Visualization of phosphoinositides that bind pleckstrin homology domains: calcium- and agonist-induced dynamic changes and relationship to myo-[3H]inositol-labeled phosphoinositide pools.
Visualization of phosphoinositides that bind pleckstrin homology domains: calcium- and agonist-induced dynamic changes and relationship to myo-[3H]inositol-labeled phosphoinositide pools.
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DOI：
10.1083/jcb.143.2.501
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发表时间：
1998-10-19
影响因子：
7.8
通讯作者：
Balla, T
中科院分区：
文献类型：
--
作者：
Várnai, P;Balla, T
通过在活细胞中表达磷脂酶C (PLC)δ PH结构域-绿色荧光蛋白融合构建和共聚焦图像分析，可视化了结合pleckstrin同源（PH）结构域的磷脂酰肌醇4,5-二磷酸（PtdIns[4,5]P2）池。荧光探针的质膜定位需要在已知与PtdIns(4,5)P2形成关键接触的PLCδ PH结构域中存在三个基本残基。通过离子载体或受体刺激激活内源性plc会产生荧光信号从膜到胞浆的快速重新分配，这在Ca2+螯合后被逆转。在离子霉素和激动剂刺激的细胞中，荧光探针的分布与PtdIns绝对质量的变化密切相关（4,5）P2。槲皮素或氧化苯larsin对PtdIns(4,5)P2合成的抑制阻止了离子霉素处理的细胞中Ca2+螯合或激动剂刺激时荧光探针在膜上的重新定位。相比之下，PH结构域成像的PtdIns(4,5)P2的合成对wortmannin的浓度不敏感，wortmannin被发现抑制myo-[3H]肌醇标记的PtdIns(4,5)P2的合成。与PH结构域相互作用的磷酸肌苷的鉴定和动态成像将进一步加深我们对肌醇磷脂对这些蛋白质的调节的理解。
Phosphatidylinositol 4,5-bisphosphate (PtdIns[4,5]P2) pools that bind pleckstrin homology (PH) domains were visualized by cellular expression of a phospholipase C (PLC)δ PH domain–green fluorescent protein fusion construct and analysis of confocal images in living cells. Plasma membrane localization of the fluorescent probe required the presence of three basic residues within the PLCδ PH domain known to form critical contacts with PtdIns(4,5)P2. Activation of endogenous PLCs by ionophores or by receptor stimulation produced rapid redistribution of the fluorescent signal from the membrane to cytosol, which was reversed after Ca2+ chelation. In both ionomycin- and agonist-stimulated cells, fluorescent probe distribution closely correlated with changes in absolute mass of PtdIns(4,5)P2. Inhibition of PtdIns(4,5)P2 synthesis by quercetin or phenylarsine oxide prevented the relocalization of the fluorescent probe to the membranes after Ca2+ chelation in ionomycin-treated cells or during agonist stimulation. In contrast, the synthesis of the PtdIns(4,5)P2 imaged by the PH domain was not sensitive to concentrations of wortmannin that had been found inhibitory of the synthesis of myo-[3H]inositol– labeled PtdIns(4,5)P2. Identification and dynamic imaging of phosphoinositides that interact with PH domains will further our understanding of the regulation of such proteins by inositol phospholipids.
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影响因子：
56.9
作者：
Franke, TF;Kaplan, DR;Toker, A
通讯作者：
Toker, A
影响因子：
3.3
作者：
BALLA, T;SIM, SS;CATT, KJ
通讯作者：
CATT, KJ
DOI：
10.1073/pnas.92.23.10472
发表时间：
1995-11-07
影响因子：
11.1
作者：
LEMMON, MA;FERGUSON, KM;SCHLESSINGER, J
通讯作者：
SCHLESSINGER, J
影响因子：
11.4
作者：
Falasca, M;Logan, SK;Schlessinger, J
通讯作者：
Schlessinger, J
DOI：
10.1073/pnas.92.12.5317
发表时间：
1995-06-06
影响因子：
11.1
作者：
NAKANISHI, S;CATT, KJ;BALLA, T
通讯作者：
BALLA, T