Real-time visualization of a fluorescent GαS:: Dissociation of the activated G protein from plasma membrane
Real-time visualization of a fluorescent GαS:: Dissociation of the activated G protein from plasma membrane
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DOI：
10.1124/mol.61.2.352
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发表时间：
2002-02-01
影响因子：
3.6
通讯作者：
Rasenick, MM
中科院分区：
文献类型：
--
作者：
Yu, JZ;Rasenick, MM
为了研究激活的Galpha(s)在活细胞中的行为，将绿色荧光蛋白（GFP）插入到Galpha(s)的内部氨基酸序列中，生成Galpha(s)-GFP融合蛋白。融合蛋白保持亮绿色荧光，用抗Galpha(s)或GFP抗体进行免疫印迹鉴定。Galpha(s)-GFP的细胞分布与内源性Galpha(s)相似。Galpha(s)-GFP与β肾上腺素能受体紧密结合，激活Galpha(s)效应物腺苷酸环化酶。霍乱毒素激活Galpha(s)-GFP导致融合蛋白逐渐从质膜转移到活细胞的整个细胞质。与霍乱毒素诱导的Galpha(s)-GFP从细胞膜缓慢释放不同，β -肾上腺素能激动剂异丙肾上腺素引起融合蛋白快速部分释放到细胞质中。异丙肾上腺素处理后1min，质膜部位的Galpha -GFP释放量最大；然而，Galpha(s)-GFP在其他膜位点的插入也在同一时期发生。异丙肾上腺素诱导的Galpha(s)-GFP融合蛋白易位表明，Galpha(s)的内化可能通过在多个细胞位点与效应分子和细胞骨架元件相互作用，在信号转导中发挥作用。
To study behavior of activated Galpha(s) in living cells, green fluorescent protein (GFP) was inserted within the internal amino acid sequence of Galpha(s) to generate a Galpha(s)-GFP fusion protein. The fusion protein maintained a bright green fluorescence and was identified by immunoblotting with antibodies against Galpha(s) or GFP. The cellular distribution of Galpha(s)-GFP was similar to that of endogenous Galpha(s). Galpha(s)-GFP was tightly coupled to the beta adrenergic receptor to activate the Galpha(s) effector, adenylyl cyclase. Activation of Galpha(s)-GFP by cholera toxin caused a gradual displacement of the fusion protein from the plasma membrane throughout the cytoplasm in living cells. Unlike the slow release of Galpha(s)-GFP from the membrane induced by cholera toxin, the beta-adrenergic agonist isoproterenol caused a rapid partial release of the fusion protein into the cytoplasm. At 1 min after treatment with isoproterenol, the extent of Galpha(s)-GFP release from plasma membrane sites was maximal; however, insertion of Galpha(s)-GFP at other membrane sites occurred during the same time period. Translocation of Galpha(s)-GFP fusion protein induced by isoproterenol suggested that the internalization of Galpha(s) might play a role in signal transduction by interacting with effector molecules and cytoskeletal elements at multiple cellular sites.