Real-time Analysis of Conformation-sensitive Antibody Binding Provides New Insights into Integrin Conformational Regulation
Real-time Analysis of Conformation-sensitive Antibody Binding Provides New Insights into Integrin Conformational Regulation
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DOI：
10.1074/jbc.m901178200
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发表时间：
2009-05-22
影响因子：
4.8
通讯作者：
Sklar, Larry A.
中科院分区：
文献类型：
--
作者：
Chigaev, Alexandre;Waller, Anna;Sklar, Larry A.
整合素是调节免疫细胞黏附的异二聚体黏附受体。整合素依赖的黏附是由多种构象状态控制的，这些构象状态包括与配体具有不同亲和力的状态、具有不同程度的分子不弯曲的状态等。亲和力变化和分子去弯曲在细胞黏附调节中起主要作用。整合素的不同构象状态之间的关系尚不清楚。在这里，我们使用构象敏感的抗体和含有LDV的小配体来研究通过甲酰肽受体和CXCR4由内向外的信号在调节α(4)β(1)整合素构象中的作用。我们发现，在没有配体的情况下，甲酰肽或SDF-1的激活不会导致HUTS-21表位的显著暴露。在没有细胞激活的情况下占据配体结合口袋足以诱导表位暴露。在LDV存在下，HUTS-21结合的EC50与先前报道的静息和激活后的配体平衡解离常数相同。此外，HUTS-21的结合速率还与VLA-4的激活状态有关，即使在饱和配体浓度下也是如此。我们认为，整合素分子在鸟嘌呤核苷酸结合蛋白偶联受体诱导的信号转导后的伸展可以解释HUTS-21结合率的提高。综上所述，目前的结果支持存在由内向外信号和配体结合独立调节的多种构象状态。我们的数据表明，VLA-4整合素杂合域的移动不依赖于配体结合口袋的亲和力状态。
Integrins are heterodimeric adhesion receptors that regulate immune cell adhesion. Integrin-dependent adhesion is controlled by multiple conformational states that include states with different affinity to the ligand, states with various degrees of molecule unbending, and others. Affinity change and molecule unbending play major roles in the regulation of cell adhesion. The relationship between different conformational states of the integrin is unclear. Here we have used conformationally sensitive antibodies and a small LDV-containing ligand to study the role of the inside-out signaling through formyl peptide receptor and CXCR4 in the regulation of alpha(4)beta(1) integrin conformation. We found that in the absence of ligand, activation by formyl peptide or SDF-1 did not result in a significant exposure of HUTS-21 epitope. Occupancy of the ligand binding pocket without cell activation was sufficient to induce epitope exposure. EC50 for HUTS-21 binding in the presence of LDV was identical to a previously reported ligand equilibrium dissociation constant at rest and after activation. Furthermore, the rate of HUTS-21 binding was also related to the VLA-4 activation state even at saturating ligand concentration. We propose that the unbending of the integrin molecule after guanine nucleotide-binding protein-coupled receptor-induced signaling accounts for the enhanced rate of HUTS-21 binding. Taken together, current results support the existence of multiple conformational states independently regulated by both inside-out signaling and ligand binding. Our data suggest that VLA-4 integrin hybrid domain movement does not depend on the affinity state of the ligand binding pocket.