Date of Award
Doctor of Philosophy
The extracellular levels of the neurotransmitter glycine in the brain are tightly regulated by the high-affinity glycine transporter 1 (GlyT1) and the clearance of glycine depends on its rate of transport and the levels of cell surface GlyT1. Over the past years, it has been shown that PKC activation diminishes the activity and promoted phosphorylation of several neurotransmitter transporters including the dopamine, serotonin and norepinephrine transporters however, its role is unknown for the glycine transporter. To get insights into the role of PKC activation on GlyT1 regulation, we used three N-terminus GlyT1 isoforms stably expressed in porcine aortic endothelial (PAE) cells and assaying for [32P]-orthophosphate metabolic labeling. We demonstrated that the isoforms GlyT1a, GlyT1b, and GlyT1c were constitutively phosphorylated, and that phosphorylation was dramatically enhanced, in a time-dependent fashion, after PKC activation by phorbol ester (PMA). The phosphorylation was PKC-dependent, since pre-incubation of the cells with bisindolylmaleimide I (BIM), a selective PKC inhibitor, abolished the phorbol ester-induced phosphorylation. Moreover, blotting of a purified GlyT1 fraction with specific antibodies to phosphorylated tyrosine residues did not yield any signal that could correspond to GlyT1 phosphorylation, suggesting that the phosphorylation occurs at serine and/or threonine residues. In addition, by using more specific inhibitors to the different PKC isoenzymes, we were able to determine the PKC isoenzyme(s) involved in downregulation of glycine uptake and GlyT1 phosphorylation. Specifically, we found that pre-incubation of the cells with the selective PKCalpha/beta inhibitor GÃ¶6976 completely abolished the effect of phorbol ester on uptake and phosphorylation. On the other hand, incubation with either selective PKCbeta inhibitors (PKCbeta inhibitor or LY333531) prevented the PKC-dependent phosphorylation of GlyT1 without affecting the downregulation triggered by PMA. Taken together, this data suggest that conventional PKCalpha/beta regulates the uptake of glycine and probably its efflux, whereas PKCbeta is responsible for GlyT1 phosphorylation.
In order to determine the sites of phosphorylation, we mutated all the threonine and serine residues found at either, the N- or C-terminus to alanine residues. Incubation of cells stably expressing the GlyT1 N- or C-mutants with PMA triggered phosphorylation at similar levels to those obtained for the wild-type transporter, suggesting that both N- and C-terminal tails in the GlyT1 are phosphorylated. Although the results demonstrate GlyT1 phosphorylation, the role of this modification still remains to be elucidated.
Additionally, in order to obtain structural information about the potential glycine-binding site, a model of three-dimensional structure of the GlyT1 was built based on the atomic coordinates of the related bacterial leucine transporter (LeuT). The resulting model showed a helical bundle and a substrate binding-pocket similar to the LeuT. This model was used to analyze the reactivity of cysteine residues in the GlyT1 to fluorescently-labeled maleimides. The reactivity of the cysteine residues was assayed by incubation of cells expressing the wild-type transporter with the hydrophilic fluorescein maleimide resulting in labeling of transporter without any significant effect on glycine uptake. By contrast, incubation with either hydrophobic pyrenyl or coumarin maleimides resulted in rapid inhibition of glycine uptake. These results suggest that inhibition of the activity could be due a modification of a cysteine residue(s) lying in or near to the glycine-binding site. In agreement with this hypothesis, our model suggests the presence of the Cys-152 in the putative substrate-pocket. Further mutagenesis analysis combined with chemical modification should pave the way for studying the glycine-binding site and translocation pathway in the GlyT1.
Received from ProQuest
Javier Vargas Medrano
Vargas Medrano, Javier, "Phosphorylation of the Glycine Transporter 1" (2010). Open Access Theses & Dissertations. 2607.