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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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Tie1 does not affect the agonist activity of Ang2. (A) Endothelial cells were treated with control vehicle or 10 ng/ml PMA before addition of 200 ng/ml Ang2 for 30 min as indicated. Cells were lysed and immunoprecipitated with anti-phosphotyrosine antibodies (Py-IP). Tie2 in immunoprecipitates and Tie2 and Tie1 in whole cell lysates (Wcl) were detected by immunoblotting following SDS/PAGE. Tie1 ectodomain cleavage is indicated by loss of the higher molecular mass Tie1 immunoreactive band corresponding to surface expressed Tie1 (arrow). The effect of PMA on Ang2-induced Tie2 phosphorylation was determined in three independent experiments by immunoblotting and densitometric quantification of blots. Data are presented as means and SEM. ⁎ indicates Ang2 significantly increased Tie2 phosphorylation (p < 0.05, Student's t test). (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 Ang2 for 30 min. Cells were lysed and immunoprecipitated with anti-phosphotyrosine antibodies (Py-IP). Tie2 detected in immunoprecipitates and Tie2 and Tie1 in whole cell lysates (Wcl) were detected by immunoblotting following SDS/PAGE. The effect of control and Tie1 siRNA on the ability of Ang2 to stimulate Tie2 phosphorylation was determined in three independent experiments by immunoblotting and densitometric quantification of blots. Data are presented as means and SEM. ⁎ indicates Ang2 significantly increased Tie2 phosphorylation compared with control Sc Si transfected cells (p < 0.05, Student's t test), however loss of Tie1 did not enhance Ang2-activation of Tie2.
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fig4: Tie1 does not affect the agonist activity of Ang2. (A) Endothelial cells were treated with control vehicle or 10 ng/ml PMA before addition of 200 ng/ml Ang2 for 30 min as indicated. Cells were lysed and immunoprecipitated with anti-phosphotyrosine antibodies (Py-IP). Tie2 in immunoprecipitates and Tie2 and Tie1 in whole cell lysates (Wcl) were detected by immunoblotting following SDS/PAGE. Tie1 ectodomain cleavage is indicated by loss of the higher molecular mass Tie1 immunoreactive band corresponding to surface expressed Tie1 (arrow). The effect of PMA on Ang2-induced Tie2 phosphorylation was determined in three independent experiments by immunoblotting and densitometric quantification of blots. Data are presented as means and SEM. ⁎ indicates Ang2 significantly increased Tie2 phosphorylation (p < 0.05, Student's t test). (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 Ang2 for 30 min. Cells were lysed and immunoprecipitated with anti-phosphotyrosine antibodies (Py-IP). Tie2 detected in immunoprecipitates and Tie2 and Tie1 in whole cell lysates (Wcl) were detected by immunoblotting following SDS/PAGE. The effect of control and Tie1 siRNA on the ability of Ang2 to stimulate Tie2 phosphorylation was determined in three independent experiments by immunoblotting and densitometric quantification of blots. Data are presented as means and SEM. ⁎ indicates Ang2 significantly increased Tie2 phosphorylation compared with control Sc Si transfected cells (p < 0.05, Student's t test), however loss of Tie1 did not enhance Ang2-activation of Tie2.

Mentions: The reason why Ang2 is a partial rather than full agonist is not known. It is possible that Tie1 could suppress full agonist activity of Ang2. Therefore to examine the influence of Tie1 ectodomain and full-length Tie1 on the agonist activity of Ang2 we examined the effects of Tie1 ectodomain cleavage on the ability of Ang2 to activate Tie2 phosphorylation. Endothelial cells were treated with Ang2 in the presence or absence of PMA and phosphorylated Tie2 immunoprecipitated. Ang2 induced only a marginal increase in the amount of Tie2 immunoprecipitated by anti-phosphotyrosine antibodies demonstrating a mild agonist activity. However, induction of Tie1 ectodomain cleavage did not increase the ability of Ang2 to activate Tie2 (Fig. 4A). The impact of Tie1 on Ang2 activation of Tie2 was further examined by suppression of Tie1 expression using siRNA. Endothelial cells transfected with siRNA directed against Tie1 expressed undetectable levels of this receptor (Fig. 4B). Again, Ang2 exhibited very low agonist activity and this was not increased by removal of Tie1 (Fig. 4B). These data demonstrate that Tie1 does not affect the agonist activity of Ang2.


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)

Tie1 does not affect the agonist activity of Ang2. (A) Endothelial cells were treated with control vehicle or 10 ng/ml PMA before addition of 200 ng/ml Ang2 for 30 min as indicated. Cells were lysed and immunoprecipitated with anti-phosphotyrosine antibodies (Py-IP). Tie2 in immunoprecipitates and Tie2 and Tie1 in whole cell lysates (Wcl) were detected by immunoblotting following SDS/PAGE. Tie1 ectodomain cleavage is indicated by loss of the higher molecular mass Tie1 immunoreactive band corresponding to surface expressed Tie1 (arrow). The effect of PMA on Ang2-induced Tie2 phosphorylation was determined in three independent experiments by immunoblotting and densitometric quantification of blots. Data are presented as means and SEM. ⁎ indicates Ang2 significantly increased Tie2 phosphorylation (p < 0.05, Student's t test). (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 Ang2 for 30 min. Cells were lysed and immunoprecipitated with anti-phosphotyrosine antibodies (Py-IP). Tie2 detected in immunoprecipitates and Tie2 and Tie1 in whole cell lysates (Wcl) were detected by immunoblotting following SDS/PAGE. The effect of control and Tie1 siRNA on the ability of Ang2 to stimulate Tie2 phosphorylation was determined in three independent experiments by immunoblotting and densitometric quantification of blots. Data are presented as means and SEM. ⁎ indicates Ang2 significantly increased Tie2 phosphorylation compared with control Sc Si transfected cells (p < 0.05, Student's t test), however loss of Tie1 did not enhance Ang2-activation of Tie2.
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fig4: Tie1 does not affect the agonist activity of Ang2. (A) Endothelial cells were treated with control vehicle or 10 ng/ml PMA before addition of 200 ng/ml Ang2 for 30 min as indicated. Cells were lysed and immunoprecipitated with anti-phosphotyrosine antibodies (Py-IP). Tie2 in immunoprecipitates and Tie2 and Tie1 in whole cell lysates (Wcl) were detected by immunoblotting following SDS/PAGE. Tie1 ectodomain cleavage is indicated by loss of the higher molecular mass Tie1 immunoreactive band corresponding to surface expressed Tie1 (arrow). The effect of PMA on Ang2-induced Tie2 phosphorylation was determined in three independent experiments by immunoblotting and densitometric quantification of blots. Data are presented as means and SEM. ⁎ indicates Ang2 significantly increased Tie2 phosphorylation (p < 0.05, Student's t test). (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 Ang2 for 30 min. Cells were lysed and immunoprecipitated with anti-phosphotyrosine antibodies (Py-IP). Tie2 detected in immunoprecipitates and Tie2 and Tie1 in whole cell lysates (Wcl) were detected by immunoblotting following SDS/PAGE. The effect of control and Tie1 siRNA on the ability of Ang2 to stimulate Tie2 phosphorylation was determined in three independent experiments by immunoblotting and densitometric quantification of blots. Data are presented as means and SEM. ⁎ indicates Ang2 significantly increased Tie2 phosphorylation compared with control Sc Si transfected cells (p < 0.05, Student's t test), however loss of Tie1 did not enhance Ang2-activation of Tie2.
Mentions: The reason why Ang2 is a partial rather than full agonist is not known. It is possible that Tie1 could suppress full agonist activity of Ang2. Therefore to examine the influence of Tie1 ectodomain and full-length Tie1 on the agonist activity of Ang2 we examined the effects of Tie1 ectodomain cleavage on the ability of Ang2 to activate Tie2 phosphorylation. Endothelial cells were treated with Ang2 in the presence or absence of PMA and phosphorylated Tie2 immunoprecipitated. Ang2 induced only a marginal increase in the amount of Tie2 immunoprecipitated by anti-phosphotyrosine antibodies demonstrating a mild agonist activity. However, induction of Tie1 ectodomain cleavage did not increase the ability of Ang2 to activate Tie2 (Fig. 4A). The impact of Tie1 on Ang2 activation of Tie2 was further examined by suppression of Tie1 expression using siRNA. Endothelial cells transfected with siRNA directed against Tie1 expressed undetectable levels of this receptor (Fig. 4B). Again, Ang2 exhibited very low agonist activity and this was not increased by removal of Tie1 (Fig. 4B). These data demonstrate that Tie1 does not affect the agonist activity of Ang2.

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