Glycine is the major inhibitory neurotransmitter in the spinal cord and brainstem and is also required for the activation of NMDA receptors. The extracellular concentration of this neuroactive amino acid is regulated by at least two glycine transporters (GLYT1 and GLYT2). To study the localization and properties of these proteins, sequence- specific antibodies against the cloned glycine transporters have been raised. Immunoblots show that the 50–70 kDa band corresponding to GLYT1 is expressed at the highest concentrations in the spinal cord, brainstem, diencephalon, and retina, and, in a lesser degree, to the olfactory bulb and brain hemispheres, whereas it is not detected in peripheral tissues. Pre-embedding light and electron microscopic immunocytochemistry show that GLYT1 is expressed in glial cells around both glycinergic and nonglycinergic neurons except in the retina, where it is expressed by amacrine neurons, but not by glia. The expression of a 90–110 kDa band corresponding to GLYT2 is restricted to the spinal cord, brain-stem, and cerebellum; in addition, very low levels occur in the diencephalon. GLYT2 is found in presynaptic elements of neurons thought to be glycinergic. However, in the cerebellum, GLYT2 is expressed both in terminal boutons and in glial elements. The physiological consequences of the regional and cellular distributions of these two proteins as well as the possibility of the existence of an unidentified neuronal form of GLYT1 are discussed.

The high-affinity glycine transporter in neurons and glial cells is the primary means of inactivating synaptic glycine. Previous molecular cloning studies have indicated heterogeneity of glycine transporters in the CNS. Here the distribution of glycine transporter GLYT1 and GLYT2 transcripts and proteins in different regions and developmental stages of the rat brain were analysed by Northern, Western and in situ hybridization techniques. Sequence-specific riboprobes and two specific antibodies raised against fusion proteins were used, containing either 76 or 193 amino acids of the C or N terminus of the GLYT1 and GLYT2 transporters respectively. High levels of GLYT1 transcripts were found in the spinal cord, brainstem and cerebellum, and moderate levels in forebrain regions such as the cortex or hippocampus. GLYT2 transcripts are restricted to the spinal cord, brainstem and cerebellum. The onset of both GLYT1 and GLYT2 expression in the brainstem occurred in late fetal life, and full expression of these proteins was observed before weaning. There was a stepwise increase in the levels of mRNA and protein for these two transporters, reaching a maximum by the second postnatal week, followed by a slight decrease until adult values were reached by the fourth postnatal week. These data reveal interesting parallelism between the distribution of different glycine transporters and glycine receptor subunits, and suggest discrete roles for distinct glycine transporters.

In this study, we present evidence that a glycine transporter, GLYT1, is expressed in neurons and that it is associated with glutamatergic synapses. Despite the presence of GLYT1 mRNA in both glial cells and in glutamatergic neurons, previous studies have mainly localized GLYT1 immunoreactivity to glial cells in the caudal regions of the nervous system. However, using novel sequence specific antibodies, we have identified GLYT1 not only in glia, but also in neurons. The immunostaining of neuronal elements could best be appreciated in forebrain areas such as the neocortex or the hippocampus, and it was found in fibers, terminal boutons and in some dendrites. Double labeling confocal microscopy with the glutamatergic marker vGLUT1 revealed an enrichment of GLYT1 in a subpopulation of glutamatergic terminals. Moreover, through electron microscopy, we observed an enrichment of GLYT1 in both the presynaptic and the postsynaptic aspects of putative glutamatergic terminals that established asymmetric synapses. In addition, we demonstrated that GLYT1 was physically associated with the NMDA receptor in a biochemical assay. In conclusion, the close spatial association of GLYT1 and glutamatergic synapses strongly supports a role for this protein in neurotransmission mediated by NMDA receptors in the forebrain, and perhaps in other regions of the CNS.

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Background:The glutamate transporter GLT-1 is regulated by PKC, which promotes its ubiquitination and subsequent endocytosis. Results: Phosphorylation of GLT-1 occurs at Ser-520, whereas ubiquitination is mediated by the ubiquitin ligase Nedd4-2. Conclusion: PKC-promoted endocytosis of GLT-1 requires Nedd4-2-dependent ubiquitination but not its phosphorylation. Significance: Intracellular trafficking of glutamate transporters seems to play a major role in the pathophysiology of the nervous system.

To elucidate the role of N-glycosylation in the function of the high affinity glycine transporter GLYT1, we have investigated the effect of the glycosylation inhibitor tunicamycin as well as the effect of the disruption of the putative glycosylation sites by site-directed mutagenesis. SDS-polyacrylamide gel electrophoresis of proteins from GLYT1-transfected COS cells reveals a major band of 80-100 kDa and a minor one of 57 kDa. Treatment with tunicamycin produces a 40% inhibition in transport activity and a decrease in the intensity of the 80-100-kDa band, whereas the 57-kDa band decreases in size to yield a 47-kDa protein corresponding to the unglycosylated form of the transporter. Simultaneous mutation of Asn-169, Asn-172, Asn-182, and Asn-188 to Gln also produces the 47-kDa form of the protein, indicating that there are no additional sites for N-glycosylation. Progressive mutation of the potential glycosylation sites produces a progressive decrease in transport activity and in size of the protein, indicating that the four putative glycosylation sites are actually glycosylated. N-Glycosylation of the GLYT1 is not indispensable for the transport activity itself, as demonstrated by enzymatic deglycosylation of the transporter. Analysis of surface proteins by biotinylation and by immunofluorescence demonstrates that a significant portion of the unglycosylated GLYT1 mutant remains in the intracellular compartment. This suggests that the carbohydrate moiety of glycine transporter GLYT1 is necessary for the proper trafficking of the protein to the plasma membrane.

A theoretical 12-transmembrane segment model based on the hydrophobic moment has been proposed for the transmembrane topology of the glycine transporter GLYT1 and all other members of the sodium-and chloridedependent transporter family. We tested this model by introducing N-glycosylation sites along the GLYT1 sequence as reporter for an extracellular localization and by an in vitro transcription/translation assay that allows the analysis of the topogenic properties of different segments of the protein. The data reported herein are compatible with the existence of 12 transmembrane segments, but support a rearrangement of the first third of the protein. Contrary to prediction, hydrophobic domain 1 seems not to span the membrane, and the loop connecting hydrophobic domains 2 and 3, formerly believed to be intracellular, appears to be extracellularly located. In agreement with the theoretical model, we provide evidence for the extracellular localization of loops between hydrophobic segments 5 and 6, 7 and 8, 9 and 10, and 11 and 12.Glycine is a major inhibitory neurotransmitter in the spinal cord and the brain stem of vertebrates. In addition, glycine can potentiate the action of glutamate, the main excitatory neurotransmitter in the brain, on postsynaptic N-methyl-D-aspartate receptors. The re-uptake of glycine into presynaptic nerve terminals or the neighboring fine glial processes provides one way of clearing the extracellular space of this neuroactive substance and so constitutes an efficient mechanism by which the postsynaptic action can be terminated (1-3). This process is carried out by two different glycine transporters, named GLYT1 and GLYT2, which belong to the Na ϩ -and Cl Ϫ -dependent neurotransmitter transporter family (4 -9). GLYT1 and GLYT2 present a differential expression pattern among central nervous system cells (7, 10 -14).The hydropathic profiles of the Na ϩ -and Cl Ϫ -dependent neurotransmitter transporters reveal the presence of 12 hydrophobic segments that have been suggested to form transmembrane ␣-helices (15). With the aid of sequence-specific antibodies, immunofluorescence, and electron microscopy, it has been shown that both the amino-and carboxyl-terminal ends of GLYT1 are intracellularly located (11,16). Additional topological data have been obtained from the study of the glycosylation pattern of GLYT1. Site-directed mutagenesis has shown that GLYT1 is heavily glycosylated at four asparagine residues (Asn 169 , Asn 172 , Asn 182 , and Asn 188 ) (17). This fact involves an extracellular localization of the hydrophilic loop placed between hydrophobic segments 3 and 4.In this report, we present an extensive experimental evaluation of a neurotransmitter carrier protein transmembrane topology. The data included are compatible with a 12-membrane spanning segment model, with evidence for the extracellular localization of loops connecting hydrophobic domains 5 and 6, 7 and 8, 9 and 10, and 11 and 12. However, data herein favor an extracellular localization of the loop connecting HD2 1 and HD3,...

The activity of the main glutamate transporter in the CNS, GLT1, can be regulated by protein kinase C (PKC). It is known that activation of PKC by phorbol esters promotes the clathrin-dependent internalization of the transporter, followed by its lysosomal degradation. However, the molecular mechanisms that link PKC activation and the internalization of GLT1 are not fully understood. In this article, we show that this internalization process is dependent on the ubiquitylation of lysine residues located in the C-terminal tail of GLT1. Exposure to PMA increases the ubiquitylation of GLT1 in transfected cells and in the rat brain, and this ubiquitylated GLT1 accumulates in the intracellular compartment. However, internalization of ubiquitylated GLT1 was blocked with a dominant negative dynamin 2 mutant, indicating that the addition of ubiquitin moieties to the transporter in the membrane precedes its endocytosis. The elimination of lysines from the C-terminus of the transporter (lysines 497, 517, 526, 550, 558, 570, and 573) blocked GLT1 ubiquitylation and endocytosis. However, reintroduction of lysine 517 alone into this mutant was sufficient to restore PMA dependent ubiquitylation and internalization of GLT1. Similarly, reintroduction of lysine 526 restored the endocytosis, while this was only partially recovered after the individual reintroduction of lysines 550 or 570. These data suggest that the activation of PKC induces the ubiquitylation of these C-terminal lysine residues in GLT1 and that this modification mediates the interaction of the transporter with the endocytic machinery.