Date of Award

January 2014

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Department

Biomedical Sciences

First Advisor

Roxanne A. Vaughan

Abstract

The dopamine transporter (DAT) is a plasma membrane protein that clears extraneuronal dopamine (DA) and thus controls the spatio-temporal dynamics of dopaminergic neurotransmission. Also, DAT is the major target for psychostimulant substrates, amphetamine (AMPH) and methamphetamine (METH), and psychostimulant uptake blocker, cocaine (COC). DAT is a phosphoprotein with both the N- and C-termini facing toward the cytosol, with multiple phosphorylation sites on the N-terminus. DAT has a closely spaced 6-serine cluster on the distal N-terminus that is phosphorylated in a protein kinase C (PKC)-dependent manner and a recently identified proline-directed site, Thr (T) 53, that is phosphorylated in vitro by the MAP kinases ERK, JNK and p38. Current studies indicate that COC and AMPH impact DAT regulatory properties including uptake activity and surface expression. Although the mechanism of drug action on DAT is not completely known, phosphorylation conditions of DAT have been found to be associated with altered surface expression and activity of DAT. In this study, we examined the effect of several psychostimulant drugs on the phosphorylation of DAT using a newly developed phospho-specific antibody against phosphorylated T53 (pT53) on DAT. A detailed analysis of pT53 on DAT was performed in LLC-PK1 cells expressing rat DAT (rDAT), rat striatal synaptosomes and in vivo in male Sprague-Dawley rats. Our studies revealed psychostimulant substrates but not uptake blockers significantly stimulated pT53. Pretreatment with COC blocked the AMPH stimulation of pT53 indicating that AMPH stimulates pT53 in a DAT-dependent manner. In rat striatal synaptosomes, METH-stimulation of pT53 was very fast; occurring within 60 sec. Subcutaneous injections of METH in rats stimulated pT53 in a time-dependent manner. Our study demonstrates that the proline-directed phosphorylation site, pT53 is subject to differential regulation by psychostimulant drugs. Prolyl cis-trans isomerase, Pin1 catalyzes the cis-trans isomerization of pThr-Pro peptides allowing the dephosphorylation by conformation-specific phosphatases. We investigated the regulation of pT53 by Pin1 using Juglone (Jug), a small molecule inhibitor of Pin1. Treatment with Jug in LLC-PK1 rDAT cells and rat striatal synaptosomes revealed significant stimulation of pT53. Jug treatment enhances [3H]DA efflux from rat striatal synaptosomes. ELISA indicated interaction between the N-terminus of DAT and Pin1. This is the first evidence of DAT regulation by Pin1. Our study demonstrates the regulation of DAT pT53 by Pin1 and psychostimulant drugs under physiological conditions.

Download

Share

COinS