Constitutive and Regulated Endocytosis of the Glycine Transporter GLYT1b Is Controlled by Ubiquitination

The glycine transporter GLYT1 regulates both glycinergic and glutamatergic neurotransmission by controlling the reuptake of glycine at synapses. Trafficking of GLYT1 to and from the cell surface is critical for its function. Activation of PKC down-regulates the activity of GLYT1 through a mechanism that has so far remained uncharacterized. Here we show that GLYT1b undergoes fast constitutive endocytosis that is accelerated by phorbol esters. Both constitutive and regulated endocytosis occur through a dynamin 2- and clathrin-dependent pathway, accumulating in the transporter in transferrin-containing endosomes. A chimera with the extracellular and transmembrane domains of the nerve growth factor receptor and the COOH-terminal tail of GLYT1 was efficiently internalized through this clathrin pathway, suggesting the presence of molecular determinants for GLYT1b endocytosis in its COOH-terminal tail. Extensive site-directed mutagenesis in this region of the chimera highlighted the involvement of lysine residues in its internalization. In the context of the full-length transporter, lysine 619 played a prominent role in both the constitutive and phorbol 12-myristate 13-acetate-induced endocytosis of GLYT1b, suggesting the involvement of ubiquitin modification of GLYT1b during the internalization process. Indeed, we show that GLYT1b undergoes ubiquitination and that this process is stimulated by phorbol 12-myristate 13-acetate. In addition, this endocytosis is impaired in an ubiquitination-deficient cell line, further evidence that constitutive and regulated endocytosis of GLYT1b is ubiquitin-dependent. It remains to be determined whether GLYT1b recycling might be affected in pathologies involving alterations to the ubiquitin system, thereby interfering with its influence on inhibitory and excitatory neurotransmission.Glycine fulfills a dual role in neurotransmission by mediating inhibition through the strychnine-sensitive glycine receptor and excitation as a co-agonist of the NMDA² receptors (1, 2). Although it was initially believed that the concentration of glycine in the synaptic cleft would be sufficient to saturate the glycine sites on NMDA receptors, recent pharmacological and electrophysiological evidence indicates that due to the activity of the GLYT1 (glycine transporter-1) glycine transporter, this is probably not the case. Three isoforms of GLYT1 exist that differ in their NH₂-terminal sequence (GLYT1a, GLYT1b, and GLYT1c), and they are strongly expressed in glycinergic areas of the nervous system, predominantly in glial cells (3). Indeed, mice lacking GLYT1 have impaired glycinergic neurotransmission, which has been attributed to an increase in extracellular glycine close to the strychnine-sensitive glycine receptor (4). Moreover, GLYT1 has been identified in neuronal elements closely associated with the glutamatergic pathways throughout the brain (5, 6). GLYT1 is enriched in presynaptic buttons, where it largely co-localizes with the vesicular glutamate transporter vGLUT1. It is also present in the postsynaptic densities of asymmetric synapses, and complexes containing both NMDA receptor and GLYT1 have been shown to exist (5). In these postsynaptic sites, the distribution of GLYT1 is partially controlled through its interaction with the scaffolding protein PSD-95 (7). Accordingly, GLYT1 is believed to play a role in controlling the concentration of glycine in the microenvironment around the NMDA receptor. Indeed, functional studies have shown that a specific GLYT1 inhibitor, N-3-(4′-fluorophenyl)-3-(4′-phenylphenoxy)propyl]sarcosine, potentiates NMDA-mediated responses in vitro and in vivo (8–10). The potential role of GLYT1 in glutamatergic neurotransmission has also been confirmed in heterozygous Glyt1^+/− animals that express only 50% of the normal levels of GLYT1 as well as when GLYT1 expression is disturbed in forebrain neurons. In these animals, hippocampal NMDA receptor function is enhanced, and the mice appear to display better memory retention than wild type mice (11–13).The mechanisms responsible for the insertion of GLYT1 into glutamatergic synapses are unknown. However, recent studies indicate that the movement of transporters within the cell is highly organized and that a number of ancillary proteins control their intracellular trafficking by interacting with targeting motifs in the transporter. Indeed, like other neurotransmitter transporters, GLYT1 is asymmetrically distributed in polarized cells (14, 15). The asymmetric distribution of sodium-dependent neurotransmitter transporters (NSS) requires a number of steps that commence with their efficient exit from the endoplasmic reticulum. This is followed by sorting processes in the Golgi complex, insertion into the plasma membrane, and the retention of the transporter at functional synaptic sites. Moreover, the amount of transporter in the plasma membrane is also regulated by endocytosis and recycling mechanisms. Like several other members of the NSS family, GLYT1 is subjected to regulation by protein kinase C. Activation of PKC by phorbol esters down-regulates GLYT1, which is endocytosed from the plasma membrane to intracellular compartments in several cell lines (16–18). For years, the molecular mechanisms that mediate phorbol 12-myristate 13-acetate (PMA)-stimulated endocytosis of the NSS family members have remained elusive. However, recent evidence obtained for the dopamine transporter (DAT) has revealed the importance of ubiquitination of DAT for its endocytosis (19). DAT is ubiquitinated by the Nedd4-2 ligase at several intracellular lysines. Indeed, mutation of these lysines abolished both ubiquitination and phorbol ester-stimulated endocytosis, indicating that the associated ubiquitin molecules serve as a platform to recruit endocytotic adaptors (20–23). Ubiquitination has also been implicated in the endocytosis of other membrane proteins, including the main transporter for glutamate, GLT1, and the system A transporter SNAT2 (24–26).Ubiquitin coupling can involve either mono- or polyubiquitination. Monoubiquitination occurs when a single ubiquitin molecule is coupled to one or more lysine residues on a target protein, such that the final stoichiometry is one ubiquitin per lysine. Polyubiquitination refers to the coupling of a chain of ubiquitins to a lysine on the target protein, with a final stoichiometry of four or more ubiquitins per lysine. Whereas monoubiquitinated proteins are degraded in lysosomes, polyubiquitinated proteins are recognized by and subsequently degraded by the 26 S proteasome (27).In this study, we show that GLYT1b is endocytosed through a clathrin-dependent mechanism, a process that is accelerated by phorbol esters. Through a mutational analysis, we have identified a lysine residue in the COOH-terminal tail of the protein as the major determinant for GLYT1b internalization through both constitutive and PMA-stimulated pathways. Ubiquitination GLYT1b is stimulated by PMA, a finding compatible with ubiquitin being the platform on which the clathrin network is assembled.