酸敏感G蛋白偶合受體功能上探討

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姓名

蘇禹軒(Yeu-shiuan Su) 查詢紙本館藏

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系統生物與生物資訊研究所

論文名稱

酸敏感G蛋白偶合受體功能上探討
(Functional analysis of proton-sensing G-protein-coupled receptors)

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摘要(中)

G蛋白偶合受體 (GPCRs)屬於一種具有七個穿膜區域的受體，並傳遞細胞外來各種不同的刺激至細胞內產生反應。目前市面上約有五成的藥物是以GPCRs為目標但這些作為目標的GPCRs只佔了全部GPCR家族數量的百分之十，表示仍然有百分之九十的GPCRs是有潛力可以做為未來藥物的目標。而無法在細胞外獲得足量的GPCRs蛋白並獲得其結晶的結構，一直是研究GPCRs結構上很大的障礙。更複雜的是，G蛋白偶合受體被發現會以形成同源或是異源聚合體的方式來行使其生理功能。而這些同源和異源聚合體之間，或是單體和聚合體之間在受到相同配位體刺激後，卻會有不同的反應產生。這些聚合體的形成表示G蛋白偶合受體可以透過這種方式來接受不同配位體的刺激，產生不同的訊息傳遞路徑。有4個被認為是酸敏感的G蛋白偶合受體 (OGR1、GPR4、G2A、TDAG8) 被發現會在pH6.8至pH6.4時被完全的活化，且在生理pH值的恆定以及酸所引起的疼痛上有著重要的角色。在這些受體中，DNA序列比對的結果發現G2A與其他三個受體相似性較低，且相較於OGR1受體在被認有五個與接受氫離子相關的histidne胺基酸中，G2A有四個是被其他胺基酸所取代的。且以酸刺激G2A受體大量表現的細胞，意外地發現G2A受體並不會產生任何明顯的反應，但OGR1受體卻可以。因此，對於G2A受體是否會接受氫離子刺激或是可能與其他受體 (例如:OGR1)以形成異源聚合體的方式來行使其功能，至經仍然不清楚。本論文的目的是 (1) 於細菌中表現酸敏感GPCRs並純化供結構分析及 (2) 利用與配位體結合後產生內在化的現象來探討G2A受體是否會受酸所刺激並透過FRET 接受體光漂白的技術來觀察在酸的刺激下G2A與OGR1受體是否會以形成異源聚合體來產生反應。在這邊發現 (1) 酸敏感GPCRs無法順利的在細菌系統中表現；(2) G2A受體不會受酸所刺激並內在化到細胞質內；(3) G2A與OGR1受體會形成異源聚合體並對酸刺激產生反應。

摘要(英)

G-protein-coupled receptors (GPCRs) that belong to seven transmembrane receptors, mediate a variety of extracellular signals to induce intracellular responses. Only 10% of GPCRs are targeted bt 50% of current marked drugs, emphasizing the potential of the remaining 90% of the GPCR superfamily for drug targets. The difficulties in large producing GPCRs in vitro and in generating crystal structures hinder structural studies of GPCRs. More complicatedly, GPCRs can form homo- or heteromers for function. The responses between homomers and heteromers or between oligomers and monomers are different while using the same agonist to stimulate. The oligomeric potential of GPCRs allows for more complex ligand-receptor relationships and signaling pathways. Four proton-sensing GPCRs (OGR1, GPR4, TDAG8, G2A) are identified to have fully activation at pH6.4~pH6.8 and important to pH homeostasis and acid-induced pain. Among these genes, primary sequence of G2A is less close to the others, and four of five critical histidine residues that are involved in pH sensing of OGR1 are replaced by other amino acids in G2A. Unexpectedly, G2A is the only proton-sensing GPCR that does not generate any significant responses after acid stimulation in cells overexpressing G2A gene, but did so in OGR1. Whether G2A does respond proton or forms heteromers with other family receptors (such as OGR1) to be functional, remains unclear. The objective of this thesis are (1) to purify proton-sensing GPCRs using bacteria expression system for structural analysis and (2) to explore whether G2A responds acid stimulation using ligand-mediated internalization technique and whether G2A form a functional heteromer with OGR1 to respond acid stimulation using FRET acceptor photobleaching technique. I have found that (1) bacteria expression system was not suitable for in vitro expression of proton-sensing GPCRs; (2) G2A did not internalize into the cytosol in response to acid stimulation, but OGR1 and TDAG8 did so; (3) G2A and OGR1 can form heteromers in resonse to acid stimulation.

關鍵字(中)

★ 酸敏感G蛋白偶合受體
★ 內在化
★ 聚合體

關鍵字(英)

★ oligomerization
★ FRET
★ internalization
★ g protein coupled receptors

論文目次

目錄
中文摘要 i
英文摘要 ii
致謝辭 iii
目錄 ……………………………………………………………………………..iv
圖目錄 ix
表目錄 x
縮寫與全名對照表 xi
第一章緒論 1
1-1 G蛋白偶合受體 (G-protein-coupled receptors, GPCRs) 1
1-1-1重要性 1
1-1-2結構與種類 1
1-1-3訊號傳遞路徑 2
1-1-4構型改變 (conformational change) 3
1-1-5內在化 (internalization) 4
1-1-6 寡聚物 (Oligomerization) 4
1-2痛覺 5
1-2-1組織發炎與酸化 6
1-3酸敏感G蛋白偶合受體 (proton-sensing G-protein-coupled receptors) 6
1-3-1 酸敏感G蛋白偶合受體家族 7
1-3-1-1 OGR1(ovarian cancer G-protein-coupled receptor 1) 7
1-3-1-2 GPR4 (G-protein-coupled receptor 4) 7
1-3-1-3 TDAG8 (T cell death-associated gene 8) 8
1-3-1-4 G2A (G2 phase accumulation) 8
1-3-2 酸敏感G蛋白偶合受體家族結構的差異 8
1-4 G蛋白偶合受體結構的分析 9
1-4-1 G蛋白偶合受體結構的分析方法 9
1-4-2 G蛋白偶合受體的表現與純化 10
1-5 研究動機 11
第二章實驗材料與方法 13
2-1實驗材料 13
2-1-1菌株 13
2-1-2表現載體 (Expression vector) 13
2-1-3藥品 13
2-1-4其他 13
2-1-5溶液 13
2-2實驗方法 14
2-2-1大腸桿菌轉型作用 (E. coli Transformation) 14
2-2-1-1勝任細胞的製備 14
2-2-1-2轉型作用 14
2-2-2細菌培養 14
2-2-2-1液體培養 14
2-2-2-2菌種保存 14
2-2-3質體DNA的製備 15
2-2-3-1小量質體製備方法 (mini-pre) 15
2-2-3-2大量質體製備方法 (midi-pre) 15
2-2-4基因轉殖 16
2-2-4-1引子 (primer)的設計 16
2-2-4-2聚合酵素連鎖反應 (Polymerase chain reaction, PCR) 17
2-2-4-3瓊脂膠體的製備及電泳 17
2-2-4-4膠體萃取法 (gel extraction) 17
2-2-4-5載體的製備 18
2-2-4-6轉殖基因的製備 18
2-2-4-7接合作用 (ligation) 18
2-2-5酸敏感G蛋白偶合受體蛋白在E. coli的表現 19
2-2-5-1 IPTG誘導作用 (Isopropylβ-D-1-thiogalactopyranoside induction) 19
2-2-5-2粗抽蛋白樣品製備 19
2-2-5-3 SDS蛋白質丙烯醛基氨基膠體電泳(SDS-PAGE) 19
2-2-5-4 SDS蛋白質丙烯醛基氨基膠體的染色 20
2-2-5-5西方墨點法 (for Bacteria) 20
2-2-6酸敏感G蛋白偶合受體蛋白在HEK293T細胞的表現 21
2-2-6-1細胞培養 21
2-2-6-2轉染作用(transfection) 21
2-2-6-2-1玻片(coverslip)的前處理 21
2-2-6-2-1轉染作用 21
2-2-6-3西方墨點法 (for HEK293T cell) 22
2-2-6-3-1樣品的製備 22
2-2-7酸敏感G蛋白偶合受體蛋白內在化現象實驗 22
2-2-7-1免疫染色法 (immunostaining) 22
2-2-7-2內在化 ( Internalization)的觀測方法 23
2-2-7-3內在化的計算方式 23
2-2-8 FRET (Fluorescence resonance energy transfer) by acceptor phtobleaching 23
第三章結果 25
3-1 pET32 與pET21表現載體在Rosetta-DE3宿主細胞表現的結果 25
3-2 mOGR1基因的長度可能影響到其蛋白質的表現 25
3-3 mTDAG8無法在同樣的表現載體pET32表現 26
3-4 pET21a載體也無法表現mTDAG8蛋白質 27
3-5接有HA-tag的酸敏感G蛋白偶合受體家族 27
3-6 Anti-HA的單株抗體確實可辨認接有HA-tag的酸敏感G蛋白偶合受體 27
3-7 mOGR1受體在pH值6.8的環境下刺激30分鐘相較pH7.6的原點有顯著內在化 (internalization)的情形 28
3-8 mTDAG8受體在pH 6.4環境下刺激45分鐘後相較pH 7.6原點有顯著的內在化情況產生 29
3-9 mGPR4受體在pH 7.6，pH 7.2，pH 6.8的環境下，無顯著內在化的情況出現 29
3-10 mG2A受體受pH 6.8、pH 6.4以及20μM SPC刺激30分鐘後，相較原點pH 7.6，無顯著內在化的情況產生 30
3-11共同表現mOGR1-pEGFP和mG2A-DsRed2蛋白的細胞受pH6.8緩衝溶液刺激時FRET Efficiency的值相較於原點pH7.6時有顯著的增加 31
3-12共同表現mOGR1-pEGFP和mG2A-DsRed2蛋白的細胞其FRET efficiency值與 donor/acceptor的比例無明顯的相關性 32
3-13共同表現mOGR1-pEGFP和mG2A-DsRed2蛋白的細胞其FRET efficiency值與acceptor的螢光值大小無明顯的相關性 32
第四章討論 33
4-1裝有酸敏感G蛋白偶合受體的pET32與pET21載體，無法在Rosetta與 33
BL21-DE3宿主細胞中表現 33
4-2 mOGR1，mTDAG8與mGPR4受到持續的氫離子刺激後，會產生內在化的 34
現象 34
4-3 mG2A與mOGR1受體共同表現在HEK293細胞時會形成異源聚合體，且數量會隨著氫離子濃度增加而上升 37
第五章參考文獻 40
附錄 74

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指導教授

孫維欣(Wei-hsin Sun)

審核日期

2010-8-9

推文