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dc.contributor.advisor吳億秀-
dc.contributor.author최성문-
dc.creator최성문-
dc.date.accessioned2016-08-26T12:08:06Z-
dc.date.available2016-08-26T12:08:06Z-
dc.date.issued2004-
dc.identifier.otherOAK-000000009791-
dc.identifier.urihttps://dspace.ewha.ac.kr/handle/2015.oak/191135-
dc.identifier.urihttp://dcollection.ewha.ac.kr/jsp/common/DcLoOrgPer.jsp?sItemId=000000009791-
dc.description.abstractThe syndecans are known to form homologous oligomers, which may be important for their functions. I have therefore determined the role of oligomerization of syndecan-2 and -4. A series of glutathione-S-transferase-syndecan-2 and -4 chimeric proteins showed that all syndecan constructs containing the transmembrane domain formed SDS-resistant dimers, but not those lacking it. SDS-resistant dimer formation was hardly seen in the syndecan chimeras where each transmembrane domain was substituted with that of platelet derived growth factor receptor (PDGFR). Increased MAP kinase activity was detected in HEK293T cells transfected with syndecan/PDGFR chimeras in a syndecan transmembrane domain-dependent fashion. The chimera-induced MAP kinase activation was independent of both ligand and extracellular domain, implying that the transmembrane domain is sufficient to induce dimerization/oligomerization in vivo. Furthermore, the syndecan chimeras were defective in syndecan-4-mediated focal adhesion formation, protein kinase Ca activation, and development of Xenopus laevis embryos, or syndecan-2-mediated cell migration. Taken together, these data suggest that the transmembrane domains are sufficient for inducing dimerization and that transmembrane domain-induced oligomerization is crucial for syndecan-2 and -4 functions.;Syndecan은 focal adhesion에 널리 존재하며 ECM이 매개하는 신호 전달을 조절하는 transmembrane heparin sulfate proteoglycan이다. Syndecan은 homologous dimer나 oligomer를 형성하는 것으로 알려져 있으며 이는 syndecan 의 기능을 조절하는데 매우 중요한 역할을 한다. 본 논문에서는syndecan-2와 syndecan-4의 transmembrane domain을 포함하는 여러 GST- fusion construct들이 SDS-resistant dimer를 형성하는 것을 in vitro에서 확인하였다. 이러한 SDS-resistant dimer 형성이 syndecan transmembrane 고유의 역할임을 증명하기 위해 syndecan-2와 -4의 transmembrane domain을 Platelet Derived Growth Factor (PDGFR)의 그것으로 각각 치환시켜준 경우 SDS- resistant dimer가 사라지는 것을 확인할 수 있었고, 또한 syndecan-2와 syndecan-4의 transmembrane domain에 있는 conserved glycines을 leucines으로 치환해주면 마찬가지 결과를 나타내는 것을 밝혀내었다. 이를 통해 syndecan-2와 syndecan-4의 dimerization에 있어서 transmembrane domain만으로도 충분히 이를 형성할 수 있다는 것을 확인할 수 있었다. In vivo에서 이를 증명하기 위해 syndecan-2와 -4의 transmembrane domain을 PDGFR의 그것으로 각각 치환시켜준 constructs를 Rat Embryo Fibroblasts (REF)와 Rat small Intestinal Epithelial cells 1 (RIE1) 에 transfection하여 syndecan 고유의 기능을 수행하는지 확인하였다. Transmembrane domain을 치환시켜 준 이 oligomerization mutants는 모두 wild type에 비해 제대로 된 syndecan 기능을 하지 못한다는 결과를 통해 in vivo에서도 syndecan-2와 -4의 dimerization뿐만 아니라 oligomerization에 transmembrane의 역할이 필수적인 것을 확인할 수 있었다. 또한 Xenopus laevis embryos에 syndecan-4 oligomerization mutant를 injection하면 posterior truncation을 관찰 할 수 있었고 이는 in vivo에서 transmembrane domain이 syndecan의 oligomerization을 조절할 수 있다는 큰 증거가 될 수 있었다. 이러한 모든 결과를 통해, in vitro 와 in vivo에서 syndecan-2와 syndecan-4의 dimerization과 oligomerization 형성에 transmembrane domain이 필수적이라는 것을 확인할 수 있었고, 무엇보다도 이 논문에서 보여주는 가장 중요한 것은 transmembrane domain 자체가 분명한 specificity를 갖는다는 것을 확인할 수 있었다는 데에 있다.-
dc.description.tableofcontentsCONTENTS Ⅰ. INTRODUCTION = 1 Ⅱ. MATERIALS AND METHODS = 8 1. Materials and antibodies = 8 2. cell culture and treatment = 8 3. Expression and purification of recombinant GST syndecan-2 and -4 core proteins = 9 4. Construction of expression vectors and transfection = 12 5. Analysis of proteins = 13 5.1 Immunoprecipitation and immunoblotting = 13 5.2 Protein quantification by BCA assay = 14 5.3 SDS-PAGE = 14 5.4 Western blotting = 15 6. Cell adhesion assay = 16 7. Microscopic analysis = 16 8. In vitro PKC activity assay = 17 9. GST-pulldown assays = 18 10. Migration assay = 18 11. Embryo injection = 18 Ⅲ. RESULTS = 20 1. Transmembrane domain-induced SDS-resistant dimerization of syndecan-2 and -4 = 20 1-1. Transmembrane domains are essential for SDS-resistant dimerization of syndecan-4 and syndecan-2 core proteins = 20 1-2. Conserved glycine residues in the transmembrane domain are crucial for SDS-resistant dimerization of both syndecan-2 and -4 = 23 1-3. The replacement of transmembrane domain of syndecans to that of PDGFR fails to form SDS-resistant dimerization = 26 2. Transmembrane domain-induced dimerization is independent of the extracellular domain = 29 3. Transmembrane domain-induced oligomerization is crucial for syndecan functions = 32 3-1. Transmembrane domain-induced oligomerization is crucial for syndecan-4 mediated focal adhesion formation and localization onto the cytoskeleton = 32 3-2. Transmembrane domain-induced oligomerization is crucial for the interaction with syntenin of syndecan-2 and -4 = 34 3-3. Syndecan-4 transmembrane domain-induced oligomerization is essential to interact and activate PKCα = 36 3-4. Transmembrane domain-induced oligomerization is crucial for the migratory ability of syndecan-2 = 38 3-5. Syndecan-4 oligomerization mutant causes posterior truncation in Xenopus embryos = 40 Ⅳ. DISCUSSION = 43 Ⅴ. REFERENCES = 50 논문개요 = 55-
dc.formatapplication/pdf-
dc.format.extent1086120 bytes-
dc.languageeng-
dc.publisher이화여자대학교 대학원-
dc.titleSpecific Role of Syndecan Transmembrane Domain-
dc.typeMaster's Thesis-
dc.title.translatedSyndecan transmembrane domain의 고유한 역할-
dc.format.pageviii, 58 p.-
dc.identifier.thesisdegreeMaster-
dc.identifier.major대학원 분자생명과학부-
dc.date.awarded2005. 2-
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