Date of Award
Doctor of Philosophy (PhD)
The dopamine transporter (DAT) is a presynaptic membrane phosphoprotein that terminates dopaminergic synaptic transmission by clearing dopamine (DA) back into presynaptic neuron by a reuptake process. DAT is also a molecular target for psychostimulants such as cocaine, amphetamine (AMPH) and methamphetamine (METH) that increase DA synaptic levels either by blocking reuptake or by inducing DA efflux. DAT phosphorylation and transport down regulation are best demonstrated with the treatment of Protein kinase C (PKC) activators such as (phorbol 12-myristate 13-acetate) PMA. Serine(s) and threonine(s) are known to be involved in DAT phosphorylation.
Pretreatment with psychostimulant substrates such as AMPH and METH have been shown to regulate DAT phosphorylation and DA transport activity. Here we have examined the effects of DA, cocaine and various other psychoactive transport blockers on DAT phosphorylation and regulation in rDAT expressing LLC-PK1 cells by using 32PO4 metabolic labeling and [3H] DA uptake assays. Pretreatment with cocaine or methylphenidate (MPH) had no effect on basal or PMA stimulated DAT phosphorylation and DA transport. GBR 12909 suppressed PMA-induced DAT phosphorylation and internalization. Treatment with DA did not affect DAT phosphorylation while inducing PKC mediated DA transport down regulation. These results provide information on the potential for endogenous and psychoactive compounds to modulate DAT phosphorylation-mediated regulatory mechanisms that may contribute to drug behavioral or therapeutic properties.
N-terminal phosphorylation of DAT is involved in DA efflux, but the DAT amino acid residues, protein kinases and protein phosphatases involved in this process are not known. In this study, we showed that a variety of protein kinases including PKCα, PKA, CaMKII, and MAPKs phosphorylate a recombinantly expressed N-terminal tail of DAT (NDAT) in vitro. Phosphoaminoacid analysis specifically showed that PKCα phosphorylates on serine(s) and ERK1 phosphorylates on threonine(s) of NDAT. Protein phosphatases (PP1 and PP2B) were able to dephosphorylate the PKCα phosphorylated NDAT; however, none of the tested protein phosphatases were able to dephosphorylate the ERK1 phosphorylated NDAT. Further, we have identified T53 as a MAPK phosphorylation site on NDAT in vitroand localized threonine phosphorylation on the N-terminal portion of striatal DAT in vivo. These studies implicate the role of multiple kinases or phosphatases in differential phosphorylation or dephosphorylation of DAT. Further these studies may increase the current understanding of N-terminal phosphorylation ofDAT that has been shown to regulate the DA efflux.
Gorentla, Balachandra Kumar, "Molecular Characterization of Dopamine Transporter Phosphorylation and Regulation" (2008). Theses and Dissertations. 705.