The C-terminal PDZ-ligand motif of the neuronal glycine transporter GlyT2 is required for efficient synaptic localization

Abstract

The neuronal glycine transporter 2 (GlyT2) belongs to the large SLC6 family of Na⁺/Cl⁻-dependent neurotransmitter transporters. At its extreme C-terminus, GlyT2 carries a type III PDZ domain binding motif (PDZ-ligand motif), which interacts with the PDZ domain protein syntenin-1. Here, we investigated the physiological role of the GlyT2 PDZ-ligand motif by a loss-of-function approach. Inactivation of the PDZ-ligand motif did not impair the localization, glycosylation and transport function of recombinant GlyT2 expressed in HEK293T cells. However, in transfected hippocampal neurons, the synaptic localization of GlyT2 was significantly reduced upon PDZ-ligand motif inactivation. Co-localization of GlyT2 with marker proteins of excitatory and inhibitory synapses was decreased by down to 50% upon PDZ-ligand motif deletion as compared to the wild-type protein. These data indicate that the C-terminal PDZ-ligand motif of GlyT2 plays an important role in transporter trafficking to and/or stabilization at synaptic sites.

Introduction

Glycine is the major inhibitory neurotransmitter of interneurons in spinal cord and brain stem, where it activates strychnine-sensitive postsynaptic glycine receptors (Betz and Laube, 2006, Lynch, 2004). To allow for high-frequency synaptic transmission, glycine concentrations in the synaptic cleft have to be tightly regulated. This is accomplished by two high-affinity glycine transporters (GlyTs), GlyT1 and GlyT2 (Liu et al., 1992, Lopez-Corcuera et al., 1991, Smith et al., 1992). The GlyTs belong to the SLC6a family of Na⁺/Cl⁻-dependent neurotransmitter transporters, which share a common topology with twelve transmembrane domains and intracellular N- and C-termini (Eulenburg et al., 2005, Gether et al., 2006). The transmembrane domains 3 and 4 are connected by a large extracellular loop that harbors multiple N-glycosylation sites (Martinez-Maza et al., 2001). Although GlyT1 and GlyT2 share overlapping expression domains in caudal regions of the CNS, they differ in their cellular distributions. GlyT1 is a predominantly glial transporter mainly expressed by astrocytes (Adams et al., 1995, Zafra et al., 1995a, Zafra et al., 1995b). In addition, it has been found in glycinergic amacrine cells of the retina (Pow and Hendrickson, 1999) and, more recently, in a subset of hippocampal neurons (Cubelos et al., 2005a). In contrast, GlyT2 is concentrated at glycinergic nerve terminals in close proximity to the presynaptic release sites (Mahendrasingam et al., 2003, Zafra et al., 1995a) and constitutes the only specific marker protein of glycinergic interneurons known presently (Poyatos et al., 1997).

The importance of GlyTs for inhibitory neurotransmission has been proven by the generation of GlyT-deficient mice (Gomeza et al., 2003a, Gomeza et al., 2003b). Their analysis disclosed that GlyT1 and GlyT2 have vital but complementary functions in the nervous system of young postnatal mice. GlyT1 is essential for the removal of glycine from the synaptic cleft and hence important for terminating glycinergic neurotransmission, whereas GlyT2 is required for the reuptake of glycine into the presynaptic terminal and its subsequent loading into synaptic vesicles by the vesicular inhibitory amino acid transporter (VIAAT). In addition to its function at inhibitory synapses, GlyT1 has been shown to be involved in the regulation of extracellular glycine concentrations at NMDA receptor containing excitatory synapses (Gabernet et al., 2005, Tsai et al., 2004, Yee et al., 2006).

Although the transport activities of both GlyTs are thought to be tightly controlled, GlyT2 appears to be subject to particular regulatory mechanisms (Eulenburg et al., 2005). Different lines of evidence indicate that the surface availability of GlyT2 determines neuronal glycine uptake activity. In synaptosomal preparations, only about 5–10% of the total GlyT2 protein are present in the plasma membrane, whereas its major fraction is localized intracellularly, most likely in vesicles (Geerlings et al., 2002). One protein involved in the regulation of GlyT2 plasma membrane insertion is syntaxin 1A, which has been found to indirectly interact with the N-terminal domain of GlyT2 (Geerlings et al., 2000). In transfected cells, coexpression of GlyT2 with syntaxin 1A results in a down-regulation of transport activity. However, in synaptosomal preparations inactivation of syntaxin 1A reduced GlyT2-mediated substrate uptake (Lopez-Corcuera et al., 2001), suggesting that syntaxin 1A is involved in the regulation of both incorporation into and retrieval from the plasma membrane. Phorbol ester treatment causes enhanced GlyT2 internalization, showing that protein kinase C (PKC) contributes to the regulation of GlyT2 (Gomeza et al., 1995). Although direct phosphorylation of the transporter by PKC appears to be unlikely, residues required for PKC-induced down-regulation of GlyT2 have been identified within intracellular loop 2 (Fornes et al., 2004).

The presynaptic localization of GlyT2 implies that specific targeting and anchoring proteins must exist. Consistent with this view, the putative membrane trafficking protein Ulip6, a member of the unc-33 like phosphoprotein/collapsin response mediator protein family, has been reported to interact with the long N-terminal domain of GlyT2 (Horiuchi et al., 2000, Horiuchi et al., 2005). In addition, the C-terminal intracellular tails of transporters of the SLC6a family are thought to contain motifs that are important for the trafficking and/or activity of these membrane proteins (Farhan et al., 2004). Besides residues that have been suggested to regulate protein exit from the endoplasmic reticulum, the extreme C-terminus of GlyT2 comprises a class III PDZ (postsynaptic density 95/disc large/zona occludens 1) domain binding motif (termed PDZ-ligand motif here) to which syntenin-1, a synaptically enriched protein harboring two PDZ domains, binds (Ohno et al., 2004). In different studies, PDZ domain containing proteins have been shown to play crucial roles in the trafficking and anchoring of membrane proteins at synaptic sites (Bezprozvanny and Maximov, 2001, Kim and Sheng, 2004).

Here, we inactivated the PDZ-ligand motif of GlyT2 by either truncation or addition of an alanine at its C-terminal end. The resulting mutant transporters were indistinguishable from wild-type GlyT2 in surface expression and uptake activity but, upon expression in neuronal cells, displayed significantly reduced synaptic localization. Thus, the PDZ-ligand motif of GlyT2 is required for its efficient recruitment to or stabilization at the nerve terminal.