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Betaine-γ-aminobutyric Acid Transporter 1 (BGT-1/mGAT2) Interacts with the PDZ Domain of Munc-18 Interacting Proteins (Mints)

Betaine-γ-aminobutyric acid transporter 1 (BGT-1/mGAT2)과 Munc-18-interacting (Mint) 단백질의 PDZ 결합

  • Kim, Sang-Jin (Department of Neurology, College of Medicine, Inje University) ;
  • Jeong, Young-Joo (Department of Biochemistry, College of Medicine, Inje University) ;
  • Choi, Sun-Hee (Department of Biochemistry, College of Medicine, Inje University) ;
  • Choi, Chun-Yeon (Department of Biochemistry, College of Medicine, Inje University) ;
  • Jun, Hee-Jae (Departments of Thoracic and Cardiovascular Surgery, College of Medicine, Inje University) ;
  • Moon, Il-Soo (Departments of Anatomy, College of Medicine, Dongguk University) ;
  • Seog, Dae-Hyun (Department of Biochemistry, College of Medicine, Inje University) ;
  • Jang, Won-Hee (Department of Biochemistry, College of Medicine, Inje University)
  • 김상진 (인제대학교 의과대학 신경과학교실) ;
  • 정영주 (인제대학교 의과대학 생화학교실) ;
  • 최선희 (인제대학교 의과대학 생화학교실) ;
  • 최춘연 (인제대학교 의과대학 생화학교실) ;
  • 전희재 (인제대학교 의과대학 흉부외과학교실) ;
  • 문일수 (동국대학교 의과대학 해부학교실) ;
  • 석대현 (인제대학교 의과대학 생화학교실) ;
  • 장원희 (인제대학교 의과대학 생화학교실)
  • Received : 2012.08.23
  • Accepted : 2012.09.07
  • Published : 2012.09.30

Abstract

The action of neuronally released ${\gamma}$-aminobutyric acid (GABA) is terminated by uptake into the neurons by GABA transporters (GATs). The mechanism underlying the stabilization and regulation of GAT2 has not yet been elucidated. We used the yeast two-hybrid system to identify proteins that interact with and, thereby, regulate betaine-${\gamma}$-aminobutyric acid transporter 1 (BGT-1/mGAT2). We found an interaction between BGT-1/mGAT2 and Munc-18-interacting proteins (Mints). The "T-H-L" motif at the C-terminal end of BGT-1/mGAT2 was essential for the interaction with Mint2 in the yeast two-hybrid assay. Mint2 bound to the tail region of BGT-1/mGAT2, but not to other GAT members. When co-expressed in HEK-293T cells, Mint2 was co-immunoprecipitated with BGT-1/mGAT2. In addition, we demonstrated the cellular co-localization of BGT-1/mGAT2 and Mint2 in the cells. These results suggest that Mint2 contributes to the regulation of BGT-1/mGAT2.

${\gamma}$-Aminobutyric acid (GABA)는 신경세포 밖으로 분비된 후 GABA 수송체들(GATs)에 의하여 다시 신경세포 안으로 재흡수 된다. 그러나, GABA 수송체들이 어떻게 연접전막의 위치에 안정적으로 존재하는지 또한 어떤 단백질과 결합하여 조절을 받는지는 알려져 있지 않다. 본 연구에서 효모 two-hybrid system을 이용하여 betaine-${\gamma}$-aminobutyric acid transporter 1 (BGT-1/mGAT2)의 C-말단과 특이적으로 결합하는 Munc-18-interacting (Mint) 단백질을 분리하였다. BGT-1/mGAT2의 C-말단에 존재하는 "T-H-L" 아미노산배열은 Mint2와의 결합에 필수적으로 관여하였다. Mint2은 BGT-1/mGAT2와는 결합하지만, 다른 종류의 GAT와는 결합하지 않았다. 또한 HEK-293T 세포에 Mint2와 BGT-1/mGAT2을 동시에 발현시켜 면역침강한 결과 두 단백질은 같이 면역침강하였으며, 두 단백질은 세포 내에서 세포막 부위에 같이 존재함도 확인하였다. 이러한 결과들은 Mint2가 BGT-1/mGAT2와 결합하여 BGT-1/mGAT2을 조절하는 역할을 함을 시사한다.

Acknowledgement

Supported by : Inje University

References

  1. Ahn, J., Mundigl, O., Muth, T. R., Rudnick, G. and Caplan, M. J. 1996. Polarized expression of GABA transporters in Madin-Darby canine kidney cells and cultured hippocampal neurons. J. Biol. Chem. 271, 6917-6924. https://doi.org/10.1074/jbc.271.12.6917
  2. Amara, S. G. and Kuhar, M. J. 1993. Neurotransmitter transporters: recent progress. Annu. Rev. Neurosci. 16, 73-93. https://doi.org/10.1146/annurev.ne.16.030193.000445
  3. Biederer, T., Cao, X., Südhof, T. C. and Liu, X. 2002. Regulation of APP-dependent transcription complexes by Mint/X11s: differential functions of Mint isoforms. J. Neurosci. 22, 7340-7351.
  4. Biederer, T., Sara, Y., Mozhayeva, M., Atasoy, D., Liu, X., Kavalali, E. T. and Südhof, T. C. 2002. SynCAM, a synaptic adhesion molecule that drives synapse assembly. Science 297, 1525-1531. https://doi.org/10.1126/science.1072356
  5. Brown, A., Muth, T. and Caplan, M. 2004. The COOH-terminal tail of the GAT-2 GABA transporter contains a novel motif that plays a role in basolateral targeting. Am. J. Physiol. Cell Physiol. 286, C1071-C1077.
  6. Burnham, C. E., Buerk, B., Schmidt, C. and Bucuvalas, J. C. 1996. A liver-specific isoform of the betaine/GABA transporter in the rat: cDNA sequence and organ distribution. Biochim. Biophys. Acta. 1284, 4-8. https://doi.org/10.1016/0005-2736(96)00118-6
  7. Clausen, R. P., Frølund, B., Larsson, O. M., Schousboe, A., Krogsgaard-Larsen, P. and White, H. S. 2006. A novel selective gamma-aminobutyric acid transport inhibitor demonstrates a functional role for GABA transporter subtype GAT2/BGT-1 in the CNS. Neurochem. Int. 48, 637-642. https://doi.org/10.1016/j.neuint.2005.12.031
  8. Deken, S. L., Beckman, M. L., Boos, L. and Quick, M. W. 2000. Transport rates of GABA transporters: regulation by the N-terminal domain and syntaxin 1A. Nat. Neurosci. 3, 998-1003. https://doi.org/10.1038/79939
  9. Doyle, D. A., Lee, A., Lewis, J., Kim, E., Sheng, M. and MacKinnon, R. 1996. Crystal structures of a complexed and peptide-free membrane protein-binding domain: molecular basis of peptide recognition by PDZ. Cell 85, 1067-1076. https://doi.org/10.1016/S0092-8674(00)81307-0
  10. Gether, U., Andersen, P. H., Larsson, O. M. and Schousboe, A. 2006. Neurotransmitter transporters: molecular function of important drug targets. Trends Pharmacol. Sci. 27, 375-383. https://doi.org/10.1016/j.tips.2006.05.003
  11. Gomperts, S. N. 1996. Clustering membrane proteins: It's all coming together with the PSD-95/SAP90 protein family. Cell 84, 659-662. 2 https://doi.org/10.1016/S0092-8674(00)81043-0
  12. Guastella, J., Nelson, N., Nelson, H., Czyzyk, L., Keynan, S., Miedel, M. C., Davidson, N., Lester, H. A. and Kanner, B. I. 1990. Cloning and expression of a rat brain GABA transporter. Science 249, 1303-1306. https://doi.org/10.1126/science.1975955
  13. Iversen, L. 2006. Neurotransmitter transporters and their impact on the development of psychopharmacology. Br. J. Pharmacol. 147, Suppl. 1, S82-S88.
  14. Kennedy, M. B. 2000. Signal-processing machines at the postsynaptic density. Science 290, 750-754. https://doi.org/10.1126/science.290.5492.750
  15. Kim, S. J., Lee, C. H., Park, H. Y., Yea, S. S., Jang, W. H., Lee, S. K., Park, Y. H., Cha, O. S., Moon, I. S. and Seog, D. H. 2007. JSAP1 interacts with kinesin light chain 1 through conserved binding segments. J. Life Sci. 17, 889-895. https://doi.org/10.5352/JLS.2007.17.7.889
  16. Lau, K. F., McLoughlin, D. M., Standen, C. and Miller, C. C. 2000. X11 alpha and x11 beta interact with presenilin-1 via their PDZ domains. Mol. Cell Neurosci.16, 557-565. https://doi.org/10.1006/mcne.2000.0898
  17. Liu, Q. R., Mandiyan, S., Nelson, H. and Nelson, N. 1992. A family of genes encoding neurotransmitter transporters. Proc. Natl. Acad. Sci. USA 89, 6639-6643. https://doi.org/10.1073/pnas.89.14.6639
  18. Maximov, A., Südhof, T. C. and Bezprozvanny, I. 1999. Association of neuronal calcium channels with modular adaptor proteins. J. Biol. Chem. 274, 24453-24456. https://doi.org/10.1074/jbc.274.35.24453
  19. Nelson, N. 1998. The family of Na+/Cl- neurotransmitter transporters. J. Neurochem. 71, 1785-1803.
  20. Okamoto, M. and Südhof, T. C. 1997. Mints, Munc-18-interacting proteins in synaptic vesicle exocytosis. J. Biol. Chem. 272, 31459-31464. https://doi.org/10.1074/jbc.272.50.31459
  21. Radian, R., Ottersen, O. P., Storm-Mathisen, J., Castel, M. and Kanner, B. I. 1990. Immunocytochemical localization of the GABA transporter in rat brain. J. Neurosci. 10, 1319-1330.
  22. Roche, K. W., Standley, S., McCallum, J., Dune Ly, C., Ehlers, M. D. and Wenthold, R. J. 2001. Molecular determinants of NMDA receptor internalization. Nat. Neurosci. 4, 794-802. https://doi.org/10.1038/90498
  23. Roettger, V. R. and Amara, S. G. 1999. GABA and glutamate transporters: therapeutic and etiologic implications for epilepsy. Adv. Neurol.79, 551-560.
  24. Sambrook, J., Fritsch, E. F. and Maniatis, T. 1989. Molecular cloning: a laboratory manual. Cold Spring Habor Laboratory, Cold Spring Habor, New York.
  25. Schousboe, A., Larsson, O. M., Sarup, A. and White, H. S. 2004. Role of the betaine/GABA transporter (BGT-1/GAT2) for the control of epilepsy. Eur. J. Pharmacol. 500, 281-287. https://doi.org/10.1016/j.ejphar.2004.07.032
  26. Setou, M., Nakagawa, T., Seog, D. H. and Hirokawa, N. 2000. Kinesin superfamily motor protein KIF17 and mLin-10 in NMDA receptor-containing vesicle transport. Science 288, 1796-1802. https://doi.org/10.1126/science.288.5472.1796
  27. Sheng, M. and Sala, C. 2001. PDZ domains and the organization of supramolecular complexes. Annu. Rev. Neurosci. 24, 1-29. https://doi.org/10.1146/annurev.neuro.24.1.1
  28. Verhage, M., Maia, A. S., Plomp, J. J., Brussaard, A. B., Heeroma, J. H., Vermeer, H., Toonen, R. F., Hammer, R. E., van den Berg, T. K., Missler, M., Geuze, H. J. and Südhof, T. C. 2000. Synaptic assembly of the brain in the absence of neurotransmitter secretion. Science 287, 864-869. https://doi.org/10.1126/science.287.5454.864
  29. White, H. S., Watson, W. P., Hansen, S. L., Slough, S., Perregaard, J., Sarup, A., Bolvig, T., Petersen, G., Larsson, O. M., Clausen, R. P., Frolund, B., Falch, E., Krogsgaard-Larsen, P. and Schousboe, A. 2005. First demonstration of a functional role for central nervous system betaine/gamma- aminobutyric acid transporter (mGAT2) based on synergistic anticonvulsant action among inhibitors of mGAT1 and mGAT2. J. Pharmacol. Exp. Ther. 312, 866-874.