G_β,G_(βγ)与腺苷酸环化酶Ⅱ在酵母双杂交和三杂交系统中的相互作用  

Roles of G-Proteinβ andγ Subunits in the Interaction of G_(βγ) with Adenylyl CyclasesⅡ

作　　者：李旌军[1] 鲁宁[1] 黄秉仁[1] 

机构地区：[1]中国医学科学院中国协和医科大学基础医学研究所医学分子生物学国家重点实验室,北京100005

出　　处：《中国医学科学院学报》2001年第2期115-118,共4页Acta Academiae Medicinae Sinicae

基　　金：国家自然科学基金!( 39630120)&&

摘　　要：目的 探讨 G蛋白β和γ亚基在 G_(βγ)与效应器相互作用中的地位 ,特别是γ亚基在此相互关系中的作用。方法 利用酵母双杂交和自行构建的酵母三杂交系统分别研究 G_(β 1)和 G_(β 1)γ 2与腺苷酸环化酶Ⅱ (ACⅡ )的相互作用。 结果 发现酵母三杂交系统中 AD-β 1，γ 2和 BD-ACⅡ间的相互作用明显强于双杂交系统中 AD-β 1和 BD-ACⅡ的相互作用。对 BD-ACⅡ Q和 AD-β 1在酵母双杂交系统和三杂交系统中的表达进行免疫印迹杂交分析 ,发现其表达水平与β-半乳糖苷酶活力大小无关 ,即酵母三杂交系统和双杂交系统中相互作用的显著差别不是由 BD-ACⅡ Q和 AD-β 1蛋白表达水平的差异所造成。结论 β亚基对维持 G_(βγ)与 ACⅡ的相互作用十分重要 ,而γ亚基的存在显著增强了β亚基与 ACⅡ的相互作用 ,对维持 G_(βγ)与 ACⅡ的高亲和性很重要。Objective To explore the individual roles of G proteinβ andγ subunit in the interactions with effectors. Methods We investigated the interactions of G_(β 1) and G_(β 1)γ 2 with adenylyl cyclaseⅡ (ACⅡ ) using the yeast two-hybrid and three-hybrid systems. Results When assayed for the abilities to activate the reporter gene, the interactions among AD-β 1,γ 2 and BD-ACⅡ Q in the three-hybrid system were more potent than the interactions between AD-β 1 and BD-ACⅡ Q in the two-hybrid system. The expressions of BD-ACⅡ Q and AD-β 1 in transformants coexpressed AD-β 1 and BD-ACⅡ Q, and transformants coexpressed AD-β 1,γ 2 and BD-ACⅡ Q were respectively detected. The comparisons between the reporter activity and the expression levels of BD-ACⅡ Q and AD-β 1 in the yeast cells show there was no correlations, i.e. The difference in the reporter activity was not a reflection of differential expression level of the hybrid proteins. Conclutions All these results suggest that G proteinβ 1 subunit is sufficient to maintain the basic interaction between G_(β 1)γ 2 and ACⅡ Q, andγ 2 subunit plays an important role in the high affinity interaction of G_(β 1)γ 2 with ACⅡ Q.

关 键 词：Gβγ 腺苷酸环化酶Ⅱ 酵母三杂交系统 酵母双杂交系统 

分 类 号：Q503[生物学—生物化学]