Javier Vargas Medrano - Phosphorylation of the glycine transporter 1

Version 1

      Publication Details (including relevant citation   information):

      Javier Vargas Medrano, "Phosphorylation of the glycine   transporter 1" (January 1, 2010). ETD Collection for   University of Texas, El Paso. Paper AAI3433515.
      http://digitalcommons.utep.edu/dissertations/AAI3433515

      Abstract:

      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 PKCα/β inhibitor Go6976 completely   abolished the effect of phorbol ester on uptake and   phosphorylation. On the other hand, incubation with either   selective PKCβ inhibitors (PKCβ inhibitor or LY333531) prevented   the PKC-dependent phosphorylation of GlyT1 without affecting the   downregulation triggered by PMA. Taken together, this data   suggest that conventional PKCα/β regulates the uptake of glycine   and probably its efflux, whereas PKCβ 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.

      Address (URL): http://digitalcommons.utep.edu/dissertations/AAI3433515/