A Proteomic Analysis of the Norepinephrine Transporter

Yale University, New Haven CT

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Abstract

[unreadable] DESCRIPTION (provided by applicant): The norepinephrine transporter (NET) terminates noradrenergic signals by clearing released NE from extracellular synapses. Stimulation of presynaptic receptors linked to pathways involving second messengers, kinases and phosphatases regulate the activity of NET by modulating surface trafficking and catalytic activity. Further clarification of these pathways may suggest candidates for novel therapeutics as well as identify mechanisms that may be altered in disease states. NET interacts with proteins such as syntaxin 1A, protein phosphatase 2A, PICK 1 and Hic-5. The interactions are known to influence NET activity and trafficking, and participate in cellular responses to NET-targeted medications/ psychostimulants. To better understand these regulatory mechanisms, I propose a proteomic analysis to elucidate a more complete picture of NET protein complexes. In this effort, NET will be immunoprecipitated from stably transfected neuroblastoma cell lines and the proteins that co-immunoprecipitate with NET will be analyzed by mass spectrometry (MS) using LC-MS/MS (liquid chromatography coupled tandem mass spectrometry) method. Identified proteins will be validated for the interactions with NET and further studied for influences on co-localization, trafficking and activity of NET. These approaches will identify novel interacting proteins that can elucidate regulatory pathways controlling NE clearance capacity and provide information on new targets for pharmacological manipulation in neurological and psychiatric disorders. The relevance to public health: The norepinephrine transporter (NET) is a target of antidepressants. I propose a proteomic analysis to better understand regulatory mechanisms of NET. This approach will identify novel interacting proteins that can elucidate regulatory pathways controlling NE clearance capacity and provide information on new targets for pharmacological manipulation in neurological and psychiatric disorders. [unreadable] [unreadable]