Rin GTPase And Dopamine Transporter Trafficking: Linking Mechanisms To Behavior

Univ Of Massachusetts Med Sch Worcester, Worcester MA

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Abstract

? DESCRIPTION (provided by applicant): Dopamine (DA) signaling in the CNS is essential for modulating complex behaviors such as movement, cognition, reward, and motivation. Aberrant DAergic transmission is directly linked to Parkinson's disease, attention-deficit hyperactivity disorder, schizophrenia and addiction. The plasma membrane dopamine transporter (DAT) is central to DAergic signaling, and both limits extracellular DA availability and maintains presynaptic DA stores. Importantly, DAT is the primary target for both addictive and therapeutic psychoactive drugs, such as cocaine, amphetamine, methylphenidate (Ritalin), and the antidepressant bupropion (Wellbutrin), all of which competitively inhibit DAT activity. A wealth of data supports the premise that DAT constitutively traffics to and from the cell surface, and that protein kinase C (PKC) activation rapidly decreases DAT cell surface expression by acutely modulating DAT trafficking rates. While some progress has been made investigating the molecular mechanisms that govern regulated DAT trafficking, a detailed understanding of this complex process has yet to be achieved. Further, the physiological relevance that DAT trafficking imposes onto DAergic function is poorly understood. Our laboratory reported that neuronal GTPase, Rin (RIT2), specifically binds to the DAT carboxy terminus and is required for PKC-stimulated DAT internalization. In the proposed studies, I plan to investigate the molecular underpinnings required for DAT/Rin interactions, and how these interactions potentially mediate synergy between cytosolic DAT domains. Aim 1 studies will use co-immunoprecipitations and FRET microscopy approaches with chimeric DAT proteins to define the intracellular DAT domains that are necessary and sufficient to confer DAT/Rin interactions. Chimeric and point mutant transporters will further reveal the potential synergistic requirement of DAT amino- and carboxy-termini for PKC-stimulated DAT internalization. Aim 2 studies will directly test the potential role of Rin-mediated DAT trafficking in DAergic behaviors in vivo, using cell-specific, AAV2-mediated Rin knockdown in DAergic terminal regions. Taken together, these studies will provide the first investigations that directly examine how DAT trafficking potentially impacts rewarding behavior. Moreover, completion of this investigative line will provide me with outstanding training in molecular and behavioral neuroscience.