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研究生:姜念漪
研究生(外文):Nien Yi Chiang
論文名稱:探討人類腦部前額疾病BFPP的致病基因GPR56的功能與結構的特性
論文名稱(外文):Structural and functional analysis of GPR56 protein in human frontal cortex developmental disease BFPP: The role of disease-associated mutations in receptor trafficking and functions
指導教授:林錫賢
指導教授(外文):H. H. Lin
學位類別:碩士
校院名稱:長庚大學
系所名稱:基礎醫學研究所
學門:醫藥衛生學門
學類:醫學學類
論文種類:學術論文
論文出版年:2008
畢業學年度:96
論文頁數:114
中文關鍵詞:G蛋白質聯繫接受器56雙側額頂多小腦迴畸形G蛋白質聯繫接受器蛋白構造自動裂解點配體
外文關鍵詞:GPR56bilateral frontoparietal polymicgyriaBFPPGPSligand
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G蛋白質聯繫接受器56 (GPR56) 又稱為TM7XN1,GPR56系屬於G蛋白質聯繫接受器家族中LNB-TM7次家族最近新發現的成員,其重要角色是參與神經前驅細胞 (neural progenitor cells) 的發育,而且也被發現和人類腦部發育畸形有關。GPR56的蛋白質結構不同於典型的G蛋白質聯繫接受器是擁有一個很長N端的細胞外區域 (~381胺基酸) 和一個包含四個多胱胺酸 (Cysteine) 的G蛋白質聯繫接受器蛋白構造自動裂解點 (GPS motif),其位置很靠近第一個穿細胞膜區域。GPR56的N端細胞外區域包含很多可能的N-和O-醣苷化點,此特徵非常相似黏蛋白 (mucin-like proteins)。這些GPR56的特徵被認為具有參與細胞和細胞或細胞和細胞外基質的交互作用的功能。GPR56基因的突變會引起人類腦部發育畸形,此疾病稱為雙側額頂多小腦迴畸形 (bilateral frontoparietal polymicgyria, BFPP)。雙側額頂多小腦迴畸形疾病的特徵是會嚴重破壞前額皮質層,雙側額頂多小腦迴畸形病人的病徵會有癲癇、智能缺陷、語言損傷和行動發育遲緩。目前為止,這些GPR56基因突變如何影響正常GPR56的功能仍然是不清楚。我們已經製備了野生型GPR56的cDNA和疾病相關GPR56 N端細胞外區域的點突變 (R38W, Y88C, and C91S), 穿膜蛋白細胞外環區域的點突變 (R565W and L640R)和G蛋白質聯繫接受器蛋白構造自動裂解區域的點突變 (C346S and W349S)。我們主要目標是研究這些和疾病相關的GPR56點突變是如何影響GPR56細胞內的運輸和細胞表面的表現。到目前為止,我們已經解釋了野生型GPR56和點突變GPR56其生化特性跟蛋白質運輸的不同。我們發現點突變GPR56會降低細胞內運輸的效率, 降低細胞表面表現量和破壞G蛋白質聯繫接受器蛋白構造自動裂解效率而導致GPR56蛋白質都停留在內質網。此外,我們已經製造GPR56細胞外區域的重組蛋白質當作探針去從各種細胞株中找出可能的配體 (ligands)。更重要地是,我們已經發展出一個鍵結分析法 (Binding assay)可以確認GPR56新的可能配體。我們也已經證明此新GPR56的配體是不同於已知的GPR56配體轉谷氨酰胺酶2 (transglutaminase 2,Tg2)。在為來的研究中我們想去確認此新GPR56的配體是否和GPR56引起雙側額頂多小腦迴畸形疾病有關聯。
G protein coupled receptor 56 (GPR56, also known as TM7XN1) is a newly discovered G-protein-coupled receptor (GPCR) of the LNB-TM7 family that has a role in the development of neural progenitor cells and has been linked to the developmental malformations of human brain. GPR56 diverges from other typical GPCRs in that it has an extremely large N-terminal extracellular region (381 amino acids) and contains a novel protein motif, consisting of four cysteine residues that define a GPCR proteolytic site (GPS motif) located just before the first transmembrane spanning domain. The rest of the amino-terminal domain contains a large number of possible N- and O-linked glycosylation sites similar to mucin-like proteins. These features suggest a role in cell–cell, or cell–matrix interactions. Mutations in the gene encoding GPR56 cause a human brain cortical malformation called bilateral frontoparietal polymicrogyria (BFPP). BFPP is characterized by disorganized cortical lamination that is most severe in frontal cortex. Individuals with BFPP suffer from epilepsy, mental retardation, language impairment and motor developmental delay. However, it remains unclear how these mutations affect GPR56 function. We cloned the wild type GPR56 cDNA and disease-associated GPR56 mutants in the tip of the N terminal domain R38W, Y88C, and C91S, two mutations in the extracellular loop domain R565W, L640R, and the two mutations in the GPS domain C346S and W349S. We aim to investigate how the BFPP-associated mutations affect the intracellular trafficking and cell surface expression of GPR56. To this end, we examine the biochemical properties and protein trafficking of wild type and mutant GPR56. We found that disease-associated GPR56 missense mutations produce proteins with reduced intracellular trafficking and poor cell surface expression or impaired GPS cleavage that fail to traffic beyond the ER. Furthermore, we has produced recombinant GPR56 extracellular domain as a protein probe for ligand search on various cell lines. More importantly, we have developed a binding assay that has identified a potential novel cellular ligand for GPR56. We have tentative evidence that it is different from the known GPR56 ligand, TG2. We hope to reveal the identity of the novel ligand and its role in BFPP.
指導教授推薦書
口試委員會審定書
授權書 iii
致謝 iv
中文摘要 v
Abstract vii
Abbreviations xiii

CHAPTER I Introduction - 1 -
1.1 G protein-coupled receptors - 1 -
1.2 LNB-TM7 receptors - 2 -
1.3 The autoproteolysis at the GPS domain of the LNB-TM7 receptors - 4 -
1.4 GPR56 - 5 -
1.5 Bilateral frontoparietal polymicrogyria (BFPP) - 8 -
1.6 GRP78/BIP and ER Stress-induced Cell Death - 9 -
1.7 Tissue transglutaminase - 10 -
1.8 Specific Aims - 11 -
CHAPTER II Materials and methods - 13 -
2.1. General Materials - 13 -
2.1-1. General chemicals and solution - 13 -
2.1-2. Molecular biology reagents - 13 -
2.1-3. General solutions - 13 -
2.2. DNA methods - 15 -
2.2-1. Generation of GPR56 site directed mutant constructs - 15 -
2.2-2. Polymerase chain reaction (PCR) - 16 -
2.2-3. Colony PCR - 17 -
2.2-4. Advantage high-fidelity PCR - 18 -
2.2-5. Agarose gel electrophoresis - 19 -
2.2-6. TOPO cloning ligation - 19 -
2.2-7. Restriction enzyme digestion - 20 -
2.2-8. Plasmid DNA precipitation and purification - 21 -
2.2-9. Purification of DNA from agarose gels - 22 -
2.2-10. DNA ligation - 22 -
2.2-11. Competent cells preparation - 23 -
2.2-12. Transformation - 23 -
2.2-13. Sequencing - 24 -
2.2-14. Mini and Midi/Maxi plasmid DNA precipitation - 24 -
2.2-15. Freezing transformated bacteria - 24 -
2.3 DNA expression constructs - 24 -
2.3-1. Generation of site directed mutant constructs (overlapping PCR) - 24 -
2.3-2. Construction of expression vectors - 25 -
2.4. Cell culture and transfection - 28 -
2.4-1. Cell lines - 28 -
2.4-2. Thawing cells - 30 -
2.4-3. Freezing cells - 30 -
2.4-4. Cell line subculture - 31 -
2.4-5. Transient transfection of cell lines using calcium phosphate precipitation - 32 -
2.4-6. Transient transfection of cell lines using LipofectamineTM - 33 -
2.5. Protein methods - 33 -
2.5-1. Cell lysates with modified RIPA lysis buffer - 33 -
2.5-2. Protein quantification - 34 -
2.5-3. Cell conditioned medium collection - 34 -
2.5-4. Western blotting - 34 -
2.5-5. Purification of mFc-fusion protein by Protein G Affinity Chromatography - 35 -
2.5-6. Deglycosylation treatment - 36 -
2.5-7. Protease inhibitor treatment - 37 -
2.6. Flow cytometry (FACS) analysis - 38 -
2.7. Dynabead Protein A Cell binding assay - 39 -
2.8. Cellular ligand-binding assay - 39 -
CHAPTER III Results - 41 -
3.1. Wild-type GPR56 protein is indeed cleaved at the GPS site and the BFPP-causing point mutations either affect the GPS cleavage or affect the expression level but not GPS cleavage - 41 -
3.2. BFPP-causing point GPR56 mutations affect cell surface expression - 43 -
3.3. N terminal fragment (α-subunit) Glycosylation of GPR56 - 44 -
3.4. Endo Hf digestion of GPR56 protein - 46 -
3.5. Putative GPR56 ligand(s) on glioma cells - 47 -
3.6. Identification of a putative cellular ligand using the GPR56-ECD-mFc protein probes in a FACS-based assay - 47 -
3.7. The mutant GPR56-mFc proteins do not interact with the novel GPR56 ligan - 48 -
3.8. Divalent cations-dependent binding of GPR56 to its novel cellular ligand - 49 -
3.9. GPR56 binds well to mouse TG2 it does not bind to human TG2 on GPR56-ligand positive cells HT-1080 and M059K cells - 50 -
3.10. Wild-type GPR56 is detected in conditioned media - 50 -
CHAPTER IV Discussion - 53 -
CHAPTER V References - 59 -
CHAPTER VI Figures - 62 -
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