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研究生:高又雅
研究生(外文):Yu-Ya Kao
論文名稱:第六亞型腺苷酸環化酶之分子內交互作用
論文名稱(外文):Intramolecular interaction of type VI adenylyl cyclase
指導教授:陳儀莊陳儀莊引用關係
指導教授(外文):Yijuang Chern
學位類別:博士
校院名稱:國立陽明大學
系所名稱:神經科學研究所
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:2004
畢業學年度:92
語文別:中文
中文關鍵詞:腺苷酸環化酶
外文關鍵詞:Adenylyl Cyclase
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摘要
腺苷酸環化酶的功能是將細胞內的ATP轉成 cAMP。目前共發現了九種位於細胞膜及一種存在於細胞質中的腺苷酸環化酶。所有位於細胞膜的腺苷酸環化酶均具有三個伸入細胞質的蛋白質片段(N,C1a/b,C2)。本研究中我們發現當第六亞型腺苷酸環化酶的N端的前八十六個胺基酸被移除時,所做出的突變蛋白質(ACVI-ΔA87)與原生型第六亞型腺苷酸環化酶相比較時較易受與抑制性G蛋白連結的受體及活化的抑制性G蛋白□次單元所抑制。以第五亞型腺苷酸環化酶的N端取代第六亞型腺苷酸環化酶的N端也可顯著改變其受到與抑制性G蛋白連結的受體所抑制的程度。並且,我們也利用免疫法偵測到全長的N端 (胺基酸1到160) 及另一段截短的N端蛋白質(胺基酸1到86)均可與C1a片段結合,表示N端蛋白質上與C1a片段作用的位置位於第一到第八十六個胺基酸之間。此發現十分有趣,因為C1a片段是腺苷酸環化酶上可與抑制性G蛋白□次單元結合的位置。突變分析方法進一步指出N端可能結合於C1a片段上第434到505胺基酸之間的位置,此位置也包含C1a片段與抑制性G蛋白□次單元作用的區域。若將與N端作用的C1a片段上的兩個胺基酸(Leu-472 及Val-476)突變之後,此C1a片段突變蛋白與N端蛋白的結合即會受到抑制。而此突變也會明顯降低ACVI-ΔA87受抑制性G蛋白□次單元所抑制的程度。進一步地以生化方法分析Leu-472 及Val-476的突變對原生型第六亞型腺苷酸環化酶與ACVI-ΔA87受抑制性G蛋白□次單元抑制的影響,分析結果顯示當抑制性G蛋白□次單元的量較低的時候(約在IC50值上下時),N端是藉由結合到C1a片段來調控抑制性G蛋白□次單元對第六亞型腺苷酸環化酶的抑制作用,而在抑制性G蛋白□次單元的量較高的時候(約在IC50值的大約十到二十倍時),則是較為複雜的干擾模式,雖仍與N端有關,但與其結合到C1a片段無關。綜合以上的結果顯示N端在抑制性G蛋白□次單元對第六亞型腺苷酸環化酶的抑制作用上具有重要的功能。
Abstract
Adenylyl cyclase (AC) is an enzyme that synthesizes cAMP from ATP upon activation of G protein-coupled receptor. The mammalian AC superfamily consists of nine membrane-bound isoforms and one soluble form. All of membrane-bound ACs possess three large cytosolic domains (N, C1, and C2 domains).We show herein that removal of the first 86 amino acids (a.a.) of the N terminus (designated N) of rat type VI adenylyl cyclase (ACVI) caused the resultant ACVI mutant (ACVI-ΔA87) to be more greatly inhibited by a Giα-coupled receptor or activated Giα protein. Replacement of the entire N terminus of ACVI with that of the type V adenylyl cyclase (ACV) also dramatically altered D2-R-evoked inhibition. Moreover, in vitro binding of the full-length N and C1a domains (designated C1a), which interacts with Giα, was detected. A truncated N terminus (a.a. 1~86) also interacted with C1a, suggesting that the C1a-interacting region is located within a.a. 1~86. Mutation analyses further revealed that N might interact with C1a in the region (a.a. 434~505) where Giα is bound. Mutations of 2 residues (Leu-472 and Val-476) located in this N-binding region of C1a suppressed the interaction between recombinant N and C1a and markedly reduced Giα-mediated inhibition of ACVI-ΔA87. Further biochemical analyses of the effect of internal mutations of Leu-472/Val-476 on Giα-mediated inhibition of wildtype ACVI and ACVI-ΔA87 suggested that N modulates the Giα-mediated inhibition of ACVI via binding to C1a when the level of Giα is low (i.e., around the IC50 value), and that a more complicated interfering mode results when the level of Giα is high (i.e., approximately 10- to 20-fold of the IC50 value). Data presented herein suggest a novel function of the N terminus of ACVI in Giα-mediated regulation.
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