Chigaev Alexandre, Zwartz Gordon J, Buranda Tione, Edwards Bruce S, Prossnitz Eric R, Sklar Larry A
Department of Pathology and the Cancer Center, University of New Mexico Health Sciences Center, Albequerque, 87131, USA.
J Biol Chem. 2004 Jul 30;279(31):32435-43. doi: 10.1074/jbc.M404387200. Epub 2004 May 27.
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 Mn(2+) 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.
α(4)β(1)整合素（极迟抗原-4（VLA-4），CD49d/CD29）是一种黏附受体，参与淋巴细胞、树突状细胞和干细胞与细胞外基质及内皮细胞的相互作用。这种整合素及其他整合素能够通过一种称为“由内向外”信号传导的过程来调节其对配体的亲和力，该过程会影响细胞黏附亲和力。已知有几种机制可调节整合素的亲和力和构象：C末端尾巴分离、二价离子和还原剂诱导的构象变化。最近，我们描述了一种含LDV的荧光小分子，用于监测活细胞中VLA-4亲和力的变化（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）。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检测方法，以探测激活时VLA-4的“弹簧刀样”打开。在此，我们实时研究了还原剂对VLA-4整合素亲和力和构象状态的影响，同时研究了活细胞中通过G蛋白偶联受体或Mn(2+)由内向外信号传导引发的细胞激活。我们发现还原剂（二硫苏糖醇和2,3-二巯基-1-丙磺酸钠）诱导VLA-4出现多种高亲和力状态，其中亲和力变化伴随着整合素分子的伸展。杆菌肽是质膜还原功能的抑制剂，它减弱了二硫苏糖醇的作用，但对由内向外信号传导没有影响。基于这一结果以及整合素激活动力学的差异，我们得出结论，由内向外信号传导引起的VLA-4构象激活独立于还原调节的整合素激活，且二者具有叠加性。
