Full metadata record
DC Field | Value | Language |
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dc.contributor.author | 이은정 | - |
dc.creator | 이은정 | - |
dc.date.accessioned | 2016-08-26T03:08:33Z | - |
dc.date.available | 2016-08-26T03:08:33Z | - |
dc.date.issued | 2003 | - |
dc.identifier.other | OAK-000000003485 | - |
dc.identifier.uri | https://dspace.ewha.ac.kr/handle/2015.oak/194873 | - |
dc.identifier.uri | http://dcollection.ewha.ac.kr/jsp/common/DcLoOrgPer.jsp?sItemId=000000003485 | - |
dc.description.abstract | Syndecan는 ECM이 매개하는 신호전달을 조절하는 transmembrane heparan sulfate proteoglycan이다. 이러한 syndecan는 그 기능에 중요한 homologous monomer나 dimer 를 형성하는 것이 알려져 있다. 본 논문에서는 transmembrane domain을 포함하는glutathione-S-transferase-syndecan-2와 syndecan-4 core 단백질이 SDS-resistant dimer를 형성하는 것을 보여준다. 다시 말해, syndecan-2와 syndecan-4의 transmembrane domain이 SDS-resistant dimerization 에 중요하다는 것을 보여준다. 이 때extracellular domain과 cytoplasmic domain은 SDS-resistant dimerization 에 관여하지 않았다. In vitro에서 syndecan의 dimerization 에 관여하는 domain 을 연구한 데 이어 in vivo에서 중요하게 작용하는 domain을 알아보기 위해 syndecan과 platelet derived growth factor (PDGF) receptor 로 구성된 chimera를 제작하였다. syndecan-2와 syndecan-4의 cytoplasmic domain을 PDGF receptor로 치환한 cDNA를 세포에 과발현 시켰을 때 transmembrane domain을 통한 dimerization에 의해서 유도되는 MAP kinase 활성이 증가하였다. 이 chimera에 의한 MAP kinase의 활성 증가는 ligand와 extracellular domain에 무관했다. 이는 다음의 세 가지 실험을 통해서 알 수 있었다. (1) 특이적인 항체에 의한 oligomerization이나 PDGF가 chimera에 의한 MAP kinase활성에 영향을 주지 않았다. (2) chimera에 의한MAP kinase 활성은 suspension 상태의 세포에서도 비슷한 양상을 나타내었다. (3) PDGFR의 transmembrane domain만 syndecan-4로 치환한 chimera에서도 chimera에 의해서 유도되는 MAP kinase 활성은 증가하였다. 이러한 모든 결과를 통해, in vitro와 in vivo에서 syndecan-2와 syndecan-4의 dimerization 형성에 transmembrane domin이 필수적이라는 것을 알 수 있다. ; The syndecans, a major family of transmembrane cell surface heparan sulfate proteoglycans, regulate ECM-mediated signal transduction. They are known to form homologous dimers or multimers, which may be important for their functions. Here, I demonstrated that glutathione-S-transferase-syndecan-2 and -4 core proteins containing the transmembrane domain can form SDS-resistant dimers, implying that the transmembrane domain is sufficient for inducing SDS-resistant dimerization. Neither the extracellular domain nor the cytoplasmic domain was involved in SDS-resistant dimerization. Cells transfected with syndecan-2 and -4 cDNA of which each cytoplasmic domain was substituted with that of platelet derived growth factor (PDGF) receptor showed increased MAP kinase activity through the transmembrane domain induced dimerization. This chimera-induced MAP kinase activation was both ligand and extracellular domain-independent, since (1) neither the specific antibody-induced oligomerization nor PDGF affected the chimera-induced MAP kinase activation, (2) the chimera-induced MAP kinase activation was similarly increased in suspended cells, and (3) PDGF receptor chimera containing the transmembrane domain of syndecan-4 in itself was sufficient to induce MAP kinase activation. Therefore, it is likely that the transmembrane domains are sufficient for inducing functional dimer formation of syndecan-2 and -4. | - |
dc.description.tableofcontents | I. INTRODUCTION = 1 II. MATERIALS AND METHODS = 8 1. Materials and antibodies = 8 2. Instruments = 9 3. Cell culture and treatment = 9 4. Construction of expression vectors and transfection = 10 4.1 Construction of expression vectors = 10 4.2 Transient transfection = 11 5. Analysis of proteins = 11 5.1 Immunoprecipitation and immunoblotting = 11 5.2 Protein quantification by BCA assay = 12 5.3 SDS-PAGE = 13 5.4 Western blotting = 14 6. Cell proliferation assay : MTT assay 15 7. Expression and purification of GST fusion protein = 16 8. Plating experiment = 20 9. Cell free transcription and translation = 20 III. RESULTS = 22 1. Transmembrane domains are essential for SDS-resistant dimerization of syndecan-4 and syndecan-2 core proteins = 22 2. The ectodomain flanking regions are not essential for transmembrane domain induced dimerization = 27 3. Transmembrane domain is sufficient for inducing dimer formation of chimeric proteins = 28 4. The chimera enhances MAP kinase activity through MEK kinase activation = 35 5. Chimera-induced MAP kinase activation enhances cell proliferation = 39 6. Transmembrane domain-induced dimerization is independent of the extracellular domain = 42 7. Chimera-induced MAP kinase activation requires cytoskeleton organization = 48 IV. DISCUSSION = 51 V. REFERENCES = 58 논문개요 = 65 | - |
dc.format | application/pdf | - |
dc.format.extent | 1342275 bytes | - |
dc.language | eng | - |
dc.publisher | 이화여자대학교 대학원 | - |
dc.title | The function of transmembrane domain in syndecan-2 and -4 dimerization | - |
dc.type | Master's Thesis | - |
dc.identifier.thesisdegree | Master | - |
dc.identifier.major | 대학원 분자생명과학부 | - |
dc.date.awarded | 2003. 2 | - |