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研究生:項聖軍
研究生(外文):Hsiang, Sheng-Chun
論文名稱:大白鼠第五亞型蕈毒乙醯膽鹼受體之內化作用和G蛋白質偶合有關
論文名稱(外文):Requirement of G protein coupling for the internalization of the rat M5 muscarinic acetylcholine receptor
指導教授:廖欽峰
指導教授(外文):Liao, Ching-Fong
學位類別:碩士
校院名稱:國立陽明大學
系所名稱:生理學研究所
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:1994
畢業學年度:82
語文別:中文
中文關鍵詞:生理學第五亞型蕈毒乙醯膽鹼內化作用
外文關鍵詞:PHYSIOLOGY
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本研究旨在探討激活劑Carbachol(CCh)所引發之第五亞型蕈毒乙醯膽鹼受體(m5受體)之內化作用(internaliztion)的形成機轉,並進而探討m5受體之內化作用和protein kinasaa A(PKA)之間的關係。我們成功地將m5-WT基因轉殖,並且穩定地表現於小鼠之Ltk-細胞上(LM5-WT細胞),利用親水性之拮抗體 N-[3H]nethylscopolamine測細胞表面之受體密度,利用親油性之拮抗體[3H]quinuclidinyl benzilate測量細胞之總受體密度。
針對CCh所引發之m5受體內化作用的形成機轉,首先我們先針對CCh刺激LM5-WT後所產生的兩個二級訊息傳遞分子來探討。結果顯示 (1)Protien kinase C(PKC)之激活劑 phorbol 12-myristatet 13-acetate(PMA,1μM)不能促進m5受體之內化作用,但卻抑制了CCh(100 μM)所引發之m5受體內化作用,而這種抑制現象可被PKC之抑制劑staurosporine(1μM)所阻斷,而且將LM5-WT以PMA(HM)做24小時處理,而使胞內的PKC被dOWN-reguLATION,則發現此種PMA抑制CCh所引發之m5受體內化作用現象可被阻斷,並且PKC down-regulation並不影響CCh所引發之m5受體的內化作用,(2)Ca2+ionophore A23187(1μM)和Ca2+/Calmodulin dependent protein kinase(CDPK)抑制劑KN-62(10μM)本身對m5受體的內化作用也無影響。綜合上述結果,CCh所引發之m5受體內化作用並非因m5受體激活pKc或CDPK所造成的。
前人報告CCh所引發之內化作用可能和G蛋白有關 [Thompson,A.K.,S.P.Mastafapour,L.C.Denlinger,J.E.Bleasdale,andS.K.F;Sher.J.Biol.Chem.266:2856-23862,1991]。因此利用定位突變技術,我們構築了一株可能影響其G蛋白偶合作用之突變種m5-51(Tyr216→Ser),於表現於Ltk細胞上後(LM5-51.2),此株突變種顯示其影響G蛋白偶合之作用,同時也影響了CCh所引發了m5受體內化作用。因此確認了G蛋白偶合於CCh所引發內化作 用之重要性,但是對於CCh 引發m5受體內化作用中,是 因G蛋白激活本身,或激活後由其它和phosphomnosinde (PI)水解後無關之後續步驟所造的,則仍需進一步探討。
1993年,Lee和Fraser[Lee,N.H.,andC.M.Fraser.J.Biol.Chem.268:7949-7957,1993]指出第一至型蕈毒乙醯膽鹼受體(ml受體)之第三內環中段,可能含有類似β2-adrenergic receptor kinase,及PKA之辨識區,而此辨識區之切除,便會影響CCh所引發之ml受體內化作用和PKA之激活於ml受體內化作用的影響。因此構築了三株突變種,m5-45(del 269-290),m5-SE(del 268-364)及m5-SHP(del 269-448),皆去除第三內環中段潛在之蛋白質激酉每辨識區,待表現於Ltk-細胞上後(LM5-45.24,LM5-SE.1,LM5-SHP),這些突變種顯示並不影響CCh所引發之m5受體的內化作用 ,且對於PKA激活後於m5受體內化作用之影響也反有明顯的差異。故本實驗之結論為 G蛋白偶合是CCh引發m5受體內化作用的一項重要因子,至於其間之 關聯性,仍需更進一步的探討。
The purpose of this investigation was to identify the signal transduction pathways leading to internalization of the m5 muscarinic acetylchoine receptor (m5 mAChR) and the relationship between protein kinase A (PKA) activation and internalization of cell surface m5 mAChR. Genes encoding the m5 mAChR were stably expressed in Ltk- cells. Cell surface receptor density was measured with a hydrophihc tracer, N-[3H] methyl scopolamine, while total receptor content was measured with [3H] quinuclidinyl benzilate.
Firstly, protein kinase C (PKC) and Ca2+/calrnodulin pathways activated by CCh were examined. The results showed that (1) m5 receptor internalization could not be induced by phorbol 12-myristate 13 acetate (PMA, 1μM), a PKC activator. However, the CCh-induced m5 receptor Internalization was inhibited by PMA, and the inhibition was reversed by staurosporine (1μM). (2) Ca2+ ionophore A23187 (1 μM) and Ca2+/calmodulin dependent protein kinase (CDPK.) inhibitor KN-62 (10 μM) had no effect on m5 receptor internalization. The results suggest that internalization of the m5 receptor are not induced through PKC or Ca2+/calmodulin pathway activation.
For further investigation on the relationship between G protein coupling and receptor internalization, an m5 receptor with a point mutation at the N terminal ot the intracellular 3rd loop (i3 loop) of the m5 receptor (Y216→S), named m5- 51, was constructed. The results shelved that CCh- induced m5-51 internalization and G protein coupling were completely abolished.
In 1993, Lee and Fraser [Lee, N. H., and C. M. Fraser. J. Biol. Chem. 268:7949-7957, 1993]suggested that the region located in the middle part of the i3 loop of ml mAChR, containing a potential β2-adrenergic receptor kinase (PARK) or PKA phosphorylation sites, might be responsible for agonist-induced receptor internahzation and PKA-activated heterologous receptor internalization. Therefore, three mutants, m5-45 (del 269-290), m5-SE (del 268-364), and m5- SHP (del 269-448), with deletion of the potential β ARK/PKA sites in the 13 loop were constructed and expressed in Ltk- cells (LM5-45.24, LM5-SE. l, LM5-SHP). Internalization of these mutated receptors was tested and the results showed that CCh-induced receptor internalization and PKA-activated heterologous receptor internalization were not changed.
In summary, CCh-induced receptor internalization of m5 mAChR in Ltk cells requires G protein coupling. Whether G protein coupling alone activates receptor internalization, or additional mediators are needed remains unknown. However, the involvement of PKC, Ca2+/ calmodulin, and PKA pathways have been excluded in this study.



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