Interaction of GAT1 with Ubiquitin-Specific Protease Usp14 in Synaptic Terminal

국문 초록 (Abstract)

γ-aminobutyric acid (GABA)는 중추신경계에서 억제성으로 작용하는 주요한 신경전달물질이다. GABA 수송체(GAT)는 연접간격에 존재하는 GABA를 세포 내로 재 흡수하여 GABA의 농도를 조절한다. 그런데 GABA 수송체가 어떻게 조절되는지는 아직 밝혀지지 않았다. 본 연구에서는 효모 two-hybrid system을 사용하여 뇌의 주요 GABA 수송체인 GAT1의 C-말단과 특이적으로 결합하는 ubiquitin-specific protease 14 (Usp14)를 분리하였다. Usp14는 GABA 수송체 GAT1및 GAT2와는 결합하지만, 다른 GAT isoform과는 결합하지 않았다. GAT1과의 결합에는 Usp14의 C-말단부위가 필수적으로 관여함을 확인하였다. 또한 이 단백질간의 결합을 GST pull-down assay로 확인하였으며, 생쥐 뇌 균질액의 co-immunoprecipitation을 통하여 in vivo에서도 GAT1과 Usp14가 결합함을 확인하였다. 이러한 결과들은 Usp14가 GAT1과 결합하여 세포막에 존재하는 GAT1의 수를 조절하는 역할을 할 가능성을 시사한다.

다국어 초록 (Multilingual Abstract)

γ-aminobutyric acid (GABA) is the major inhibitory neurotransmitter in the central nervous system. GABA transporters (GATs) control extracellular GABA levels by reuptake of released GABA from the synaptic cleft. However, how GATs are regulated has not yet been elucidated. Here, we used the yeast two-hybrid system to identify the specific binding protein(s) that interacts with the carboxyl (C)-terminal region of GAT1, the major isoform in the brain and find a specific interaction with the ubiquitin-specific protease 14 (Usp14), a deubiquitinating enzyme. Usp14 protein bound to the tail region of GAT1 and GAT2 but not to other GAT members in the yeast two-hybrid assay. The C-terminal region of Usp14 is essential for interaction with GAT1. In addition, these proteins showed specific interactions in the glutathione S-transferase (GST) pull-down assay. An antibody to GAT1 specifically co-immunoprecipitated Usp14 from mouse brain extracts. These results suggest that Usp14 may regulate the number of GAT1 at the cell surface.

목차 (Table of Contents)

• Introduction
• Materials and Methods
• Results
• Discussion
• Acknowledgement
• References
• 초록

참고문헌 (Reference)

• 1 Mukherjee, S., 77 : 759-803, 1997
• 2 Lappe-Siefke, C., "ataxia (axJ) mutation causes abnormal GABAA receptor turnover in mice" 9 : e1000631-, 2009
• 3 DiAntonio, A., "Ubiquitination-dependent mechanisms regulate synaptic growth and function" 412 : 449-452, 2001
• 4 Buttner, C., "Ubiquitination precedes internalization and proteolytic cleavage of plasma membrane-bound glycine receptors" 276 : 42978-42985, 2001
• 5 Hicke, L., "Ubiquitination of a yeast plasma membrane receptor signals its ligand-stimulated endocytosis" 84 : 277-287, 1996
• 6 Alves-Rodrigues, A., "Ubiquitin, cellular inclusions, and their role in neurodegeneration" 21 : 516-520, 1998
• 7 Ehlers, M. D., "Ubiquitin and synaptic dysfunction: ataxic mice highlight new common themes in neurological disease" 26 : 4-7, 2003
• 8 Yi, J. J., "Ubiquitin and protein turnover in synapse function" 47 : 629-632, 2005
• 9 Deken, S., "Transport rates of GABA transporters: regulation by the N-terminal domain and syntaxin 1A" 3 : 998-1003, 2000
• 10 Crimmins, S., "Transgenic rescue of ataxia mice with neuronal-specific expression of ubiquitin-specific protease 14" 26 : 11423-11431, 2006
• 11 Speese, S. D., "The ubiquitin proteasome system acutely regulates presynaptic protein turnover and synaptic efficacy" 13 : 899-910, 2003
• 12 Sato, K., "The recombinant GABA transporter GAT1 is downregulated upon activation of protein kinase C" 375 : 99-102, 1995
• 13 Nelson, N., "The family of Na+/Cl− neurotransmitter transporters" 71 : 1785-1803, 1998
• 14 Wilson, S. M., "Synaptic defects in ataxia mice result from a mutation in Usp14, encoding a ubiquitin-specific protease" 32 : 420-425, 2002
• 15 Hu, M., "Structure and mechanisms of the proteasome-associated deubiquitinating enzyme USP14" 24 : 3747-3756, 2005
• 16 Quick, M., "Second messengers, trafficking-related proteins, and amino acid residues that contribute to the functional regulation of the rat brain GABA transporter GAT1" 17 : 2967-2979, 1997
• 17 Haase, J., "Regulation of the serotonin transporter by interacting proteins" 29 : 722-728, 2001
• 18 Park, M., "Recycling endosomes supply AMPA receptors for LTP" 305 : 1972-1975, 2004
• 19 Beckman, M., "Protein kinase C regulates the interaction between a GABA transporter and syntaxin 1A" 18 : 6103-6112, 1998
• 20 Corey, J., "Protein kinase C modulates the activity of a cloned gamma-aminobutyric acid transporter expressed in Xenopus oocytes via regulated subcellular redistribution of the transporter" 269 : 14759-14767, 1994
• 21 Chiu, C., "Number, density, and surface/cytoplasmic distribution of GABA transporters at presynaptic structures of knock-in mice carrying GABA transporter subtype 1-green fluorescent protein fusions" 22 : 10251-10266, 2002
• 22 Kwong, W., "Neurotransmitters, neuropeptides and calcium binding proteins in developing human cerebellum: a review" 32 : 521-534, 2000
• 23 Bennett, E. R., "Mutation of arginine 44 of GAT-1, a (Na+ + Cl-)-coupled γ-aminobutyric acid transporter from rat brain, impairs net flux but not exchange" 275 : 34106-34113, 2000
• 24 Lin, C. I., "Modulation of the neuronal glutamate transporter EAAC1 by the interacting protein GTRAP3-18" 410 : 84-88, 2001
• 25 Augood, S., "Localization of GAT-1 GABA transporter mRNA in rat striatum: cellular coexpression with GAD67 mRNA, GAD67 immunoreactivity, and parvalbumin mRNA" 15 : 865-874, 1995
• 26 Isaacson, J., "Local and diffuse synaptic actions of GABA in the hippocampus" 10 : 165-175, 1993
• 27 Alwan, H. A., "Ligand-induced lysosomal epidermal growth factor receptor (EGFR) degradation is preceded by proteasome-dependent EGFR de-ubiquitination" 278 : 35781-35790, 2003
• 28 Engel, D., "Laminar difference in GABA uptake and GAT-1 expression in rat CA1" 512 : 643-649, 1998
• 29 Takeda, S., "Kinesin superfamily protein 3 (KIF3) motor transports fodrin-associating vesicles important for neurite building" 148 : 1255-1265, 2000
• 30 Setou, M., "Kinesin superfamily motor protein KIF17 and mLin-10 in NMDA receptor-containing vesicle transport" 288 : 1796-1802, 2000
• 31 Mager, S., "Ion binding and permeation at the GABA transporter GAT1" 16 : 5405-5414, 1996
• 32 Radian, R., "Immunocytochemical localization of the GABA transporter in rat brain" 10 : 1319-1330, 1990
• 33 Brown, A., "GIPC associates with GAT2 and may regulate its targeting by a novel mechanism.Mol.Biol.Cell 12,345a" 12 : 345a-, 2001
• 34 Minelli, A., "GAT-1, a high-affinity GABA plasma membrane transporter, is localized to neurons and astroglia in the cerebral cortex" 15 : 7734-7746, 1996
• 35 Chiu, C. S., "GABA transporter deficiency causes tremor, ataxia, nervousness, and increased GABA-induced tonic conductance in cerebellum" 25 : 3234-3245, 2005
• 36 Law, R., "Functional regulation of gamma-aminobutyric acid transporters by direct tyrosine phosphorylation" 23986-23991, 2000
• 37 Torres, G. E., "Functional interaction between monoamine plasma membrane transporters and synaptic PDZ domain-containing protein PICK1" 30 : 121-134, 2001
• 38 Soboleva, T. A., "Deubiquitinating enzymes: their functions and substrate specificity" 5 : 191-200, 2004
• 39 Chung, C. H., "Deubiquitinating enzymes: their diversity and emerging roles" 266 : 633-640, 1999
• 40 Sloan, J., "Cloning and functional expression of a human Na(+) and Cl(−)-dependent neutral and cationic amino acid transporter B(0+)" 274 : 23740-23745, 1999
• 41 Blakely, R. D., "Biogenic amine transporters: regulation in flux" 10 : 328-336, 2000
• 42 Clark, J., "Amino acid neurotransmitter transporters: structure, function, and molecular diversity" 15 : 323-333, 1993
• 43 Saliba, R. S., "Activity-dependent ubiquitination of GABA(A) receptors regulates their accumulation at synaptic sites" 27 : 13341-13351, 2007
• 44 Borodovsky, A., "A novel active site-directed probe specific for deubiquitylating enzymes reveals proteasome association of USP14" 20 : 5187-5196, 2001
• 45 Terrell, J., "A function for monoubiquitination in the internalization of a G protein-coupled receptor" 1 : 193-202, 1998
• 46 Liu, Q. R., "A family of genes encoding neurotransmitter transporters" 6639-6643, 1992