The N-terminal tail of the glycine transporter: role in transporter phosphorylation.
Main Article Content
Abstract
In the vertebrate nervous system, glycinergic neurotransmission is tightly regulated by the action of the glycine transporters 1 and 2 (GlyT1 and GlyT2). Unlike GlyT1, the GlyT2 is characterized by the presence of a cytosolic 201 amino acids N-terminal tail of unknown function. In the present study we stably expressed a set of GlyT2 N-terminal deletion mutants and characterized the effect on uptake, trafficking and PKC-dependent post-translational modifications. The deletion of the first 43, 109 or 157 amino acids did not affect the trafficking and maturation of GlyT2. Similarly, a chimeric protein replacing GlyT2 N-terminus by the corresponding tail of GlyT1b or deletion of the entire N-terminus domain (D201), were able to reach the plasma membrane but showed faster turnover compared to the wild-type. All of these mutant proteins appeared as a mature, glycosylated transporter and a lower protein band corresponding to the non-glycosylated transporter. Interestingly, glycine uptake and PKC-mediated endocytosis and further reduction of transport were not affected by the deletions of 43–157 amino acids. Moreover, the analysis of PKC-dependent post-translational modifications for the mutants demonstrated that GlyT2 transporter ubiquitination was not affected; however, PKC-dependent transporter phosphorylation was completely abolished in the deletions of 157 and 201 amino acids. Altogether, these results suggest that the GlyT2 N-terminus does not directly participate in the modulation of transport properties but it could be relevant for scaffold formation and retention at active zones or communication to an intracellular signaling pathway.
Keywords:
phosphorylation, ubiquitination, glycine, transporter, PKC
Article Details
How to Cite
GIROTTO, Gentil Luciana et al.
The N-terminal tail of the glycine transporter: role in transporter phosphorylation..
Medical Research Archives, [S.l.], v. 8, n. 5, may 2020.
ISSN 2375-1924.
Available at: <https://esmed.org/MRA/mra/article/view/2085>. Date accessed: 23 apr. 2024.
doi: https://doi.org/10.18103/mra.v8i5.2085.
Section
Research Articles
The Medical Research Archives grants authors the right to publish and reproduce the unrevised contribution in whole or in part at any time and in any form for any scholarly non-commercial purpose with the condition that all publications of the contribution include a full citation to the journal as published by the Medical Research Archives.
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2. Liu, Q. R., Lopez-Corcuera, B., Mandiyan, S., Nelson, H., and Nelson, N. (1993) Cloning and expression of a spinal cord- and brain-specific glycine transporter with novel structural features. J Biol Chem 268, 22802-22808
3. Morrow, J. A., Collie, I. T., Dunbar, D. R., Walker, G. B., Shahid, M., and Hill, D. R. (1998) Molecular cloning and functional expression of the human glycine transporter GlyT2 and chromosomal localisation of the gene in the human genome. FEBS Lett 439, 334-340
4. Gether, U., Andersen, P. H., Larsson, O. M., and Schousboe, A. (2006) Neurotransmitter transporters: molecular function of important drug targets. Trends in Pharmacological Sciences 27, 375-383
5. Aragon, C., and Lopez-Corcuera, B. (2003) Structure, function and regulation of glycine neurotransporters. Eur J Pharmacol 479, 249-262
6. Eulenburg, V., Armsen, W., Betz, H., and Gomeza, J. (2005) Glycine transporters: essential regulators of neurotransmission. Trends Biochem Sci 30, 325-333
7. Gomeza, J., Ohno, K., Hulsmann, S., Armsen, W., Eulenburg, V., Richter, D. W., Laube, B., and Betz, H. (2003) Deletion of the mouse glycine transporter 2 results in a hyperekplexia phenotype and postnatal lethality. Neuron 40, 797-806
8. Szoke, K., Hartel, K., Grass, D., Hirrlinger, P. G., Hirrlinger, J., and Hulsmann, S. (2006) Glycine transporter 1 expression in the ventral respiratory group is restricted to protoplasmic astrocytes. Brain Res 1119, 182-189
9. Zafra, F., Aragon, C., Olivares, L., Danbolt, N. C., Gimenez, C., and Storm-Mathisen, J. (1995) Glycine transporters are differentially expressed among CNS cells. J Neurosci 15, 3952-3969
10. Lee, E. J., Mann, L. B., Rickman, D. W., Lim, E. J., Chun, M. H., and Grzywacz, N. M. (2006) AII amacrine cells in the distal inner nuclear layer of the mouse retina. J Comp Neurol 494, 651-662
11. Mohler, H., Boison, D., Singer, P., Feldon, J., Pauly-Evers, M., and Yee, B. K. (2011) Glycine transporter 1 as a potential therapeutic target for schizophrenia-related symptoms: evidence from genetically modified mouse models and pharmacological inhibition. Biochem Pharmacol 81, 1065-1077
12. Nunez, E., Perez-Siles, G., Rodenstein, L., Alonso-Torres, P., Zafra, F., Jimenez, E., Aragon, C., and Lopez-Corcuera, B. (2009) Subcellular localization of the neuronal glycine transporter GLYT2 in brainstem. Traffic 10, 829-843
13. Rees, M. I., Harvey, K., Pearce, B. R., Chung, S. K., Duguid, I. C., Thomas, P., Beatty, S., Graham, G. E., Armstrong, L., Shiang, R., Abbott, K. J., Zuberi, S. M., Stephenson, J. B., Owen, M. J., Tijssen, M. A., van den Maagdenberg, A. M., Smart, T. G., Supplisson, S., and Harvey, R. J. (2006) Mutations in the gene encoding GlyT2 (SLC6A5) define a presynaptic component of human startle disease. Nat Genet 38, 801-806
14. Eulenburg, V., Becker, K., Gomeza, J., Schmitt, B., Becker, C. M., and Betz, H. (2006) Mutations within the human GLYT2 (SLC6A5) gene associated with hyperekplexia. Biochem Biophys Res Commun 348, 400-405
15. Harvey, R. J., Topf, M., Harvey, K., and Rees, M. I. (2008) The genetics of hyperekplexia: more than startle! Trends Genet 24, 439-447
16. Miranda, M., Dionne, K. R., Sorkina, T., and Sorkin, A. (2007) Three Ubiquitin Conjugation Sites in the Amino Terminus of the Dopamine Transporter Mediate Protein Kinase C-dependent Endocytosis of the Transporter. Mol. Biol. Cell 18, 313-323
17. Dipace, C., Sung, U., Binda, F., Blakely, R. D., and Galli, A. (2007) Amphetamine induces a calcium/calmodulin-dependent protein kinase II-dependent reduction in norepinephrine transporter surface expression linked to changes in syntaxin 1A/transporter complexes. Mol Pharmacol 71, 230-239
18. Vargas-Medrano, J., Castrejon-Tellez, V., Plenge, F., Ramirez, I., and Miranda, M. (2011) PKCbeta-dependent phosphorylation of the glycine transporter 1. Neurochem Int 59, 1123-1132
19. Fernandez-Sanchez, E., Martinez-Villarreal, J., Gimenez, C., and Zafra, F. (2009) Constitutive and regulated endocytosis of the glycine transporter GLYT1b is controlled by ubiquitination. J Biol Chem 284, 19482-19492
20. Fornes, A., Nunez, E., Alonso-Torres, P., Aragon, C., and Lopez-Corcuera, B. (2008) Trafficking properties and activity regulation of the neuronal glycine transporter GLYT2 by protein kinase C. Biochem J 412, 495-506
21. Barrera, S. P., Castrejon-Tellez, V., Trinidad, M., Robles-Escajeda, E., Vargas-Medrano, J., Varela-Ramirez, A., and Miranda, M. (2015) PKC-Dependent GlyT1 Ubiquitination Occurs Independent of Phosphorylation: Inespecificity in Lysine Selection for Ubiquitination. PLoS One 10, e0138897
22. Fornes, A., Nunez, E., Aragon, C., and Lopez-Corcuera, B. (2004) The second intracellular loop of the glycine transporter 2 contains crucial residues for glycine transport and phorbol ester-induced regulation. J Biol Chem 279, 22934-22943
23. Ohno, K., Koroll, M., El Far, O., Scholze, P., Gomeza, J., and Betz, H. (2004) The neuronal glycine transporter 2 interacts with the PDZ domain protein syntenin-1. Mol Cell Neurosci 26, 518-529
24. Armsen, W., Himmel, B., Betz, H., and Eulenburg, V. (2007) The C-terminal PDZ-ligand motif of the neuronal glycine transporter GlyT2 is required for efficient synaptic localization. Mol Cell Neurosci 36, 369-380
25. Geerlings, A., Lopez-Corcuera, B., and Aragon, C. (2000) Characterization of the interactions between the glycine transporters GLYT1 and GLYT2 and the SNARE protein syntaxin 1A. FEBS Lett 470, 51-54
26. Geerlings, A., Nunez, E., Lopez-Corcuera, B., and Aragon, C. (2001) Calcium- and Syntaxin 1-mediated Trafficking of the Neuronal Glycine Transporter GLYT2. J. Biol. Chem. 276, 17584-17590
27. Quick, M. W. (2003) Regulating the conducting states of a mammalian serotonin transporter. Neuron 40, 537-549
28. Binda, F., Dipace, C., Bowton, E., Robertson, S. D., Lute, B. J., Fog, J. U., Zhang, M., Sen, N., Colbran, R. J., Gnegy, M. E., Gether, U., Javitch, J. A., Erreger, K., and Galli, A. (2008) Syntaxin 1A interaction with the dopamine transporter promotes amphetamine-induced dopamine efflux. Mol Pharmacol 74, 1101-1108
29. Horiuchi, M., Loebrich, S., Brandstaetter, J. H., Kneussel, M., and Betz, H. (2005) Cellular localization and subcellular distribution of Unc-33-like protein 6, a brain-specific protein of the collapsin response mediator protein family that interacts with the neuronal glycine transporter 2. J Neurochem 94, 307-315
30. Horiuchi, M., El Far, O., and Betz, H. (2000) Ulip6, a novel unc-33 and dihydropyrimidinase related protein highly expressed in developing rat brain. FEBS Lett 480, 283-286
31. Warrens, A. N., Jones, M. D., and Lechler, R. I. (1997) Splicing by overlap extension by PCR using asymmetric amplification: an improved technique for the generation of hybrid proteins of immunological interest. Gene 186, 29-35
32. Bradford, M. M. (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72, 248-254
33. Torres, G. E., Yao, W. D., Mohn, A. R., Quan, H., Kim, K. M., Levey, A. I., Staudinger, J., and Caron, M. G. (2001) Functional interaction between monoamine plasma membrane transporters and the synaptic PDZ domain-containing protein PICK1. Neuron 30, 121-134.
34. Macias, M. J., Wiesner, S., and Sudol, M. (2002) WW and SH3 domains, two different scaffolds to recognize proline-rich ligands. FEBS Lett 513, 30-37
35. Li, S. S. (2005) Specificity and versatility of SH3 and other proline-recognition domains: structural basis and implications for cellular signal transduction. Biochem J 390, 641-653
36. D'Amico, A., Soragna, A., Di Cairano, E., Panzeri, N., Anzai, N., Vellea Sacchi, F., and Perego, C. (2010) The surface density of the glutamate transporter EAAC1 is controlled by interactions with PDZK1 and AP2 adaptor complexes. Traffic 11, 1455-1470
37. Dinkel, H., Michael, S., Weatheritt, R. J., Davey, N. E., Van Roey, K., Altenberg, B., Toedt, G., Uyar, B., Seiler, M., Budd, A., Jödicke, L., Dammert, M. A., Schroeter, C., Hammer, M., Schmidt, T., Jehl, P., McGuigan, C., Dymecka, M., Chica, C., Luck, K., Via, A., Chatr-aryamontri, A., Haslam, N., Grebnev, G., Edwards, R. J., Steinmetz, M. O., Meiselbach, H., Diella, F., and Gibson, T. J. (2012) ELM—the database of eukaryotic linear motifs. Nucleic Acids Research 40, D242-D251
38. Saksela, K., and Permi, P. (2012) SH3 domain ligand binding: What's the consensus and where's the specificity? FEBS Lett 586, 2609-2614
39. Sorkina, T., Miranda, M., Dionne, K. R., Hoover, B. R., Zahniser, N. R., and Sorkin, A. (2006) RNA Interference Screen Reveals an Essential Role of Nedd4-2 in Dopamine Transporter Ubiquitination and Endocytosis. J. Neurosci. 26, 8195-8205
40. Vaughan, R. A., and Foster, J. D. (2013) Mechanisms of dopamine transporter regulation in normal and disease states. Trends Pharmacol Sci 34, 489-496
41. Shpak, M., Gentil, L. G., and Miranda, M. (2014) The origin and evolution of vertebrate glycine transporters. J Mol Evol 78, 188-193
42. Miranda, M., Wu, C. C., Sorkina, T., Korstjens, D. R., and Sorkin, A. (2005) Enhanced Ubiquitylation and Accelerated Degradation of the Dopamine Transporter Mediated by Protein Kinase C. J. Biol. Chem. 280, 35617-35624
43. Zeilhofer, H. U., Studler, B., Arabadzisz, D., Schweizer, C., Ahmadi, S., Layh, B., Bosl, M. R., and Fritschy, J. M. (2005) Glycinergic neurons expressing enhanced green fluorescent protein in bacterial artificial chromosome transgenic mice. J Comp Neurol 482, 123-141
44. Hornbeck, P. V., Zhang, B., Murray, B., Kornhauser, J. M., Latham, V., and Skrzypek, E. (2015) PhosphoSitePlus, 2014: mutations, PTMs and recalibrations. Nucleic Acids Res 43, D512-520
45. de Juan-Sanz, J., Zafra, F., Lopez-Corcuera, B., and Aragon, C. (2011) Endocytosis of the neuronal glycine transporter GLYT2: role of membrane rafts and protein kinase C-dependent ubiquitination. Traffic 12, 1850-1867
46. de Juan-Sanz, J., Nunez, E., Lopez-Corcuera, B., and Aragon, C. (2013) Constitutive endocytosis and turnover of the neuronal glycine transporter GlyT2 is dependent on ubiquitination of a C-terminal lysine cluster. PLoS One 8, e58863
47. Miranda, M., and Sorkin, A. (2007) Regulation of receptors and transporters by ubiquitination: new insights into surprisingly similar mechanisms. Mol Interv 7, 157-167
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