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Intramembrane binding of VE-cadherin to VEGFR2 and VEGFR3 assembles the endothelial mechanosensory complex.

Coon BG, Baeyens N, Han J, Budatha M, Ross TD, Fang JS, Yun S, Thomas JL, Schwartz MA - J. Cell Biol. (2015)

Bottom Line: We now show that the transmembrane domain of VE-cadherin mediates an essential adapter function by binding directly to the transmembrane domain of VEGFR2, as well as VEGFR3, which we now identify as another component of the junctional mechanosensory complex.Furthermore, VEGFR3 expression is observed in the aortic endothelium, where it contributes to flow responses in vivo.In summary, this study identifies a novel adapter function for VE-cadherin mediated by transmembrane domain association with VEGFRs.

View Article: PubMed Central - HTML - PubMed

Affiliation: Yale Cardiovascular Research Center and Department of Internal Medicine, Cardiovascular Medicine, Yale University School of Medicine, New Haven, CT 06510 Yale Cardiovascular Research Center and Department of Internal Medicine, Cardiovascular Medicine, Yale University School of Medicine, New Haven, CT 06510.

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VEGFRs in PI 3-kinase and integrin activation. (A) VEGFR knockdown. HUVECs transfected with the indicated siRNAs for 72 h were analyzed for VEGFR expression by immunoblotting. Three independent experiments gave similar results. (B) PI3K signaling. HUVECs transfected as in A were subjected to shear stress and p85 was immunoprecipitated. Samples were then analyzed by immunoblotting and densitometry as in Fig 2 C. Values are means ± SEM, n = 4. (C) Akt signaling. siRNA-treated HUVECs were subjected to laminar shear stress for the indicated times and lysates were analyzed by immunoblotting with the indicated antibodies to determine Akt activity. Results were quantified by densitometry, with actin serving as a loading control. Values are means ± SEM (error bars), n = 3. *, P < 0.05 relative to unstimulated cells by two-way ANOVA. IB, immunoblotting.
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fig5: VEGFRs in PI 3-kinase and integrin activation. (A) VEGFR knockdown. HUVECs transfected with the indicated siRNAs for 72 h were analyzed for VEGFR expression by immunoblotting. Three independent experiments gave similar results. (B) PI3K signaling. HUVECs transfected as in A were subjected to shear stress and p85 was immunoprecipitated. Samples were then analyzed by immunoblotting and densitometry as in Fig 2 C. Values are means ± SEM, n = 4. (C) Akt signaling. siRNA-treated HUVECs were subjected to laminar shear stress for the indicated times and lysates were analyzed by immunoblotting with the indicated antibodies to determine Akt activity. Results were quantified by densitometry, with actin serving as a loading control. Values are means ± SEM (error bars), n = 3. *, P < 0.05 relative to unstimulated cells by two-way ANOVA. IB, immunoblotting.

Mentions: These results raise questions about the relative contribution of VEGFR2 and -3 in flow signaling. Published functional analyses (Jin et al., 2003; Tzima et al., 2005) used chemical inhibitors of tyrosine kinase activity that do not discriminate between these paralogues (Eskens and Verweij, 2006), thus, it is unclear to what extent VEGFR2 and -3 have unique effectors, and function additively or redundantly. VEGFR2 is required for shear-mediated activation of PI3K and integrins, and for cell alignment. We therefore performed shear experiments with human ECs treated with siRNAs against each VEGFR, or both (Fig. 5 A). Depletion of the individual VEGFRs partially decreased phosphorylation of PI3K and had a slight effect on Akt activation, as determined by immunoblotting pS473 (Warfel et al., 2011), whereas depletion of both strongly inhibited it (Fig. 5, B and C). Therefore, VEGFR2 and -3 both contribute to shear-mediated PI3K-Akt signaling.


Intramembrane binding of VE-cadherin to VEGFR2 and VEGFR3 assembles the endothelial mechanosensory complex.

Coon BG, Baeyens N, Han J, Budatha M, Ross TD, Fang JS, Yun S, Thomas JL, Schwartz MA - J. Cell Biol. (2015)

VEGFRs in PI 3-kinase and integrin activation. (A) VEGFR knockdown. HUVECs transfected with the indicated siRNAs for 72 h were analyzed for VEGFR expression by immunoblotting. Three independent experiments gave similar results. (B) PI3K signaling. HUVECs transfected as in A were subjected to shear stress and p85 was immunoprecipitated. Samples were then analyzed by immunoblotting and densitometry as in Fig 2 C. Values are means ± SEM, n = 4. (C) Akt signaling. siRNA-treated HUVECs were subjected to laminar shear stress for the indicated times and lysates were analyzed by immunoblotting with the indicated antibodies to determine Akt activity. Results were quantified by densitometry, with actin serving as a loading control. Values are means ± SEM (error bars), n = 3. *, P < 0.05 relative to unstimulated cells by two-way ANOVA. IB, immunoblotting.
© Copyright Policy - openaccess
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4384728&req=5

fig5: VEGFRs in PI 3-kinase and integrin activation. (A) VEGFR knockdown. HUVECs transfected with the indicated siRNAs for 72 h were analyzed for VEGFR expression by immunoblotting. Three independent experiments gave similar results. (B) PI3K signaling. HUVECs transfected as in A were subjected to shear stress and p85 was immunoprecipitated. Samples were then analyzed by immunoblotting and densitometry as in Fig 2 C. Values are means ± SEM, n = 4. (C) Akt signaling. siRNA-treated HUVECs were subjected to laminar shear stress for the indicated times and lysates were analyzed by immunoblotting with the indicated antibodies to determine Akt activity. Results were quantified by densitometry, with actin serving as a loading control. Values are means ± SEM (error bars), n = 3. *, P < 0.05 relative to unstimulated cells by two-way ANOVA. IB, immunoblotting.
Mentions: These results raise questions about the relative contribution of VEGFR2 and -3 in flow signaling. Published functional analyses (Jin et al., 2003; Tzima et al., 2005) used chemical inhibitors of tyrosine kinase activity that do not discriminate between these paralogues (Eskens and Verweij, 2006), thus, it is unclear to what extent VEGFR2 and -3 have unique effectors, and function additively or redundantly. VEGFR2 is required for shear-mediated activation of PI3K and integrins, and for cell alignment. We therefore performed shear experiments with human ECs treated with siRNAs against each VEGFR, or both (Fig. 5 A). Depletion of the individual VEGFRs partially decreased phosphorylation of PI3K and had a slight effect on Akt activation, as determined by immunoblotting pS473 (Warfel et al., 2011), whereas depletion of both strongly inhibited it (Fig. 5, B and C). Therefore, VEGFR2 and -3 both contribute to shear-mediated PI3K-Akt signaling.

Bottom Line: We now show that the transmembrane domain of VE-cadherin mediates an essential adapter function by binding directly to the transmembrane domain of VEGFR2, as well as VEGFR3, which we now identify as another component of the junctional mechanosensory complex.Furthermore, VEGFR3 expression is observed in the aortic endothelium, where it contributes to flow responses in vivo.In summary, this study identifies a novel adapter function for VE-cadherin mediated by transmembrane domain association with VEGFRs.

View Article: PubMed Central - HTML - PubMed

Affiliation: Yale Cardiovascular Research Center and Department of Internal Medicine, Cardiovascular Medicine, Yale University School of Medicine, New Haven, CT 06510 Yale Cardiovascular Research Center and Department of Internal Medicine, Cardiovascular Medicine, Yale University School of Medicine, New Haven, CT 06510.

Show MeSH
Related in: MedlinePlus