Background: Interactions between tissue factor (TF) and β1-integrin induce cell signals, but the molecular mechanisms are not completely understood. The extracellular domain of TF and EGF4-βTD domains of β1-integrin were hypothesised to be the most likely domains involved in the interaction. Additionally, the interaction may induce a conformational change in β1-integrin, which results in changes in signalling. Methods: Peptide constructs corresponding to the upper (residues 1–110; UED), lower (residues 106–219; LED) or combined extracellular domain (residues 1–219; TED) of TF were produced, as well as peptides corresponding to EGF4-βTD or EGF4 domains of β1-integrin. These constructs were expressed in TF-rich MDA-MB-231 cells and TF-deficient primary endothelial cells. The association of the peptides with endogenous-TF or β1-integrin was assessed by a proximity ligation assay and co-immunoprecipitation. Additionally, the influence of the constructs on β1-integrin conformation and the outcome on ERK1/2 activation, cyclin D expression and cell proliferation was analysed. Results: In MDA-MB-231 cells, all TF-constructs were associated with β1-integrin whilst LED was co-immunopurified with β1-integrin. EGF4-βTD was associated with and co-immunopurified with endogenous TF. Additionally, the expression of UED or EGF4-βTD reduced ERK phosphorylation and cyclin D expression and suppressed proliferation. In endothelial cells, the expression of UED, and to a lesser extent, LED, reduced the proportion of β1-integrin in the active conformation and induced ERK1/2 phosphorylation but did not induce cyclin D expression or proliferation. Conclusions: Collectively, these data indicate the extracellular domains of TF function together, with the lower domain forming a robust interaction with the βTD of β1-integrin and the upper domain inducing cell signalling by regulating β1-integrin conformation.
背景：组织因子（TF）与β1整合素之间的相互作用可诱导细胞信号传导，但其分子机制尚未完全阐明。研究假设TF的胞外结构域与β1整合素的EGF4-βTD结构域最可能参与该相互作用。此外，这种相互作用可能诱导β1整合素构象变化，进而影响信号传导。 方法：本研究制备了对应TF胞外结构域上段（第1-110位氨基酸；UED）、下段（第106-219位氨基酸；LED）及完整胞外段（第1-219位氨基酸；TED）的多肽构建体，以及对应β1整合素EGF4-βTD或EGF4结构域的多肽。这些构建体在TF高表达的MDA-MB-231细胞和TF缺陷的原代内皮细胞中进行表达。通过邻近连接实验和免疫共沉淀技术评估多肽与内源性TF或β1整合素的结合情况。此外，分析了构建体对β1整合素构象的影响，及其对ERK1/2活化、细胞周期蛋白D表达和细胞增殖的作用。 结果：在MDA-MB-231细胞中，所有TF构建体均与β1整合素存在关联，其中LED可与β1整合素发生免疫共纯化。EGF4-βTD结构域与内源性TF存在关联并可免疫共纯化。UED或EGF4-βTD的表达可降低ERK磷酸化水平和细胞周期蛋白D表达，并抑制细胞增殖。在内皮细胞中，UED（及程度较轻的LED）表达可降低活性构象β1整合素的比例，诱导ERK1/2磷酸化，但未引起细胞周期蛋白D表达或细胞增殖。 结论：综合结果表明，TF胞外结构域通过协同作用发挥功能：下段结构域与β1整合素βTD形成稳定结合，而上段结构域通过调控β1整合素构象诱导细胞信号传导。
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