신경분화과정에서 Go와 Rit의 역활 규명 = Effect of Go and Rit on neuronal differentiation

다국어 초록 (Multilingual Abstract)

Heterotrimeric GTP binding proteins (G proteins) transduce signals initiated by a variety of hormones and neurotransmitters. Among G proteins, Go is one of the most abundant G proteins expressed in the brain and is classified as a member of the Gi/Go family due to its sequence homology to Gi proteins. To determine the function of the alpha subunit of Go in brain, We searched for Goα-interacting partners from human fetal cDNA library using yeast two hybrid screening. We identified a cDNA encoding 219 amino acids of Ras like protein in all tissue (Rit). We confirmed Goα::Rit interaction employing several biochemical analysis. Rit also preferentially bind the GoαQ205L, constitutively active form of Goα than wild type Goα. To determine the effects of Goα::Rit interaction on neuronal differentiation, we transfected expression vector for Goα or/and RitDN, dominant negative form of Rit. While the expression of GoαQ205L alone induced neurite outgrowth, coexpression of GoαQ205L and RitDN did not show such effect. To investigate the downstream signaling of Rit in neuronal differentiation, we search Ral-stat3 and ERK pathway. As results, Rit could not activate Ral-stat3 pathway and rather stimulated ERK phosphorylation. ERK phospholyrlation was increased by expression of GoαQ205L but decreased by coexpression of GoαQ205L and RitDN. These results suggested that activation of Goα increase the function by Rit as a ERK-dependent neuronal differentiation inducer.

목차 (Table of Contents)

• 제 1 장 서 론 1
• 제 1 절 Go 단백질 1
• 제 2 절 Ras-like protein in all tissue (Rit) 4
• 제 2 장 실험재료 및 방법 6
• 제 1 절 실험재료 6
• 1. 실험균주 및 동물 세포주, plasmid vector 6
• 1) 실험균주 6
• 2) 동물세포주 6
• 3) Plasmid vector 7
• 2. 세균배양 배지 및 조성 13
• 1) 액체 배지의 제조 13
• 2) 고체 평판 배지의 제조 14
• 3. 항체 16
• 제 2 절 실험방법 17
• 1. Plasmid DNA 순수 분리 17
• 2. 재조합 플라스미드의 제작 19
• 1) pcHA-Rit 19
• 2) GST-Rit 20
• 3) pcFLAG-Goα 21
• 4) pcFLAG-Giα 22
• 5) pcFLAG-Gsα 23
• 6) pcFLAG-Gqα 24
• 3. Site-directed mutagenesis 25
• 4. 동물세포 배양 26
• 5. Transfection 26
• 1) Calcium-phosphate method 26
• 2) PEI(Polyethylenimino) method 27
• 3) Metafectene method 27
• 6. Bradford assay 28
• 7. GST-pulldown assay 28
• 8. Co-immunoprecipitation 29
• 9. Western blot analysis 30
• 10. Neurite outgrowth 33
• 11.Yeast two hybrid 34
• 1) Bait DNA transformation 34
• 2) Library DNA transformation 35
• 3) Library DNA efficiency 측정 36
• 4) Positive colony의 선별 37
• 5) Yeast plasmid DNA isolation 37
• 6) Electro-transformation into bacterial cell 39
• 7) Bacterial plasmid DNA isolation 40
• 40
• 제 3 장 결과 및 고찰 41
• 제 1 절 Yeast two hybrid screening을 통한 Goα와 상호작용하는 단백질의 탐색 41
• 1. Yeast two hybrid screening을 통한 Rit의 발굴 41
• 2. In vitro assay를 통한 Goα와 Rit의 상호작용확인 44
• 3. Goα의 GTPase domain과 Rit의 상호작용 46
• 4. Goα의 활성상태에 따른 Rit의 상호작용 48
• 5. 다른 종류의 Gα 단백질과 Rit과의 상호작용 49
• 6. Gα 단백질과 Rit의 직접적 상호작용 50
• 제 2 절 Go/iα와 Rit의 세포내 신호전달 경로 52
• 1. Go/iα의 신경세포 분화 조절 신호경로 52
• 2. Go/iα의 신경세포 분화 조절 신호경로에서 Rit의 역할 53
• 3. Cannabinoid receptor의 신경세포 분화 조절 신호경로에서 54
• 4. RitDN에 의한 p-ERK억제 56
• 5. RitDN에 의한 신경분화 억제 57
• 제 4 장 고찰 59
• 제 5 장 결론 63
• 참 고 문 헌 64