생명과학회지 (Journal of Life Science)

  • 제18권7호
  • /
  • Pages.940-946
  • /
  • 2008
  • /
  • 1225-9918(pISSN)
  • /
  • 2287-3406(eISSN)
한국생명과학회 (Korean Society of Life Science)
권호 표지
DOI QR코드

DOI QR Code

(γ-Aminobutyric acid transporter 2와 mammalian Lin-7의 PDZ결합

(γ-Aminobutyric Acid Transporter 2 Binds to the PDZ Domain of Mammalian Lin-7

  • 석대현 (인제대학교 의과대학 생화학교실) ;
  • 문일수 (동국대학교 의과대학 해부학교실)
  • Seog, Dae-Hyun (Departments of Biochemistry, College of Medicine, Inje University) ;
  • Moon, II-Soo (Departments of Anatomy, College of Medicine, Dongguk University)
  • 발행 : 2008.07.30
PDF 다운로드
⟨ 이전 논문 다음 논문 ⟩

초록

신경전달물질을 수송하는 신경전달물질 수송체는 연접전막에서 신경전달물질의 농도를 조절한다. 신경세포에 발현하는 GATs들은 연접에서 억제성 신경전달물질인 GABA의 재흡수에 관여한다. GAT2/BGT1가 어떻게 연접전막에 안정적으로 존재하는지, 어떤 결합단백질과 결합하여 조절을 받는지는 알려져 있지 않다. 본 연구에서 효모 two-hybrid system을 사용하여 GAT2의 C-말단과 특이적으로 결합하는 mammalian Lin-7 (MALS)-2을 분리하였다. GAT2의 C-말단에 존재하는 "T-X-L"아미노산 배열이 MALS-2와의 결합에 필수적으로 관여하였다. 또한 이 단백질간의 결합을 pull-down assay로 확인한 결과 MALS는 glutathione S-transferase (GST)와는 결합하지 않으나 GST-GAT2와는 결합하였다. 또한 생쥐의 뇌 균질액에서 GAT2는 MALS와 함께 침강함을 면역침강으로 확인하였다. 이러한 결과들은 MALS가 GAT2와 결합하여 GAT2를 연접전막에서 안정화시키는 역할을 함을 시사한다.

Neurotransmitter transporters, which remove neurotransmittesr from the synaptic cleft, are regulated by second messenger such as protein kinases and binding proteins. Neuronal ${\gamma}-aminobutyric$ acid transporters (GATs) are responsible for removing the inhibitory neurotransmitter ${\gamma}-aminobutyric$ acid (GABA) from the synaptic cleft. ${\gamma}-aminobutyric$ acid transporters 2 (GAT2/BGT1) is involved in regulating neurotransmitter recycling, but the mechanism how they are stabilized and regulated by the specific binding protein has not yet been elucidated. Here, we used the yeast two-hybrid system to identify the specific binding protein(s) that interacts with the C-terminal region of GAT2 and found a specific interaction with the mammalian LIN-7b (MALS-2). MALS-2 protein bound to the tail region of GAT2 but not to other GAT members in the yeast two-hybrid assay. The "T-X-L" motif at the C-terminal end of GAT2 is essential for interaction with MALS-2. In addition, this protein showed specific interactions in the glutathione S-transferase (GST) pull-down assay. An antibody to GAT2 specifically co-immunoprecipitated MALS associated with GAT2 from mouse brain extracts. These results suggest that MALS may stabilize GAT2 in brain.

키워드

참고문헌

  1. Ahn, J., O. Mundigl, T. R. Muth, G. Rudnick and M. J. Caplan. 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 M. J. Kuhar. 1993. Neurotransmitter transporters: recent progress. Annu. Rev. Neurosci. 16, 73-93. https://doi.org/10.1146/annurev.ne.16.030193.000445
  3. Borg, J. P., S. W. Straight, S. M. Kaech, M. de Taddeo-Borg, D. E. Kroon, D. Karnak, R. S. Turner, S. K. Kim and B. Margolis. 1998. Identification of an evolutionarily conserved heterotrimeric protein complex involved in protein targeting. J. Biol. Chem. 273, 31633-31636. https://doi.org/10.1074/jbc.273.48.31633
  4. Brown, A., T. Muth and M. Caplan. 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. https://doi.org/10.1152/ajpcell.00291.2003
  5. Burnham, C. E., B. Buerk, C. Schmidt and J. C. Bucuvalas. 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
  6. Clark, J. A., A. Y. Deutch, P. Z. Gallipoli and S. G. Amara. 1992. Functional expression and CNS distribution of a beta- alanine-sensitive neuronal GABA transporter. Neuron 9, 337-348. https://doi.org/10.1016/0896-6273(92)90172-A
  7. Clausen, R. P., B. Frolund, O. M. Larsson, A. Schousboe, P. Krogsgaard-Larsen and H. S. White. 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. Conti, F., A. Minelli and M. Melone. 2004. GABA transporters in the mammalian cerebral cortex: localization, development and pathological implications. Brain Res. Brain Res. Rev. 45, 196-212. https://doi.org/10.1016/j.brainresrev.2004.03.003
  9. Deken, S. L., M. L. Beckman, L. Boos and M. W. Quick. 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
  10. Doyle, D. A., A. Lee, J. Lewis, E. Kim, M. Sheng and R. MacKinnon. 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
  11. Garner, C. C., J. Nash and R. L. Huganir. 2000. PDZ domains in synapse assembly and signalling. Trends Cell Biol. 10, 274-280. https://doi.org/10.1016/S0962-8924(00)01783-9
  12. Gether, U., P. H. Andersen, O. M. Larsson and A. Schousboe. 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
  13. Giros, B., Y. M. Wang, S. Suter, S. B. McLeskey, C. Pifl and M. G. Caron. 1994. Delineation of discrete domains for substrate, cocaine, and tricyclic antidepressant interactions using chimeric dopamine-norepinephrine transporters. J. Biol. Chem. 269, 15985-15988.
  14. Gomperts, S. N. 1996. Clustering membrane proteins: It's all coming together with the PSD-95/SAP90 protein family. Cell 84, 659-662. https://doi.org/10.1016/S0092-8674(00)81043-0
  15. Guastella, J., N. Nelson, H. Nelson, L. Czyzyk, S. Keynan, M. C. Miedel, N. Davidson, H. A. Lester and B. I. Kanner. 1990. Cloning and expression of a rat brain GABA transporter. Science 249, 1303-1306. https://doi.org/10.1126/science.1975955
  16. Hata, Y., S. Butz and T. C. Sudhof. 1996. CASK: a novel dlg/PSD95 homolog with an N-terminal calmodulin- dependent protein kinase domain identified by interaction with neurexins. J. Neurosci. 16, 2488-2494. https://doi.org/10.1523/JNEUROSCI.16-08-02488.1996
  17. Hillier, B. J., K. S. Christopherson, K. E. Prehoda, D. S. Bredt and W. A. Lim. 1999. Unexpected modes of PDZ domain scaffolding revealed by structure of nNOS-syntrophin complex. Science. 284, 812-815. https://doi.org/10.1126/science.284.5415.812
  18. Ikegaki, N., N. Saito, M. Hashima and C. Tanaka. 1994. Production of specific antibodies against GABA transporter subtypes (GAT1, GAT2, GAT3) and their application to immunocytochemistry. Brain Res. Mol. Brain Res. 26, 47-54. https://doi.org/10.1016/0169-328X(94)90072-8
  19. Iversen, L. 2006. Neurotransmitter transporters and their impact on the development of psychopharmacology. Br. J. Pharmacol. 147, Suppl. 1, S82-S88.
  20. Iversen, L. and J. S. Kelly. 1975. Uptake and metabolism of gamma-aminobutyric acid by neurones and glial cells. Biochem. Pharmacol. 24, 933-938. https://doi.org/10.1016/0006-2952(75)90422-0
  21. Jasmin, L., M. V. Wu and P. T. Ohara. 2004. GABA puts a stop to pain. Curr. Drug Targets CNS Neurol. Disord. 3, 487-505. https://doi.org/10.2174/1568007043336716
  22. Jo, K., R. Derin, M. Li and D. S. Bredt. 1999. Characterization of MALS/Velis-1, -2, and -3: a family of mammalian LIN-7 homologs enriched at brain synapses in association with the postsynaptic density-95/NMDA receptor postsynaptic complex. J. Neurosci. 19, 4189-4199. https://doi.org/10.1523/JNEUROSCI.19-11-04189.1999
  23. Kandel, E. R., J. H. Schwartz and T. M. Jessell. 2000. Principles of Neural Science, Macgraw-Hill, New York.
  24. Kanner, B. I. 1989 Ion-coupled neurotransmitter transport. Curr. Opin. Cell Biol. 4, 735-738.
  25. Kennedy, M. B. 2000. Signal-processing machines at the postsynaptic density. Science 290, 750-754. https://doi.org/10.1126/science.290.5492.750
  26. Krnjevic, K. and S. Schwartz. 1967. The action of gamma- aminobutyric acid on cortical neurones. Exp. Brain Res. 3, 320-336. https://doi.org/10.1007/BF00237558
  27. Liu, Q. R., B. Lopez-Corcuera, S. Mandiyan, H. Nelson and N. Nelson. 1993. Molecular characterization of four pharmacologically distinct gamma-aminobutyric acid transporters in mouse brain. J. Biol. Chem. 268, 2106-2112
  28. Liu, Q. R., S. Mandiyan, H. Nelson and N. Nelson. 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
  29. McHugh, E. M., W. Zhu, S. Milgram and S. Mager. 2004. The GABA transporter GAT1 and the MAGUK protein Pals1: interaction, uptake modulation, and coexpression in the brain. Mol. Cell Neurosci. 26, 406-417. https://doi.org/10.1016/j.mcn.2004.03.006
  30. Nelson, H., S. Mandiyan and N. Nelson. 1990. Cloning of the human brain GABA transporter. FEBS Lett. 269, 181-184. https://doi.org/10.1016/0014-5793(90)81149-I
  31. Nelson, N. 1998. The family of Na+/Cl- neurotransmitter transporters. J. Neurochem. 71, 1785-1803. https://doi.org/10.1046/j.1471-4159.1998.71051785.x
  32. Olsen, O., K. A. Moore, R. A. Nicoll and D. S. Bredt. 2006. Synaptic transmission regulated by a presynaptic MALS/ Liprin-alpha protein complex. Curr. Opin. Cell Biol. 18, 223-227. https://doi.org/10.1016/j.ceb.2006.02.010
  33. Perego, C., A. Bulbarelli, R. Longhi, M. Caimi, A. Villa, M. J. Caplan and G. Pietrini. 1997. Sorting of two polytopic proteins, the gamma-aminobutyric acid and betaine transporters, in polarized epithelial cells. J. Biol. Chem. 272, 6584-6592. https://doi.org/10.1074/jbc.272.10.6584
  34. Perego, C., C. Vanoni, A. Villa, R. Longhi, S. M. Kaech, E. Frohli, A. Hajnal, S. K. Kim and G. Pietrini. 1999. PDZ-mediated interactions retain the epithelial GABA transporter on the basolateral surface of polarized epithelial cells. EMBO J. 18, 2384-2393. https://doi.org/10.1093/emboj/18.9.2384
  35. Radian, R., O. P. Ottersen, J. Storm-Mathisen, M. Castel and B. I. Kanner. 1990. Immunocytochemical localization of the GABA transporter in rat brain. J. Neurosci. 10, 1319-1330. https://doi.org/10.1523/JNEUROSCI.10-04-01319.1990
  36. Roche, K. W., S. Standley, J. McCallum, C. Dune Ly, M. D. Ehlers and R. J. Wenthold. 2001. Molecular determinants of NMDA receptor internalization. Nat. Neurosci. 4, 794-802. https://doi.org/10.1038/90498
  37. Roettger, V. R. and S. G. Amara. 1999. GABA and glutamate transporters: therapeutic and etiologic implications for epilepsy. Adv. Neurol. 79, 551-560.
  38. Sambrook, J., E. F. Fritsch and T. Maniatis. 1989. Molecular cloning: a laboratory manual. Cold Spring Habor Laboratory, Cold Spring Habor, New York.
  39. Scannevin, R. H. and R. L. Huganir. 2000. Postsynaptic organization and regulation of excitatory synapses. Nat. Rev. Neurosci. 1, 133-141. https://doi.org/10.1038/35039075
  40. Schousboe. A., O. M. Larsson and A. Sarup. 2004. White HS. 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
  41. Setou, M., T. Nakagawa, D. H. Seog and N. Hirokawa. 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
  42. Sheng, M. and C. Sala. 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
  43. Takeda, S., H. Yamazaki, D. H. Seog, Y. Kanai, S. Terada and N. Hirokawa. 2000. Kinesin superfamily protein 3 (KIF3) motor transports fodrin-associating vesicles important for neurite building. J. Cell Biol. 148, 1255-1265. https://doi.org/10.1083/jcb.148.6.1255
  44. Torres, G. E., W. D. Yao, A. R. Mohn, H. Quan, K. M. Kim, A. I. Levey, J. Staudinger and M. G. Caron. 2001. Functional interaction between monoamine plasma membrane transporters and the synaptic PDZ domain-containing protein PICK1. Neuron 30, 121-134. https://doi.org/10.1016/S0896-6273(01)00267-7
  45. Treiman, D. M. 2001. GABAergic mechanisms in epilepsy. Epilepsia. 42, Suppl. 3, 8-12.
  46. White, H. S., W. P. Watson, S. L. Hansen, S. Slough, J. Perregaard, A. Sarup, T. Bolvig, G. Petersen, O. M. Larsson, R. P. Clausen, B. Frolund, E. Falch, P. Krogsgaard-Larsen and A. Schousboe. 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.
  47. Wong, C. G., T. Bottiglieri and O. C. 3rd. Snead. 2003. GABA, gamma-hydroxybutyric acid, and neurological disease. Ann. Neurol. 54, Suppl. 6. S3-S12.