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Effects of angiopoietins-1 and -2 on the receptor tyrosine kinase Tie2 are differentially regulated at the endothelial cell surface.

Hansen TM, Singh H, Tahir TA, Brindle NP - Cell. Signal. (2010)

Bottom Line: In contrast to Ang1, binding of Ang2 to Tie2 was found to be not affected by Tie1.Stimulation of Tie1 ectodomain cleavage did not increase the agonist activity of Ang2 for Tie2.This provides a mechanism by which signalling through Tie2 can be modified by stimuli in the cellular microenvironment.

View Article: PubMed Central - PubMed

Affiliation: University of Leicester, Department of Cardiovascular Sciences, RKCSB, PO Box 65, Leicester LE2 7LX, UK.

ABSTRACT
Angiopoietin-1 (Ang1) and Ang2 are ligands for the receptor tyrosine kinase Tie2. Structural data suggest that the two ligands bind Tie2 similarly. However, in endothelial cells Ang1 activates Tie2 whereas Ang2 can act as an apparent antagonist. In addition, each ligand exhibits distinct kinetics of release following binding. These observations suggest that additional factors influence function and binding of angiopoietins with receptors in the cellular context. Previous work has shown that Ang1 binding and activation of Tie2 are inhibited by Tie1, a related receptor that complexes with Tie2 in cells. In this study we have investigated binding of Ang1 and Ang2 to Tie2 in endothelial cells. In contrast to Ang1, binding of Ang2 to Tie2 was found to be not affected by Tie1. Neither PMA-induced Tie1 ectodomain cleavage nor suppression of Tie1 expression by siRNA affected the ability of Ang2 to bind Tie2. Analysis of the level of Tie1 co-immunoprecipitating with angiopoietin-bound Tie2 demonstrated that Ang2 can bind Tie2 in Tie2:Tie1 complexes whereas Ang1 preferentially binds non-complexed Tie2. Stimulation of Tie1 ectodomain cleavage did not increase the agonist activity of Ang2 for Tie2. Similarly, the Tie2-agonist activity of Ang2 was not affected by siRNA suppression of Tie1 expression. Consistent with previous reports, loss of Tie1 ectodomain enhanced the agonist activity of Ang1 for Tie2. Importantly, Ang2 was still able to antagonize the elevated Ang1-activation of Tie2 that occurs on Tie1 ectodomain loss. Together these data demonstrate that Ang1 and Ang2 bind differently to Tie2 at the cell surface and this is controlled by Tie1. This differential regulation of angiopoietin binding allows control of Tie2 activation response to Ang1 without affecting Ang2 agonist activity and maintains the ability of Ang2 to antagonize even the enhanced Ang1 activation of Tie2 that occurs on loss of Tie1 ectodomain. This provides a mechanism by which signalling through Tie2 can be modified by stimuli in the cellular microenvironment.

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Suppression of Tie1 expression differentially affects binding of Ang1 and Ang2 to Tie2 in endothelial cells. (A) Endothelial cells were transfected with siRNA directed against Tie1 or control randomised siRNA (Sc) and cultured for 24 h before addition of control vehicle (C) or 200 ng/ml Ang2 (A2) for 30 min as indicated followed by cross-linking with the cell-impermeable cross-linker DTSSP. 20 mM Tris was added to quench cross-linking before washing and cell lysis. Ang2 was immunoprecipitated and immunoprecipitates or whole cell lysates (Wcl) were resolved by SDS/PAGE. Tie2 bound to Ang2 and Tie1 and Tie2 in whole cell lysates were detected by immunoblotting as indicated. (B) Endothelial cells were transfected with siRNA directed against Tie1 or control randomised siRNA (Sc) and cultured for 24 h before addition of 200 ng/ml Ang1 (A1) or Ang2 (A2) for 30 min as indicated. Cross-linking, quenching and immunoprecipitation were performed as above and Tie2 bound to Ang1 and Ang2 and Tie1 and Tie2 in whole cell lysates were detected by immunoblotting as indicated. The effect of suppression of Tie1 expression by siRNA on Ang1 and Ang2 binding to Tie2 on cells was determined in three independent experiments by immunoblotting and densitometric quantification of blots. Data are presented as means and SEM. ⁎ indicates Ang1 binding to Tie2 was significantly increased by loss of Tie1 (p < 0.05, Student's t test).
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fig2: Suppression of Tie1 expression differentially affects binding of Ang1 and Ang2 to Tie2 in endothelial cells. (A) Endothelial cells were transfected with siRNA directed against Tie1 or control randomised siRNA (Sc) and cultured for 24 h before addition of control vehicle (C) or 200 ng/ml Ang2 (A2) for 30 min as indicated followed by cross-linking with the cell-impermeable cross-linker DTSSP. 20 mM Tris was added to quench cross-linking before washing and cell lysis. Ang2 was immunoprecipitated and immunoprecipitates or whole cell lysates (Wcl) were resolved by SDS/PAGE. Tie2 bound to Ang2 and Tie1 and Tie2 in whole cell lysates were detected by immunoblotting as indicated. (B) Endothelial cells were transfected with siRNA directed against Tie1 or control randomised siRNA (Sc) and cultured for 24 h before addition of 200 ng/ml Ang1 (A1) or Ang2 (A2) for 30 min as indicated. Cross-linking, quenching and immunoprecipitation were performed as above and Tie2 bound to Ang1 and Ang2 and Tie1 and Tie2 in whole cell lysates were detected by immunoblotting as indicated. The effect of suppression of Tie1 expression by siRNA on Ang1 and Ang2 binding to Tie2 on cells was determined in three independent experiments by immunoblotting and densitometric quantification of blots. Data are presented as means and SEM. ⁎ indicates Ang1 binding to Tie2 was significantly increased by loss of Tie1 (p < 0.05, Student's t test).

Mentions: The effects of Tie1 on binding of Ang2 to Tie2 were explored further using an siRNA approach to generate endothelial cells lacking Tie1. Cells were transfected with control siRNA or siRNA directed against Tie1 and expression of Tie1 determined by immunoblotting. Tie1 siRNA effectively suppressed Tie1 expression (Fig. 2A). Interaction of Ang2 with Tie2 in endothelial cells expressing Tie1, and in which Tie1 expression was inhibited by siRNA, was determined by addition of the ligand and immunoprecipitation as before. Ang2 was able to bind Tie2 equally well in the absence and presence of Tie1 (Fig. 2A). The different effects of Tie1 on interaction of Ang1 and Ang2 with Tie2 were directly compared by examining the ability of Ang1 and Ang2 to bind and recover Tie2 from control cells and cells lacking Tie1. Loss of Tie1 did not affect the binding of Ang2 to Tie2, however, binding of Ang1 to Tie2 was increased in the absence of Tie1 (Fig. 2B).


Effects of angiopoietins-1 and -2 on the receptor tyrosine kinase Tie2 are differentially regulated at the endothelial cell surface.

Hansen TM, Singh H, Tahir TA, Brindle NP - Cell. Signal. (2010)

Suppression of Tie1 expression differentially affects binding of Ang1 and Ang2 to Tie2 in endothelial cells. (A) Endothelial cells were transfected with siRNA directed against Tie1 or control randomised siRNA (Sc) and cultured for 24 h before addition of control vehicle (C) or 200 ng/ml Ang2 (A2) for 30 min as indicated followed by cross-linking with the cell-impermeable cross-linker DTSSP. 20 mM Tris was added to quench cross-linking before washing and cell lysis. Ang2 was immunoprecipitated and immunoprecipitates or whole cell lysates (Wcl) were resolved by SDS/PAGE. Tie2 bound to Ang2 and Tie1 and Tie2 in whole cell lysates were detected by immunoblotting as indicated. (B) Endothelial cells were transfected with siRNA directed against Tie1 or control randomised siRNA (Sc) and cultured for 24 h before addition of 200 ng/ml Ang1 (A1) or Ang2 (A2) for 30 min as indicated. Cross-linking, quenching and immunoprecipitation were performed as above and Tie2 bound to Ang1 and Ang2 and Tie1 and Tie2 in whole cell lysates were detected by immunoblotting as indicated. The effect of suppression of Tie1 expression by siRNA on Ang1 and Ang2 binding to Tie2 on cells was determined in three independent experiments by immunoblotting and densitometric quantification of blots. Data are presented as means and SEM. ⁎ indicates Ang1 binding to Tie2 was significantly increased by loss of Tie1 (p < 0.05, Student's t test).
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fig2: Suppression of Tie1 expression differentially affects binding of Ang1 and Ang2 to Tie2 in endothelial cells. (A) Endothelial cells were transfected with siRNA directed against Tie1 or control randomised siRNA (Sc) and cultured for 24 h before addition of control vehicle (C) or 200 ng/ml Ang2 (A2) for 30 min as indicated followed by cross-linking with the cell-impermeable cross-linker DTSSP. 20 mM Tris was added to quench cross-linking before washing and cell lysis. Ang2 was immunoprecipitated and immunoprecipitates or whole cell lysates (Wcl) were resolved by SDS/PAGE. Tie2 bound to Ang2 and Tie1 and Tie2 in whole cell lysates were detected by immunoblotting as indicated. (B) Endothelial cells were transfected with siRNA directed against Tie1 or control randomised siRNA (Sc) and cultured for 24 h before addition of 200 ng/ml Ang1 (A1) or Ang2 (A2) for 30 min as indicated. Cross-linking, quenching and immunoprecipitation were performed as above and Tie2 bound to Ang1 and Ang2 and Tie1 and Tie2 in whole cell lysates were detected by immunoblotting as indicated. The effect of suppression of Tie1 expression by siRNA on Ang1 and Ang2 binding to Tie2 on cells was determined in three independent experiments by immunoblotting and densitometric quantification of blots. Data are presented as means and SEM. ⁎ indicates Ang1 binding to Tie2 was significantly increased by loss of Tie1 (p < 0.05, Student's t test).
Mentions: The effects of Tie1 on binding of Ang2 to Tie2 were explored further using an siRNA approach to generate endothelial cells lacking Tie1. Cells were transfected with control siRNA or siRNA directed against Tie1 and expression of Tie1 determined by immunoblotting. Tie1 siRNA effectively suppressed Tie1 expression (Fig. 2A). Interaction of Ang2 with Tie2 in endothelial cells expressing Tie1, and in which Tie1 expression was inhibited by siRNA, was determined by addition of the ligand and immunoprecipitation as before. Ang2 was able to bind Tie2 equally well in the absence and presence of Tie1 (Fig. 2A). The different effects of Tie1 on interaction of Ang1 and Ang2 with Tie2 were directly compared by examining the ability of Ang1 and Ang2 to bind and recover Tie2 from control cells and cells lacking Tie1. Loss of Tie1 did not affect the binding of Ang2 to Tie2, however, binding of Ang1 to Tie2 was increased in the absence of Tie1 (Fig. 2B).

Bottom Line: In contrast to Ang1, binding of Ang2 to Tie2 was found to be not affected by Tie1.Stimulation of Tie1 ectodomain cleavage did not increase the agonist activity of Ang2 for Tie2.This provides a mechanism by which signalling through Tie2 can be modified by stimuli in the cellular microenvironment.

View Article: PubMed Central - PubMed

Affiliation: University of Leicester, Department of Cardiovascular Sciences, RKCSB, PO Box 65, Leicester LE2 7LX, UK.

ABSTRACT
Angiopoietin-1 (Ang1) and Ang2 are ligands for the receptor tyrosine kinase Tie2. Structural data suggest that the two ligands bind Tie2 similarly. However, in endothelial cells Ang1 activates Tie2 whereas Ang2 can act as an apparent antagonist. In addition, each ligand exhibits distinct kinetics of release following binding. These observations suggest that additional factors influence function and binding of angiopoietins with receptors in the cellular context. Previous work has shown that Ang1 binding and activation of Tie2 are inhibited by Tie1, a related receptor that complexes with Tie2 in cells. In this study we have investigated binding of Ang1 and Ang2 to Tie2 in endothelial cells. In contrast to Ang1, binding of Ang2 to Tie2 was found to be not affected by Tie1. Neither PMA-induced Tie1 ectodomain cleavage nor suppression of Tie1 expression by siRNA affected the ability of Ang2 to bind Tie2. Analysis of the level of Tie1 co-immunoprecipitating with angiopoietin-bound Tie2 demonstrated that Ang2 can bind Tie2 in Tie2:Tie1 complexes whereas Ang1 preferentially binds non-complexed Tie2. Stimulation of Tie1 ectodomain cleavage did not increase the agonist activity of Ang2 for Tie2. Similarly, the Tie2-agonist activity of Ang2 was not affected by siRNA suppression of Tie1 expression. Consistent with previous reports, loss of Tie1 ectodomain enhanced the agonist activity of Ang1 for Tie2. Importantly, Ang2 was still able to antagonize the elevated Ang1-activation of Tie2 that occurs on Tie1 ectodomain loss. Together these data demonstrate that Ang1 and Ang2 bind differently to Tie2 at the cell surface and this is controlled by Tie1. This differential regulation of angiopoietin binding allows control of Tie2 activation response to Ang1 without affecting Ang2 agonist activity and maintains the ability of Ang2 to antagonize even the enhanced Ang1 activation of Tie2 that occurs on loss of Tie1 ectodomain. This provides a mechanism by which signalling through Tie2 can be modified by stimuli in the cellular microenvironment.

Show MeSH
Related in: MedlinePlus