Effect Of Mutations At Putative GlyT1 Phosphorylation Sites On Activity And Endocytosis

Glycine is an inhibitory neurotransmitter and a co-agonist of glutamate in the N-methyl-D-aspartate-(NMDA) receptor. NMDA plays a critical role in schizophrenia. The glycine transporter 1 (GlyT1) is a Na⁺/Cl^- glycoprotein that regulates the amount of glycine in the synaptic cleft. Its regulation may play an important role in the treatment of schizophrenia and other cognative/neurological disorders. Activation of protein kinase C (PKC) by phorbol 12-myristate 13-acetate (PMA) diminishes GlyT1 activity. We demonstrated PKC-dependent GlyT1 phosphorylation and that serine or threonine residues are phospho-acceptor sites. In the present study, we performed site-directed mutagenesis of GlyT1 serine and threonine residues at the amino- and/or carboxyl-terminal tails; these mutants were stably expressed in model cell lines. By immunofluorescence techniques, the GlyT1 protein was detected at the cell surface of the mutants suggesting that Ser/Thr-Ala substitutions do not affect protein folding and trafficking along the biosynthetic pathway. In addition, we identified fully glycosylated GlyT1 mutants at the predicted molecular weight by western blotting. Further analysis of PKC-dependent endocytosis for the wild-type GlyT1 showed that incubation of the cells for 1 h with PMA, a PKC activator, triggered GlyT1 endocytosis. In these cells, GlyT1 moved from the cell surface and accumulated into early endosomes labeled with the early endosomal antigen 1 (EEA1). When the different GlyT1 mutants were analyzed, no differences in endocytosis were obtained compared to the wild-type suggesting that GlyT1 phosphorylation does not play a role in endocytosis. Uptake experiments are currently underway to determine the effect of Ser/Thr mutations on activity.