Visualization of a functional Galpha q-green fluorescent protein fusion in living cells. Association with the plasma membrane is disrupted by mutational activation and by elimination of palmitoylation sites, but not be activation mediated by receptors or AlF4-.-猫眼课题宝

活细胞中功能性 Galpha q-绿色荧光蛋白融合的可视化。

DOI：

发表时间：

2001

期刊：

Journal of Biological Chemistry

影响因子：

4.8

通讯作者：

中科院分区：

生物学2区

文献类型：

作者：

T. Hughes;H. Zhang;D. Logothetis;C. Berlot

为了研究G蛋白α亚基定位在基础和活化条件下是如何调节的，我们将绿色荧光蛋白（GFP）插入到Ga α（q）的内环中。α（q）-GFP刺激磷脂酶C对激活受体的反应，并抑制基础G蛋白门控内向整流K（+）电流的β-γ依赖性激活，效果与α（q）一样。α（q）-GFP与质膜的结合通过突变激活而减少，并通过α（q）棕榈酰化位点的突变而消除，这表明α（q）必须处于无活性的棕榈酰化状态才能靶向该位置。我们测试了受体和AlF（4）（-）激活对表达α（q）-GFP和蛋白激酶C γ-红色荧光蛋白融合体的细胞中α（q）-GFP定位的影响，该融合体响应G（q）激活而易位到质膜。在明显表现出蛋白激酶C γ-红色荧光蛋白易位反应的细胞中，没有观察到α（q）-GFP的重新定位。因此，在与棕榈酸转换和β-γ解离相关的条件下，α（q）-GFP保持与质膜结合。这些结果表明，在到达质膜时，α（q）除了棕榈酰化和与β-γ缔合之外还接收锚定信号，或者在激活期间，这些因子中的一个或两个继续将α（q）保留在该位置。

To investigate how G protein alpha subunit localization is regulated under basal and activated conditions, we inserted green fluorescent protein (GFP) into an internal loop of Galpha(q). alpha(q)-GFP stimulates phospholipase C in response to activated receptors and inhibits betagamma-dependent activation of basal G protein-gated inwardly rectifying K(+) currents as effectively as alpha(q) does. Association of alpha(q)-GFP with the plasma membrane is reduced by mutational activation and eliminated by mutation of the alpha(q) palmitoylation sites, suggesting that alpha(q) must be in the inactive, palmitoylated state to be targeted to this location. We tested the effects of activation by receptors and by AlF(4)(-) on the localization of alpha(q)-GFP in cells expressing both alpha(q)-GFP and a protein kinase Cgamma-red fluorescent protein fusion that translocates to the plasma membrane in response to activation of G(q). In cells that clearly exhibit protein kinase Cgamma-red fluorescent protein translocation responses, relocalization of alpha(q)-GFP is not observed. Thus, under conditions associated with palmitate turnover and betagamma dissociation, alpha(q)-GFP remains associated with the plasma membrane. These results suggest that upon reaching the plasma membrane alpha(q) receives an anchoring signal in addition to palmitoylation and association with betagamma, or that during activation, one or both of these factors continues to retain alpha(q) in this location.