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dc.contributor.author권수진-
dc.creator권수진-
dc.date.accessioned2016-08-26T10:08:13Z-
dc.date.available2016-08-26T10:08:13Z-
dc.date.issued2003-
dc.identifier.otherOAK-000000033626-
dc.identifier.urihttp://dspace.ewha.ac.kr/handle/2015.oak/200599-
dc.identifier.urihttp://dcollection.ewha.ac.kr/jsp/common/DcLoOrgPer.jsp?sItemId=000000033626-
dc.description.abstractTransmembrane heparan sulfate proteoglycan의 일종인 syndecan은 세포 증식 및 focal adhesion 과 actin filaments 형성 등의 여러 가지 세포 내 기능 조절에 관여한다. 특히 syndecan-4는 cytoplasmic domain을 통해phosphatidylinositol-4, 5-bisphosphate (PIP2)와 결합하며 그 결합이 syndecan-4의 기능에 매우 중요하다고 알려져 있다. 그러나 syndecan-4와 PIP2 사이의 상호관계는 분명하게 밝혀져 있지 않다. PIP2와 특이적인 결합을 갖는 것으로 알려진 phospholipaseCδ(PLCδ)의 pleckstrin Homology (PH) domain에 형광을 접목한 DNA를 세포 내로 transfection 하여 형광을 통해 in vivo내 plasma membrane에서의 PIP2 양변화를 확인 함으로써 syndecan-4와 PIP2 사이의 관계를 조사하였다. Syndecan-4 mutant와 syndecan-2를 transfection 한 세포와는 달리, syndecan-4를 과발현시킨 세포의 경우 plasma membrane으로의 GFP-δ PH의 이동이 증가됨을 관찰하였다. 그리고, PI3K inhibitor (LY294002)를 통해 plasma membrane에서의 PIP2양의 부가적인 증가를 볼 수 있었으나 PLC inhibitor (U73122)의 경우에는 그런 현상이 관찰되지 않았다. 이를 통해 PI3K를 통한 PIP2 대사분해과정 보다는 PLC를 통한 분해과정에 syndecan-4가 관여함을 알 수 있었다. 마찬가지로 EGF 혹은 ionomycin에 의한 분해과정에서도 syndecan-4의 과발현이 PIP2의 대사분해를 지연시키고 있음을 보였다. 이 연구 과정의 모든 실험결과는 syndecan-4와 PIP2 사이의 결합이 plasma membrane에서의 PIP2양을 조절하며 또한 각각의 기능을 상호 조절 할 수 있음을 나타내고 있다.;Syndecan-4, a transmembrane heparan sulfate proteoglycan, participates in a variety of cellular functions including cell proliferation, formation of focal adhesion and organization of actin filaments. It has been shown that syndecan-4 cytoplasmic domain interacts with phosphatidylinositol-4, 5-bisphosphate (PIP2) and this interaction is crucial for its functions. However, the nature of interplay between syndecan-4 and PIP2 remains unclear. Data using GFP-Pleckstrin Homology (PH) domain from phospholipase Cδ(PLCδ) that interacts specifically with PIP2 showed clear correlation between amount of PIP2 and membrane localization of GFP-PH in vivo. Overexpression of syndecan-4 cDNA, but neither syndecan-4 mutants nor syndecan-2, enhanced membrane targeting of GFP-PH in exponentially growing cells, implying that syndecan-4 induces increase of PIP2 level in the plasma membrane. In syndecan-4 overexpressing cells, the level of PIP2 was further increased in response to phosphoinositide-3-kinase (PI3K) inhibitors LY294002, but not phospholipase C inhibitor U73122, indicating involvement of syndecan-4 on PLC-mediated hydrolysis of PIP2, rather than PI3K-mediated metabolic modification. Consistently, both ionomycin and epidermal growth factor (EGF) -stimulated hydrolysis of PIP2 were delayed by syndecan-4 overexpression. Therefore, syndecan-4 seems to regulate amount of PIP2 in the plasma membrane through inhibiting metabolic degradation of PIP2 by PLC. All these data suggest that there are mutual regulatory mechanisms in the plasma membrane where syndecan-4 and PIP2 cooperatively regulates their own functions.-
dc.description.tableofcontentsABSTRACT = 8 I. INTRODUCTION = 10 II. MATERIALS AND METHODS = 19 1. Materials and antibodies = 19 2. Cell culture = 19 3. DNA constructs = 20 4. Transfection and reagent treatment = 20 5. PIP2 permeabilization assay = 20 6. Fluorescence microscopy = 21 7. Subcellular fractionation and Immunoblotting = 21 8. In vitro PI3K assay = 22 9. In vitro PLC assay = 23 10. In vivo PLC activity assay = 24 III. RESULTS = 26 1. The PH domain of PLCδ indicates the amount of phosphatidylinositol-4, 5-bisphosphate. = 26 2. Syndecan-4 enhances membrane targeting of GFP-PLCδ PH domain. = 30 3. Syndecan-4 stabilizes PIP2 in the plasma membrane. = 33 4. Syndecan-4 participates in metabolism of PIP2. = 35 5. Syndecan-4 induces retardation of EGF-mediated PIP2 hydrolysis = 38 6. Syndecan-4 delays metabolic degradation of exogenous PIP2 = 41 7. Syndecan-4 might modulate the PLC-mediated degradation of PIP2. = 43 8. Syndecan-4 has little effect on either PLC or PI3K activity. = 46 9. Increased PIP2 by syndecan-4 contributes to PKCα membrane localization and focal adhesion formation. = 49 IV. DISCUSSION = 52 V. REFERENCES = 60 논문 개요 = 71-
dc.formatapplication/pdf-
dc.format.extent559778 bytes-
dc.languageeng-
dc.publisher이화여자대학교 대학원-
dc.titleThe role of syndecan-4 on metabolic degradation of Phosphatidylinositol-4,5- bisphosphate (PIP2)-
dc.typeMaster's Thesis-
dc.format.pagevi, 63 p.-
dc.identifier.thesisdegreeMaster-
dc.identifier.major대학원 분자생명과학부-
dc.date.awarded2004. 2-
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