Fig 2. Direct association between Tie1 and Tie2 and integrins using purified proteins.

(A) Purified Tie2(1–4)-Fc protein (33nM) was incubated with purified α₅ß₁ ectodomain protein (12.5nM) in the presence or absence of either full-length or recombinant fibronectin (5μg/mL) containing only the 9th and 10th fibronectin type-III repeats (9–10). All purified components were present at the indicated concentrations in 1mL of HBST buffer and incubated overnight with 25μL ProteinA resin. Following precipitation of Tie2(1–4)-Fc with Protein-A sepharose, proteins were electrophoresed by SDS-PAGE, transferred to nitrocellulose, and Tie2 was briefly visualized with PonceauS prior to western blotting with α₅-specific antibodies. The α₅ß₁ ectodomain readily precipitates with Tie2, although the addition of full-length fibronectin, but not recombinant, truncated fibronectin (9–10), greatly enhances the Tie2/integrin interaction. (B) As in (A) using Tie1(1–4)-Fc protein (33nM) incubated with purified α₅ß₁ ectodomain protein (12.5nM) in the presence or absence of full-length fibronectin (5μg/mL). Unlike the experiments shown in (A), fibronectin does not significantly enhance the Tie1/integrin interaction. (C) Surface Plasmon Resonance depiction of purified and untagged α₅ß₁ binding to immobilized Tie1 (Fc tagged full extracellular domain protein). Similar experiments were completed with immobilized Tie2, however no interaction was captured. (D) 33nM purified Tie1(1–4)-Fc protein was incubated with purified α₅ß₁ ectodomain (12.5nM) in the presence or absence of an increasing amount of Tie2(1–4) untagged protein (either 100nM or 600nM as indicated). Following precipitation of Tie1(1–4)-Fc with Protein-A sepharose, proteins were subjected to western blotting with α₅-specific antibodies. α₅ß₁ readily precipitates with Tie1, demonstrating that the Ig and EGF repeats are sufficient for α₅ß₁ recognition. Furthermore, Tie2 (1–4) effectively competes with Tie1 for integrin binding, suggesting a shared binding interface of Tie1 and Tie2 on the integrin molecule. Quantified values represent a normalized ratio of α₅ to Tie1(1–4)-Fc.