Phosphoinositide and inositol phosphate analysis in lymphocyte activation.
Phosphoinositide and inositol phosphate analysis in lymphocyte activation.
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淋巴细胞活化中的磷酸肌醇和磷酸肌醇分析。
DOI：
10.1002/0471142735.im1101s87
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发表时间：
2009
影响因子：
--
通讯作者：
Mayr,GeorgW
中科院分区：
文献类型：
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作者：
Sauer,Karsten;Huang,YinaHsing;Lin,Hongying;Sandberg,Mark;Mayr,GeorgW
淋巴细胞抗原受体参与深刻地改变了细胞内磷脂酰肌醇脂质和可溶性肌醇磷酸的含量。其中，磷酸肌醇磷脂酰肌醇4，5-二磷酸（PIP 2）和磷脂酰肌醇3，4，5-三磷酸（PIP 3）通过充当普列克底物蛋白同源（PH）结构域配体而发挥关键的信号传导作用，所述普列克底物蛋白同源（PH）结构域配体将信号传导蛋白募集到质膜。此外，PIP 2作为第二信使分子甘油二酯和可溶性肌醇1，4，5-三磷酸（IP 3）的前体，是淋巴细胞中PKC、Ras/Erk和Ca 2+信号传导的重要介质。IP 33-激酶磷酸化IP 3产生肌醇1，3，4，5-四磷酸（IP 4），这是发育中T细胞中PH结构域与PIP 3结合的重要可溶性调节剂。除了PIP 2、PIP 3、IP 3和IP 4之外，淋巴细胞还产生多种其他磷酸肌醇和可溶性肌醇磷酸，它们可能具有重要的生理功能。为了帮助他们的分析，详细的协议，允许一个同时测量淋巴细胞中的多种不同的磷酸肌醇或磷酸肌醇异构体的水平在这里提供。它们基于薄层、常规和高效液相色谱分离方法，然后进行放射性标记或非放射性金属染料检测。最后，讨论了用于检测特定磷酸肌醇或磷酸肌醇异构体的适用范围较广的非色谱方法。支持方案描述了如何获得纯的未刺激的CD 4 + CD 8+胸腺细胞群体，用于分析阳性和阴性选择期间的磷酸肌醇周转，这是T细胞发育的关键步骤。协议免疫学87：11.1.1 - 11.1.46。© 2009年由约翰威利父子公司。
Lymphocyte antigen receptor engagement profoundly changes the cellular content of phosphoinositide lipids and soluble inositol phosphates. Among these, the phosphoinositides phosphatidylinositol 4,5‐bisphosphate (PIP2) and phosphatidylinositol 3,4,5‐trisphosphate (PIP3) play key signaling roles by acting as pleckstrin homology (PH) domain ligands that recruit signaling proteins to the plasma membrane. Moreover, PIP2acts as a precursor for the second messenger molecules diacylglycerol and soluble inositol 1,4,5‐trisphosphate (IP3), essential mediators of PKC, Ras/Erk, and Ca2+signaling in lymphocytes. IP3phosphorylation by IP33‐kinases generates inositol 1,3,4,5‐tetrakisphosphate (IP4), an essential soluble regulator of PH domain binding to PIP3in developing T cells. Besides PIP2, PIP3, IP3, and IP4, lymphocytes produce multiple other phosphoinositides and soluble inositol phosphates that could have important physiological functions. To aid their analysis, detailed protocols that allow one to simultaneously measure the levels of multiple different phosphoinositide or inositol phosphate isomers in lymphocytes are provided here. They are based on thin layer, conventional and high‐performance liquid chromatographic separation methods followed by radiolabeling or non‐radioactive metal‐dye detection. Finally, less broadly applicable non‐chromatographic methods for detection of specific phosphoinositide or inositol phosphate isomers are discussed. Support protocols describe how to obtain pure unstimulated CD4+CD8+thymocyte populations for analyses of inositol phosphate turnover during positive and negative selection, key steps in T cell development.Curr. Protoc. Immunol. 87:11.1.1‐11.1.46. © 2009 by John Wiley & Sons, Inc.