Ma, Shiqi
(2018)
REGULATION OF DOPAMINE TRANSPORTER SUBCELLULAR LOCALIZATION AND TRAFFICKING.
Doctoral Dissertation, University of Pittsburgh.
(Unpublished)
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.
Share
| Citation/Export: |
|
| Social Networking: |
|
Details
| Item Type: |
University of Pittsburgh ETD
|
| Status: |
Unpublished |
| Creators/Authors: |
|
| ETD Committee: |
|
| 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 |
Metrics
Monthly Views for the past 3 years
Plum Analytics
Actions (login required)
 |
View Item |
![[feed]](/images/feed-icon-32x32.png){kind=icon}