Proteomic /Genetic Approaches to Monoamine Transporters

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DESCRIPTION (provided by applicant): Biogenic amine transporters have emerged as the indispensable molecules regulating monoamine transmission in the brain by controlling the duration and intensity of transmitter actions at specific molecular targets. Three distinct genes encode closely related plasma membrane transporter proteins: the dopamine transporter (DAT), the norepinephrine transporter (NET), and the serotonin transporter (SEPT). The primary role of these transporter proteins is to remove released transmitters from the synaptic cleft back into presynaptic terminals for subsequent vesicular storage and release. Deletion of the genes encoding biogenic amine transporters in mice results in profound neurochemical and behavioral changes, thereby, illustrating the impact of these transporters in controlling presynaptic monoamine homeostasis. A wide spectrum of neurological and psychiatric disorders, including drug abuse, affective disorders, and Parkinson's disease is thought to involve monoamine transmission and monoamine transporters. Indeed, it has been well established that biogenic amine transporters are the biological targets for potent psychostimulants such as cocaine, amphetamine, and (+)-3,4-methylenedioxymethamphetamine (MDMA or ecstasy) as well as therapeutic agents used to treat mental disorders including depression, attention deficit hyperactivity disorder, obsessive-compulsive disorder, and eating disorders. Recently, results from our and other laboratories suggest that monoamine transporters are highly regulated proteins and indicate a more complicated degree of organization for these transporters than previously anticipated. Findings include the elucidation of the oligomeric nature of DAT, the identification of domains involved in assembly and trafficking, and the identification and preliminary characterization of interacting proteins that regulate the targeting, trafficking, and function of monoamine transporters. Based on these results, I hypothesize that monoamine transporters exist as highly regulated macromolecular protein complexes in neurons. Thus, the overall goal of this research proposal is to provide a clearer understanding of the cellular regulatory mechanisms associated with monoamine transporters by identifying interacting proteins and establishing the role of such protein-protein interactions in transporter function. The following specific aims are proposed: Aim I: To determine the total complement/network of protein-protein interactions involved in biogenic amine transporter complexes in vivo using proteomic approaches. Aim II: To elucidate the physiological role of PICK1 and Hic-5 in the regulation of DAT function using genetic approaches. Aim IlI: To examine biochemically and functionally if putative interacting proteins identified previously in a yeast two-hybrid screen are real DAT interacting proteins. These studies should provide unprecedented insights into the regulation of monoamine transporters in brain and novel molecular targets for therapeutic approaches. This research proposal has also been designed to further develop the scientific career of the P.I. in preparation for an independent faculty position by acquiring expertise in proteomic and genetic approaches from internationally recognized scientists to better address the role of biogenic amine transporters in normal and abnormal brain function.