. Such results are expected to have a significant impact on future therapeutic strategies aimed at monoamine-related psychostimulant drug abuse. moreover, the outcomes will enhance our understanding of the factors contributing to monoamine availability and signaling in the brain.