Even though the abundance of GlyT1 in postsynaptic aspects of forebrain neurons is low, its previously reported interaction with postsynaptic density protein PSD95 represents a prototype of interaction, which might uncover the binding mechanism and regulation of the GlyT1 C-terminal PDZ motif. We used a phosphomimetic approach to mimic the potential phosphorylation of GlyT1 C-terminal serines 640, 643, 644, and 649 of the mouse GlyT1b subtype (mGlyT1b) (Uniprot P28571-2) and its effect on GlyT1-PSD95 PDZ interaction. Among them, only phosphomimetic mutation of serine 649 to aspartate, which resides in the minimal PDZ motif -SRI, significantly eliminated the interaction of the GlyT1 C-terminus with PSD95 PDZ domain 2. The effect was observed with recombinant fusion proteins as well as with GlyT1 and PSD95 expressed in tissue culture. Results indicate that phosphorylation of mouse GlyT1b serine 649 and equivalent serines of GlyT1a and GlyT1c subtypes might regulate the PDZ interaction of the GlyT1 C-terminal PDZ binding motif.
Keywords: GlyT1; NMDA; PDZ; PSD95; Phosphomimetic; Phosphorylation.