Identification of the Interacting Domains Between Tissue Factor and β1-Integrin and the Signalling Properties of the Two Fibronectin-like Domains of Tissue Factor

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

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.

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.

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.