In vivo interaction between RGS4 and calmodulin visualized with FRET techniques: Possible involvement of lipid raft
In vivo interaction between RGS4 and calmodulin visualized with FRET techniques: Possible involvement of lipid raft
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DOI：
10.1016/j.bbrc.2005.10.026
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发表时间：
2005-12-16
影响因子：
3.1
通讯作者：
Kurachi, Y
中科院分区：
文献类型：
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作者：
Ishii, M;Ikushima, M;Kurachi, Y
G蛋白信号调节因子（Regulators of G-protein signaling，RGS）是一类能促进异源三聚体G蛋白α亚基内源性GTP水解的蛋白质。尽管有人认为RGS 4的功能是通过膜磷脂、磷脂酰肌醇-3，4，5-三磷酸盐（PtdIns（3，4，5）P-3）和Ca 2 +/钙调蛋白（CaM）的竞争性结合相互调节的，但这些相互作用仍有待证明在体内发生。在这里，使用荧光共振能量转移（FRET）技术，我们表明，离子霉素的细胞内Ca 2+浓度的升高增加了FRET效率从ECFP（青色荧光蛋白的变体）标记的钙调素金星（黄色荧光蛋白的变体）标记的RGS 4。通过用甲基-β-环糊精预处理细胞，FRET效率的增加大大减弱，甲基-β-环糊精消耗膜胆固醇，从而破坏脂筏。这些结果提供了第一个演示RGS 4和钙调素在体内的钙依赖性之间的相互作用，并表明，在质膜脂筏协会可能参与RGS蛋白的生理调节。(c)2005年爱思唯尔公司All rights reserved.
Regulators of G-protein signaling (RGS) are a family of proteins which accelerate intrinsic GTP-hydrolysis on heterotrimeric G-protein-alpha-subunits. Although it has been suggested that the function of RGS4 is reciprocally regulated by competitive binding of the membrane phospholipid, phosphatidylinositol-3,4,5,-trisphosphate(PtdIns(3,4,5)P-3), and Ca2+/calmodulin (CaM), it remains to be shown that these interactions occur in vivo. Here, using fluorescence resonance energy transfer (FRET) techniques, we show that an elevation of intracellular Ca2+ concentration by ionomycin increased the FRET efficiency from ECFP (a variant of cyan fluorescent protein)-labeled calmodulin to Venus (a variant of yellow fluorescent protein)-labeled RGS4. The increase in FRET efficiency was greatly attenuated by pre-treating the cells with methyl-beta-cyclodextrin, which depletes membrane cholesterol and thus disrupts lipid rafts. These results provide the first demonstration of a Ca2+-dependent interaction between RGS4 and CaM in vivo and show that association in lipid rafts of the plasma membrane might be involved in this physiological regulation of RGS proteins. (c) 2005 Elsevier Inc. All rights reserved.