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REGULATION OF DOPAMINE TRANSPORTER SUBCELLULAR LOCALIZATION AND TRAFFICKING

Ma, Shiqi (2018) REGULATION OF DOPAMINE TRANSPORTER SUBCELLULAR LOCALIZATION AND TRAFFICKING. Doctoral Dissertation, University of Pittsburgh. (Unpublished)

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Abstract

Dopamine transporter (DAT) controls dopamine (DA) neurotransmission by clearing synaptically released DA. The substrate uptake function of DAT is regulated by its subcellular localization. We propose that DAT localization and trafficking are regulated by its molecular conformation and interactions of the transporter with lipids and membrane-associated proteins. We used several experimental approaches and two DAT mutants to demonstrate that accumulation of DAT in plasma membrane protrusions, such as myosin X-dependent filopodia, requires an outward facing (OF) conformation of the DAT molecule. Furthermore, using targeted plasma membrane depletion of phosphatidylinositol 4,5-bisphosphate (PIP2), a lipid that is highly enriched in the plasma membrane and proposed to bind DAT, we demonstrated that PIP2 is necessary for protein kinase C (PKC)-stimulated DAT endocytosis but not essential for maintaining steady-state levels of DAT at the cell surface. Therefore, these results suggest that PIP2 is essential for clathrin-mediated PKC-stimulated DAT endocytosis and PIP2 is not involved in the retention of DAT at the cell surface. In search of DAT interactors regulating its plasma membrane retention, distribution, subcellular localization, and function, we used quantitative mass spectrometry analysis to identify proteins that are co-precipitated with DAT isolated from the mouse striatum. A novel interaction of DAT with Gαo, the α subunit of Go proteins that are coupled to dopamine D2 receptor and other receptors, was confirmed by reciprocal co- immunoprecipitation and western blotting in mouse striatum and a heterologous expression system. While structure-functional analysis of this interaction will be performed in the future studies, our biochemical and morphological analyses led to the hypothesis that DAT-Gαo interaction may mediate the regulatory effects of D2 auto-receptors on DAT localization and activity. In summary, our data suggest that the localization of DAT in filopodia, which is regulated by DAT conformation and possibly DAT-Gαo interaction but not DAT interaction with PIP2, is the primary mechanisms of the retention of functional DAT at the cell surface.


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Details

Item Type: University of Pittsburgh ETD
Status: Unpublished
Creators/Authors:
CreatorsEmailPitt UsernameORCID
Ma, Shiqimashiqi1021@gmail.comshm90
ETD Committee:
TitleMemberEmail AddressPitt UsernameORCID
Committee ChairThiels, Eddathiels@pitt.eduthiels
Thesis AdvisorSorkin, Alexandersorkin@pitt.edusorkin
Committee MemberAmara, Susansusan.amara@nih.gov
Committee MemberDong, Yanyandong@pitt.eduyandong
Committee MemberKwiatkowski, Adamadamkwi@pitt.eduadamkwi
Committee MemberPuthenveedu, Manojkumarmap3@andrew.cmu.edu
Date: 18 January 2018
Date Type: Publication
Defense Date: 12 September 2017
Approval Date: 18 January 2018
Submission Date: 20 December 2017
Access Restriction: No restriction; Release the ETD for access worldwide immediately.
Number of Pages: 112
Institution: University of Pittsburgh
Schools and Programs: School of Medicine > Neurobiology
Degree: PhD - Doctor of Philosophy
Thesis Type: Doctoral Dissertation
Refereed: Yes
Uncontrolled Keywords: dopamine transporter, trafficking, endocytosis, filopodia
Date Deposited: 18 Jan 2018 15:18
Last Modified: 18 Jan 2018 15:18
URI: http://d-scholarship.pitt.edu/id/eprint/33655

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