Journal of Life Science (생명과학회지)

Korean Society of Life Science (한국생명과학회)
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Interaction of GAT1 with Ubiquitin-Specific Protease Usp14 in Synaptic Terminal

GAT1과 ubiquitin-specific protease Usp14의 결합

  • Seog, Dae-Hyun (Department of Biochemistry, College of Medicine, Inje University) ;
  • Kim, Sang-Jin (Department of Neurology, College of Medicine, Inje University) ;
  • Joung, Young-Ju (Department of Biochemistry, College of Medicine, Inje University) ;
  • Yea, Sung-Su (Department of Biochemistry, College of Medicine, Inje University) ;
  • Park, Yeong-Hong (Department of Biochemistry, College of Medicine, Inje University) ;
  • Kim, Moo-Seong (Department of Neurosurgery, College of Medicine, Inje University) ;
  • Moon, Il-Soo (Departments of Anatomy, College of Medicine, Dongguk University) ;
  • Jang, Won-Hee (Department of Biochemistry, College of Medicine, Inje University)
  • 석대현 (인제대학교 의과대학 생화학교실) ;
  • 김상진 (인제대학교 신경과학교실) ;
  • 정영주 (인제대학교 의과대학 생화학교실) ;
  • 예성수 (인제대학교 의과대학 생화학교실) ;
  • 박영홍 (인제대학교 의과대학 생화학교실) ;
  • 김무성 (인제대학교 신경외과학교실) ;
  • 문일수 (동국대학교 의과대학 해부학교실) ;
  • 장원희 (인제대학교 의과대학 생화학교실)
  • Received : 2010.04.27
  • Accepted : 2010.05.27
  • Published : 2010.07.30
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Abstract

$\gamma$-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.

$\gamma$-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.

Keywords

References

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