博碩士論文 982204018 詳細資訊


姓名 彭明隆(Ming-long Peng)  查詢紙本館藏   畢業系所 生命科學系
論文名稱 酸敏感G蛋白偶合受體功能上的拮抗機制
(Functional antagonism of proton-sensing G-protein-coupled-receptors)
檔案 [Endnote RIS 格式]    [Bibtex 格式]    至系統瀏覽論文 ( 永不開放)
摘要(中) G 蛋白偶合受體 (G Protein Coupled Receptor) 是人體中最大的一群表現在細胞膜上的受體家族,它與配體 (Ligand) 結合後產生的訊息傳遞路徑調控了多種重要的人體生理功能。而目前已經開發的 GPCR 作為標靶藥物的僅佔人體內所有GPCR 家族成員的 5~6%,顯示 GPCR 在新藥研究領域中仍具有非常大的開發潛力。而最近幾年研究中提出GPCR中OGR1 subfamily (TDAG8、OGR1、GPR4、G2A)能雙配對 (Double-pairing)兩種不同的化學物質,分別是質子和lysolipid。其中目前對於T-cell death-associated gene 8 (TDAG8)受體目前是受爭議性的,有人發現在單獨使用氫離子來刺激TDAG8受體時,發在在pH值接近6.2時有發現有最大量環單磷酸腺苷(cAMP)的累積;但也有人發現在透過半乳糖腦苷脂(psychosine)刺激TDAG8受體時,有發現在鈣離子的累積。所以說明氫離子和半乳糖腦苷脂為TDAG8受體的配體。是屬於促進劑(agonist)的角色。但特別的另外也有人發現在含有半乳糖腦苷脂的環境下,氫離子所引發的環單磷酸腺苷的量有受到抑制的情形,推測半乳糖腦苷脂可能為抑制劑(antagonist)的角色。所以在本篇論文主要是探討半乳糖腦苷脂在何種環境下對TDAG8受體,為促進劑或者抑制劑的角色。而在結果部分發現到,透過RNAi方式半乳糖腦苷脂確實能刺激活化TDAG8,但也發現在小鼠神經瘤母細胞上,對於半乳糖腦苷脂和氫離子的刺激,有相互抑制的情形;另外也觀察到對於氫離子的刺激主要是走Gq反應途徑,半乳糖腦苷脂則為Gi。
摘要(英) T-cell death-associated gene 8 (TDAG8) is a member of proton-sensing G-protein coupled receptors, which are sensitive to acid stimulation. TDAG8 can also respond to the lysophospholipid, 1-β-D-Galactosylacyosylsphingosine (Psychosine), an intermediate of cerebrosides biosynthesis. A model was proposed in which the receptors have two ligand-binding sites, one for protons and the other for lipids. The liplids were suggested to interact with both sites, as agonist and antagonist, respectively. The aim of this study is toinvestigate whether psychosine acts on TDAG8 as an agonist and antagonized proton response. We stimulated cells with psychosine in the presence or absence of proton. Cells were stimulated with psychosine in presence or the absence of proton. The results have demonstrated that psychosine in presence or the absence of proton. The results have demonstrated that psychosine can act on TDAG8. Psychosine can inhibit proton-induced [Ca2+] increase in N2A cells, and vice versa.
關鍵字(中) ★ G蛋白偶合受體
★ 氫離子
★ 半乳糖腦苷脂
關鍵字(英) ★ psychosine
★ G-protein-coupled-receptors
★ proton
論文目次 第一章 緒論………………………………………………………………………………1
1.1 G蛋白偶合受體 (G-protein-coupled receptors, GPCRs)…………………………2
1.1.1生理功能及其重要性………………………………………………………………2
1.1.2結構與功能性…………………………………………………………………2
1.1.3活化機制與下游反應…………………………………………………………3
1.1.4酸敏感G蛋白偶合受體(Proton sensing G-protein-coupled receptors)……4
1.2受體的雙配對(Double-paring)機制…………………………………………………4
1.2.1氫離子(Proton)………………………………………………………………5
1.2.2溶血磷脂質(Lysophospholipid)………………………………………………5
1.2.3 OGR1受體家族對氫離子的下游反應和功能性……………………………6
1.2.4 OGR1受體家族對溶血磷脂質的下游反應和功能性………………………7
1.3半乳糖腦苷脂和氫離子對於TDAG8之間的關係…………………………………8
1.3.1半乳糖腦苷脂在生理上的功能及其重要性…………………………………8
1.3.2 半乳糖腦苷脂下游的訊息傳遞……………………………………………9
1.4 研究動機 ……………………………………………………………………………9
第二章 實驗材料與方法……………………………………………………………………………11
2.1實驗材料……………………………………………………………………………12
2.1.1菌株…………………………………………………………………………12
2.1.2細胞株………………………………………………………………………12
2.1.3實驗藥品……………………………………………………………………12
2.1.3.1購自 Sigma藥廠 ……………………………………………………………………12
2.1.3.2購自 Invitrogen公司 …………………………………………………………………… 12
2.1.3.3購自 Qiagen …………………………………………………………………… 12
2.1.3.4購自 其他 …………………………………………………………………… 12
2.2實驗方法……………………………………………………………………………13
2.2.1表現質體的確認……………………………………………………………13
2.2.1.1限制酵素分析 …………………………………………………………………… 13
2.2.1.2瓊脂醣膠的製備及電泳 ……………………………………………………………………13
2.2.2大腸桿菌的轉型作用(Transformation)……………………………………13
2.2.3細菌的培養…………………………………………………………………14
2.2.3.1細菌固體培養……………………………………………………………………14
2.2.3.2液體培養 ……………………………………………………………………14
2.2.3.3菌種保存 ……………………………………………………………………14
2.2.4表現質體的製備……………………………………………………………14
2.2.4.1小量的製備(mini-prep) ……………………………………………………………………14
2.2.4.2大量的製備(midi-prep) ……………………………………………………………………15
2.2.5細胞培養(cell culture)……………………………………………………………………16
2.2.6轉染作用(Transfection)……………………………………………………………………16
2.2.6.1玻片的前處理 ……………………………………………………………………16
2.2.6.2轉染作用(transfection) ……………………………………………………………………17
2.2.7嘌呤黴素(Puromycin)篩選細胞……………………………………………17
2.2.8聚合酶連鎖反應(Polymerase chain reaction, PCR)………………………17
2.2.8.1核醣核苷酸(RNA)的萃取 ……………………………………………………………………17
2.2.8.2 DNAase I處理 ……………………………………………………………………18
2.2.8.3 cDNA的合成 ……………………………………………………………………18
2.2.8.4反轉錄-聚合酶素連鎖反應(RT-PCR) ……………………………………………………………19
2.2.9細胞內鈣離子濃度的分析…………………………………………………19
2.2.10鈣離子成像分析方法……………………………………………………20
2.2.11以流氏細胞儀觀察sh-mTDAG8的沉默效率(knockdown efficiency)……………………………………………………………………20
第三章 結果 ……………………………………………………………………21
3.1 TDAG8對Psychosine的刺激有濃度的依賴性………………………………22
3.2 N2A細胞會受到Psychosine的刺激而活化,並有濃度的依賴性……………22
3-3 TDAG8會受到Psychosine刺激而活化………………………………………24
3.4在EGTA環境下,N2A細胞在pH6.0刺激下會得到最大的反應………………24
3.5在EGTA pH6.0的環境下,OGR1和GPR4會受到刺激而活化…………………25
3.6 Proton引發的下游反應主要是Gq途徑,Psychosine則是Gi途徑……………26
3.7在含有氫離子的環境下會抑制由Psyhocsine引起的細胞內鈣離子濃度的增加……………………………………………………………………26
3.8氫離子刺激N2A細胞會抑制Psyhocsine引發的鈣離子濃度增加………………27
3.9 Psyhocsine刺激N2A細胞會抑制氫離子引發的鈣離子濃度增加………………28
3.10細胞重置五分鐘後,psychosine和proton仍然可以引發反應…………………28
3.11 Proton和Psychosine會相互抑制,且Psychosine的抑制性有濃度的依賴性……………………………………………………………………29
3.12透過流氏細胞儀無法觀察到shRNA-mTDAG8對mTDAG8基因沉默果……29
3.13透過聚合酶連鎖反應shRNA-mTDAG8無法有效的抑制TDAG8 mRNA的表現…………………………………………………………………………………30
3.14在N2A和HEK293T細胞上,利用shRNA-mTDAG8會抑制Psyhosine引發的反應………………………………………………………………………………31
3.15 shRNA-mTDAG8-B1轉染到N2A細胞,pH6.0刺激反應上有受到影響…….32
3.16以流氏細胞儀分析不含紅螢光蛋白shRNA-mTDAG8 的沉默(Knockdown)效率…………………………………………………………………………………32
3.17以流氏細胞儀分析含有紅螢光蛋白shRNA-mTDAG8 的沉默(Knockdown)效率………………………………………………………………………………33
3.18以RT-PCR分析mTDAG8基因的沉默(Knockdown)效率……………………34
第四章 討論………………………………………………………………………………35
4.1半乳糖腦苷脂為TDAG8的配體,並能有效刺激活化TDAG8受體,並走Gi的反應途徑…………………………………………………………………………36
4.2氫離子引發小鼠神經瘤母細胞內的鈣離子的累積主要為OGR1受體,並走Gq反應途徑…………………………………………………………………………37
4.3探討氫離子與半乳糖腦苷脂對N2A細胞相互抑制的情形 ……………………38
4.4利用流氏細胞儀和RT-PCR沒有明顯觀察到shRNA-mTDAG8 對TDAG8基因沉默的結果,但功能性分析上卻有影響……………………………………………41
4.5利用流氏細胞儀和RT-PCR沒有明顯觀察到shRNA-mTDAG8 對TDAG8基因沉默的結果,但功能性分析上卻有影響……………………………………………42
第五章 參考文獻 …………………………………………… 45
附錄 …………………………………………… 91
參考文獻 1. Bektas M., Barak LS., Jolly PS., Liu H., Lynch KR., Lacana E., Suhr KB., Milstien S., Spiegel S. (2003). The G protein-coupled receptor GPR4 suppresses ERK activation in a ligand-independent manner. Biochemistry. Oct 28;42(42):12181-91.
2. Bockaert J., Perroy J., Becamel C., Marin P., Fagni L. (2010). GPCR interacting proteins (GIPs) in the nervous system: Roles in physiology and pathologies. Annu Rev Pharmacol Toxicol. 50:89–109
3. Casey JR., Grinstein S., Orlowski J. (2001). Sensors and regulators of intracellular pH. Nat Rev Mol Cell Biol.Jan;11(1):50-61.
4. Choi JW., Lee SY., Choi Y. (1996). Identification of a putative G protein-coupled receptor induced during activation-induced apoptosis of T cells. Cell Immunol. Feb 25;168(1):78-84.
5. Eglen RM., Bosse R., Reisine T. (2007). Emerging concepts of guanine nucleotide-binding protein-coupled receptor (GPCR) function and implications for high throughput screening. Assay Drug Dev Technol. Jun;5(3):425-51.
6. Franco R., Ferre S., Agnati L., Torvinen M., Gines S., Hillion J., Casado V., Lledo P., Zoli M., Lluis C., Fuxe K. (2000). Evidence for adenosine/dopamine receptorinteractions: indications for heteromerization. Neuropsychopharmacology. Oct;23(4 suppl):S50-9.
7. Frang H., Cockcroft V., Karskela T., Scheinin M., Marjamaki A. (2001). Phenoxybenzamine binding reveals the helical orientation of the third transmembrane domain of adrenergic receotors. J Biol Chem. Aug 17;276(33):31279-84
8. Giri S., Jatana M., Rattan R., Won JS., Singh I., Singh AK. (2002). Galactosylsphingosine (psychosine)-induced expression of cytokine-mediated inducible nitric oxide synthases via AP-1 and C/EBP: implications for Krabbe disease. FASEB J. May;16(7):661-72.
9. Giri S., Khan M., Rattan R., Singh I., Singh AK. (2006). Krabbe disease: psychosine-mediated activation of phospholipase A2 in oligodendrocyte cell death. J Lipid Res. 2006 Jul;47(7):1478-92. Epub Apr 27.
10. Haq E., Giri S., Singh I., Singh AK. (2003). Molecular mechanism of psychosine-induced cell death in human oligodendrocyte cell line. J Neurochem. Sep;86(6):1428-40.
11. Im DS. ( 2005).Two ligands for a GPCR, proton vs lysolipid. Acta Pharmacol Sin. Dec;26(12):1435-41
12. Im DS., Heise CE., Nguyen T., O'Dowd BF., Lynch KR. (2001). Identification of a molecular target of psychosine and its role in globoid cell formation. J Cell Biol. Apr 16;153(2):429-34.
13. Ishii S., Kihara Y., Shimizu T. (2005). Identification of T cell death-associated gene 8 (TDAG8) as a novel acid sensing G-protein-coupled receptor. J Biol Chem. Mar 11;280(10):9083-7.
14. Jatana M., Giri S., Singh AK. (2002). Apoptotic positive cells in Krabbe brain and induction of apoptosis in rat C6 glial cells by psychosine. Neurosci Lett. Sep 20;330(2):183-7.
15. Kabarowski JH., Zhu K., Le LQ., Witte ON., Xu Y. (2001). Lysophosphatidylcholine as a ligand for the immunoregulatory receptor G2A. Science. Jul 27;293(5530):702-5.
16. Kabarowski JH., Xu Y., Witte ON. (2002). Lysophosphatidylcholine as a ligand for immunoregulation. Biochem Pharmacol. Jul 15;64(2):161-7.
17. Kanazawa T., Nakamura S., Momoi M., Yamaji T., Takematsu H., Yano H., Sabe H., Yamamoto A., Kawasaki T., Kozutsumi Y. (2000). Inhibition of cytokinesis by a lipid metabolite, psychosine. J Cell Biol. May 15;149(4):943-50.
18. Kim KS., Ren J., Jiang Y., Ebrahem Q., Tipps R., Cristina K., Xiao YJ., Qiao J., Taylor KL., Lum H., Anand-Apte B., Xu Y. (2005). GPR4 plays a critical role in endothelial cell function and mediates the effects of sphingosylphosphorylcholine. FASEB J. May;19(7):819-21.
19. King N., Hittinger CT., Carroll SB. (2003). Evolution of key cell signaling and adhesion protein families predates animal origins. Science. Jul 18;301(5631):361-3.
20. Kyaw H., Zeng Z., Su K., Fan P., Shell BK., Carter KC., Li Y. (1998). Cloning, characterization, and mapping of human homolog of mouse T-cell death-associated gene. DNA Cell Biol. Jun;17(6):493-500.
21. Kyaw H., Zeng Z., Su K., Fan P., Shell BK., Carter KC., Li Y. (1998). Cloning, characterization, and mapping of human homolog of mouse T-cell death-associated gene. DNA Cell Biol. Jun;17(6):493-500
22. Le LQ., Kabarowski JH., Weng Z., Satterthwaite AB., Harvill ET., Jensen ER., Miller JF., Witte ON. (2001). Mice lacking the orphan G protein-coupled receptor G2A develop a late- onset autoimmune syndrome. Immunity. May;14(5):561-71.
23. Li H., Wang D., Singh LS., Berk M., Tan H., Zhao Z., Steinmetz R., Kirmani K., Wei G., Xu Y. (2009). Abnormalities in osteoclastogenesis and decreased tumorigenesis in mice deficient forovarian cancer G protein-coupled receptor 1. PLoS One. May 28;4(5):e5705.
24. Ludwig MG., Vanek M., Guerini D., Gasser JA., Jones CE., Junker U., Hofstetter H., Wolf RM., Seuwen K. (2003). Proton-sensing G-protein-coupled receptors. Nature. Sep 4;425(6953):93-8.
25. Lum H., Qiao J., Walter RJ., Huang F., Subbaiah PV., Kim KS., Holian O. (2003). Inflammatory stress increases receptor for lysophosphatidylcholine in human microvascular endothelial cells. Am J Physiol Heart Circ Physiol. Oct;285(4):H1786-9
26. Maghazachi AA., Knudsen E., Jin Y., Jenstad M., Chaudhry FA. (2004). D-galactosyl-beta1-1'-sphingosine and D-glucosyl-beta1-1'-sphingosine induce human natural killer cell apoptosis. Biochem Biophys Res Commun. Jul 30;320(3):810-5.
27. May LT., Avlani VA., Sexton PM., Christopoulos A. (2004). Allosteric modulation of G protein-coupled receptors. Curr Pharm Des. 10(17):2003-13
28. Murakami N., Yokomizo T., Okuno T., Shimizu T. (2004). G2A is a proton-sensing G-protein-coupled receptor antagonized bylysophosphatidylcholine .. J Biol Chem. Oct 8;279(41):42484-91.
29. Nixon GF., Mathieson FA., Hunter I. (2008). The multi-functional role of sphingosylphosphorylcholine. Prog Lipid Res. Jan;47(1):62-75. Epub 2007 Nov 7.
30. Pyne S., Pyne N. (2000). Sphingosine 1-phosphate signalling via the endothelial differentiation gene family of G-protein-coupled receptors. Pharmacol Ther. Nov;88(2):115-31.
31. Radu CG., Yang LV., Riedinger M., Au M, Witte ON. (2004). T cell chemotaxis to lysophosphatidylcholine through the G2A receptor. Proc Natl Acad Sci U S A. Jan 6;101(1):245-50.
32. Radu CG., Nijagal A., McLaughlin J., Wang L., Witte ON. (2005). Differential proton sensitivity of related G protein-coupled receptors T cell death-associated gene 8 and G2A expressed in immune cells. Proc Natl Acad Sci U S A. Feb 1;102(5):1632-7.
33. Radu CG., Cheng D., Nijagal A., Riedinger M., McLaughlin J., Yang LV., Johnson J., Witte ON. (2006). Normal immune development and glucocorticoid-induced thymocyte apoptosis in mice deficient for the T-cell death-associated gene 8 receptor.Mol Cell Biol. Jan;26(2):668-77.
34. Roos A., Boron WF. (1981). Intracellular pH. Physiol Rev. Apr;61(2):296-434.
35. Sautel M., Milligan G. (2000). Molecular manipulation of G-protein-coupled receptors: a new avenue into drug discovery. Curr. Med. Chem. Sep;7(9):889-96.
36. Sin WC., Zhang Y., Zhong W., Adhikarakunnathu S., Powers S., Hoey T., An S., Yang J. (2004). G protein-coupled receptors GPR4 and TDAG8 are oncogenic and overexpressed in human cancers. Oncogene. Aug 19;23(37):6299-303.
37. Suzuki K. (1998). Twenty five years of the "psychosine hypothesis": a personal perspective of its history and present status. Neurochem Res. Mar;23(3):251-9.
38. Taniike M., Mohri I., Eguchi N., Irikura D., Urade Y., Okada S., Suzuki K. (1999). An apoptotic depletion of oligodendrocytes in the twitcher, a murine model of globoid cell leukodystrophy. J Neuropathol Exp Neurol. Jun;58(6):644-53.
39. Tosa N., Murakami M., Jia WY., Yokoyama M., Masunaga T., Iwabuchi C., Inobe M., Iwabuchi K., Miyazaki T., Onoe K., Iwata M., Uede T. (2003). Critical function of T cell death-associated gene 8 in glucocorticoid-induced thymocyte apoptosis. Int Immunol. Jun;15(6):741-9.
40. Wang JQ., Kon J., Mogi C., Tobo M., Damirin A., Sato K., Komachi M., Malchinkhuu E., Murata N., Kimura T., Kuwabara A., Wakamatsu K., Koizumi H., Uede T., Tsujimoto G., Kurose H.,Sato T., Harada A., Misawa N., Tomura H., Okajima F. (2004) TDAG8 is a proton-sensing and psychosine-sensitive G-protein-coupled receptor. J Biol Chem. Oct 29;279(44):45626-33
41. Wang L, Radu CG, Yang LV, Bentolila LA, Riedinger M, Witte ON. (2005). Lysophosphatidylcholine-induced surface redistribution regulates signaling of the murine G protein-coupled receptor G2A . Mol Biol Cell. May;16(5):2234-47.
42. Weng Z., Fluckiger AC., Nisitani S., Wahl MI., Le LQ., Hunter CA., Fernal AA., Le Beau MM., Witte ON. (1998). A DNA damage and stress inducible G protein-coupled receptor blocks cells in G2/M. Proc Natl Acad Sci U S A. Oct 13;95(21):12334-9.
43. Wenger DA., Suzuki K., Suzuki Y., Suzuki K., Galactosylceramide lipidosis: globoid cell leukodystrophy (Krabbe disease). McGraw-Hill; New York: 2001. p. 3669-3694.
44. Wilson S., Bergsma DJ., Chambers JK., Muir AI., Fantom KG., Ellis C., Murdock PR., Herrity NC., Stadel JM. (1998). Orphan G-protein-coupled receptors: the next generation of drug targets? Br. J. Pharmacol. Dec;125(7):1387-92.
45. Xiao SH., Reagan JD., Lee PH., Fu A., Schwandner R., Zhao X., Knop J., Beckmann H., Young SW. (2008). High throughput screening for orphan and liganded GPCRs. Comb Chem High Throughput Screen. Mar;11(3):195-215.
46. Xu Y., Zhu K., Hong G., Wu W., Baudhuin LM., Xiao Y., Damron DS. (2000). Sphingosylphosphorylcholine is a ligand for ovarian cancer G-protein-coupled receptor 1. Nat Cell Biol. May;2(5):261-7.
47. Xu Y. (2002). Sphingosylphosphorylcholine and lysophosphatidylcholine: G protein-coupled receptors and receptor-mediated signal transduction. Biochim Biophys Acta. May 23;1582(1-3):81-8.
48. Yang LV., Radu CG., Wang L., Riedinger M., Witte ON. (2005). Gi-independent macrophage chemotaxis to lysophosphatidylcholine via the immunoregulatory GPCR G2A. Blood. Feb 1;105(3):1127-34.
49. Yang LV., Radu CG., Roy M., Lee S., McLaughlin J., Teitell MA., Iruela-Arispe ML., Witte ON. (2007). Vascular abnormalities in mice deficient for the G protein-coupled receptor GPR4 that functions as a pH sensor. Mol Cell Biol. Feb;27(4):1334-47.
50. Zaka M., Rafi MA., Rao HZ., Luzi P., Wenger DA. (2005). Insulin-like growth factor-1 provides protection against psychosine-induced apoptosis in cultured mouse oligodendrocyte progenitor cells using primarily the PI3K/Akt pathway. Mol Cell Neurosci. Nov;30(3):398-407. Epub 2005 Sep 19.
51. Zheng H., Loh H., Law P. (2010). Agonist-selective signaling of G protein-coupled receptor: mechanisms and implications. IUBMB life. 62(2):112–119.
52. Zhu K., Baudhuin LM., Hong G., Williams FS., Cristina KL., Kabarowski JH., Witte ON., Xu Y. (2001). Sphingosylphosphorylcholine and lysophosphatidylcholine are ligands for the G protein-coupled receptor GPR4. J Biol Chem. Nov 2;276(44):41325-35.
53. Zohn IE., Klinger M., Karp X., Kirk H., Symons M., Chrzanowska-Wodnicka M., Der CJ., Kay RJ. (2000). G2A is an oncogenic G protein-coupled receptor. Oncogene. Aug 10;19(34):3866-77
指導教授 孫維欣(Wei-hsin Sun) 審核日期 2011-11-30
推文 facebook   plurk   twitter   funp   google   live   udn   HD   myshare   reddit   netvibes   friend   youpush   delicious   baidu   

若有論文相關問題,請聯絡國立中央大學圖書館推廣服務組 TEL:(03)422-7151轉57407,或E-mail聯絡