Conformational regulation of α4β1-integrin affinity by reducing agents -: "Inside-out" signaling is independent of and additive to reduction-regulated integrin activation
Conformational regulation of α4β1-integrin affinity by reducing agents -: "Inside-out" signaling is independent of and additive to reduction-regulated integrin activation
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DOI：
10.1074/jbc.m404387200
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发表时间：
2004-07-30
影响因子：
4.8
通讯作者：
Sklar, LA
中科院分区：
文献类型：
--
作者：
Chigaev, A;Zwartz, GJ;Sklar, LA
α（4）β（1）-整联蛋白（极晚期抗原-4（VLA-4），CD 49 d/CD 29）是参与淋巴细胞、树突状细胞和干细胞与细胞外基质和内皮细胞的相互作用的粘附受体。这种整合素和其他整合素能够通过影响细胞粘附亲合力的称为“由内而外”的信号传导过程来调节它们对配体的亲和力。已知有几种机制可以调节整合素的亲和力和构象：由C-末端尾部、二价离子和还原剂的分离诱导的构象变化。最近，我们描述了用于监测活细胞中VLA-4亲和力变化的含荧光LDV的小分子（Chigaev，A.，Blenc，A. M.，Braaten，J. V.，Kumaraswamy，N.，凯普利角L.，安德鲁斯河，巴西-地P.，奥利弗，J.M.，爱德华兹，B。美国，Prossnitz，E. R.，拉森河，巴西-地美国，和Sklar，L. A.（2001）J.Biol.Chem.276，48670-48678）。使用相同的分子，我们还开发了一种基于荧光共振能量转移的测定，以探测VLA-4激活后的“弹簧刀样”开放。在这里，我们研究了还原剂对VLA-4整合素的亲和力和构象状态的影响，同时通过G蛋白偶联受体或Mn 2+在活细胞中真实的时间内由内而外的信号传导启动细胞活化。我们发现，还原剂（二硫苏糖醇和2，3-二巯基-1-丙磺酸）诱导VLA-4的高亲和力的多个状态，其中亲和力的变化伴随着整联蛋白分子的延伸。杆菌肽，一种质膜还原功能的抑制剂，减少了二硫苏糖醇的作用，但对由内而外的信号传导没有影响。基于这一结果和整合素活化动力学的差异，我们得出结论，VLA-4由内而外的信号传导的构象活化是独立的，并添加到还原调节的整合素活化。
The alpha(4)beta(1)-integrin (very late antigen-4 (VLA-4), CD49d/CD29) is an adhesion receptor involved in the interaction of lymphocytes, dendritic cells, and stem cells with the extracellular matrix and endothelial cells. This and other integrins have the ability to regulate their affinity for ligands through a process termed "inside-out" signaling that affects cell adhesion avidity. Several mechanisms are known to regulate integrin affinity and conformation: conformational changes induced by separation of the C-terminal tails, divalent ions, and reducing agents. Recently, we described a fluorescent LDV-containing small molecule that was used to monitor VLA-4 affinity changes in live cells (Chigaev, A., Blenc, A. M., Braaten, J. V., Kumaraswamy, N., Kepley, C. L., Andrews, R. P., Oliver, J. M., Edwards, B. S., Prossnitz, E. R., Larson, R. S., and Sklar, L. A. (2001) J. Biol. Chem. 276, 48670-48678). Using the same molecule, we also developed a fluorescence resonance energy transfer-based assay to probe the "switchblade-like" opening of VLA-4 upon activation. Here, we investigated the effect of reducing agents on the affinity and conformational state of the VLA-4 integrin simultaneously with cell activation initiated by inside-out signaling through G protein-coupled receptors or Mn2+ in live cells in real time. We found that reducing agents ( dithiothreitol and 2,3-dimercapto-1-propanesulfonic acid) induced multiple states of high affinity of VLA-4, where the affinity change was accompanied by an extension of the integrin molecule. Bacitracin, an inhibitor of the reductive function of the plasma membrane, diminished the effect of dithiothreitol, but had no effect on inside-out signaling. Based on this result and differences in the kinetics of integrin activation, we conclude that conformational activation of VLA-4 by inside-out signaling is independent of and additive to reduction-regulated integrin activation.