Frontline Science: A flexible kink in the transmembrane domain impairs β2 integrin extension and cell arrest from rolling
Frontline Science: A flexible kink in the transmembrane domain impairs β2 integrin extension and cell arrest from rolling
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DOI：
10.1002/jlb.1hi0219-073rr
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发表时间：
2019-09-01
影响因子：
5.5
通讯作者：
Ley, Klaus
中科院分区：
文献类型：
--
作者：
Sun, Hao;Fan, Zhichao;Ley, Klaus
β 2整合素是中性粒细胞和其他白细胞中的主要粘附分子，可被由内而外的信号迅速激活，从而导致通过跨膜结构域（TMD）传递的构象变化。在这里，我们研究了在β 2整合素TMD中引入脯氨酸突变的生物学效应，以创建一个灵活的扭结，使TMD内半部的拓扑结构与外半部分离，并损害整合素的激活。β 2整合素链，α L, α M， α X和α D都包含一个插入(I)结构域，与von Willebrand因子A结构域同源。通过两种报告性单克隆抗体的均匀结合试验监测β 2的激活：KIM127报告扩展（E+）， mAb24报告β 2 I-like结构域的高亲和力（H+）构象。脯氨酸突变部分减少了趋化因子诱导的延伸，但没有减少高亲和力构象。β 2 TMD中的脯氨酸突变完全抑制了HL-60细胞对趋化因子IL-8的反应。在p -选择素和ICAM-1上滚动的TMD突变体HL-60细胞在IL-8的作用下不能降低其滚动速度。定量动态足迹活细胞成像显示，阻断TMD拓扑传递会损害趋化因子诱导的β 2激活，限制扩展高亲和力(E+H+) β 2的出现。这也导致了早期扩散缺陷（阻滞后3分钟），这可以通过使用Mn2+强制整合素激活来克服。我们得出结论，TMD脯氨酸突变严重损害β 2整合素延伸、细胞阻滞和早期扩散。
beta 2 integrins are the main adhesion molecules in neutrophils and other leukocytes and are rapidly activated by inside-out signaling, which results in conformational changes that are transmitted through the transmembrane domain (TMD). Here, we investigated the biologic effect of introducing a proline mutation in the beta 2 integrin TMD to create a flexible kink that uncouples the topology of the inner half of the TMD from the outer half and impairs integrin activation. The beta 2 integrin alpha chains, alpha L, alpha M, alpha X, and alpha D, all contain an inserted (I) domain with homology to von Willebrand factor A domain. beta 2 activation was monitored in a homogenous binding assay of 2 reporter monoclonal antibodies: KIM127 reporting extension (E+) and mAb24 reporting the high-affinity (H+) conformation of the beta 2 I-like domain. The proline mutation partially diminished chemokine-induced extension, but not the high-affinity conformation. The proline mutation in the TMD of beta 2 completely inhibited arrest of rolling HL-60 cells in response to the chemokine IL-8. TMD mutant HL-60 cells rolling on P-selectin and ICAM-1 were unable to reduce their rolling velocity in response to IL-8. Quantitative dynamic footprinting live-cell imaging showed that blocking TMD topology transmission impaired the chemokine-induced activation of beta 2, limiting the appearance of extended high-affinity (E+H+) beta 2. This also resulted in a defect in early spreading (3 min after arrest), which could be overcome by forced integrin activation using Mn2+. We conclude that the TMD proline mutation severely impairs beta 2 integrin extension, cell arrest, and early spreading.