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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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VEcad TMD in shear-mediated VEGFR2 and VEGFR3 activation. (A) VEGFR activation. HUVECs exposed to 12 dynes/cm2 laminar shear for the indicated times were immunoblotted with the indicated antibodies. The upper band on the anti-VEGFR2/3pY1054/9 blot is active VEGFR2; the lower band is active VEGFR3 (arrowheads). IB, immunoblotting. (B) Dependence on TMD. VEcad−/−, VEcadWT, and VEcadΔTMD cells were subjected to shear stress, then lysed and immunoblotted as in A. The anti-VEGFRpY1230 blots were quantified by densitometry of the ∼120-kD band with actin serving as a loading control. Values are means ± SEM (error bars), n ≥ 3. Yellow lines mark boundaries between cell types.
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fig4: VEcad TMD in shear-mediated VEGFR2 and VEGFR3 activation. (A) VEGFR activation. HUVECs exposed to 12 dynes/cm2 laminar shear for the indicated times were immunoblotted with the indicated antibodies. The upper band on the anti-VEGFR2/3pY1054/9 blot is active VEGFR2; the lower band is active VEGFR3 (arrowheads). IB, immunoblotting. (B) Dependence on TMD. VEcad−/−, VEcadWT, and VEcadΔTMD cells were subjected to shear stress, then lysed and immunoblotted as in A. The anti-VEGFRpY1230 blots were quantified by densitometry of the ∼120-kD band with actin serving as a loading control. Values are means ± SEM (error bars), n ≥ 3. Yellow lines mark boundaries between cell types.

Mentions: Flow induces ligand-independent activation of VEGFR2, which is required for subsequent activation of PI3K and integrins (Jin et al., 2003; Tzima et al., 2005). We therefore tested whether flow also transactivates VEGFR3. To allow a direct comparison in these experiments, we used an antibody that recognizes phosphorylated Y1054/9 in the kinase domain activation loops, a site that is highly conserved between VEGFR2 and -3. Thus, anti-pY1054/9 does not discriminate between the two paralogues. HUVECs were subjected to short-term laminar shear and Y1054/9 phosphorylation was analyzed. Both VEGFR2 and VEGFR3 were initially activated similarly, but VEGFR3 exhibited a more sustained response (Fig. 4 A). The activation kinetics of each receptor were confirmed with additional phospho-VEGFR antibodies, anti-VEGFR2pY1175 and anti-VEGFR3pY1230, which recognize sites commonly phosphorylated in response to ligand (Fig. 4 A). Thus, VEGFR3, like VEGFR2, shows activation by flow in the absence of ligand.


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)

VEcad TMD in shear-mediated VEGFR2 and VEGFR3 activation. (A) VEGFR activation. HUVECs exposed to 12 dynes/cm2 laminar shear for the indicated times were immunoblotted with the indicated antibodies. The upper band on the anti-VEGFR2/3pY1054/9 blot is active VEGFR2; the lower band is active VEGFR3 (arrowheads). IB, immunoblotting. (B) Dependence on TMD. VEcad−/−, VEcadWT, and VEcadΔTMD cells were subjected to shear stress, then lysed and immunoblotted as in A. The anti-VEGFRpY1230 blots were quantified by densitometry of the ∼120-kD band with actin serving as a loading control. Values are means ± SEM (error bars), n ≥ 3. Yellow lines mark boundaries between cell types.
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Related In: Results  -  Collection

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fig4: VEcad TMD in shear-mediated VEGFR2 and VEGFR3 activation. (A) VEGFR activation. HUVECs exposed to 12 dynes/cm2 laminar shear for the indicated times were immunoblotted with the indicated antibodies. The upper band on the anti-VEGFR2/3pY1054/9 blot is active VEGFR2; the lower band is active VEGFR3 (arrowheads). IB, immunoblotting. (B) Dependence on TMD. VEcad−/−, VEcadWT, and VEcadΔTMD cells were subjected to shear stress, then lysed and immunoblotted as in A. The anti-VEGFRpY1230 blots were quantified by densitometry of the ∼120-kD band with actin serving as a loading control. Values are means ± SEM (error bars), n ≥ 3. Yellow lines mark boundaries between cell types.
Mentions: Flow induces ligand-independent activation of VEGFR2, which is required for subsequent activation of PI3K and integrins (Jin et al., 2003; Tzima et al., 2005). We therefore tested whether flow also transactivates VEGFR3. To allow a direct comparison in these experiments, we used an antibody that recognizes phosphorylated Y1054/9 in the kinase domain activation loops, a site that is highly conserved between VEGFR2 and -3. Thus, anti-pY1054/9 does not discriminate between the two paralogues. HUVECs were subjected to short-term laminar shear and Y1054/9 phosphorylation was analyzed. Both VEGFR2 and VEGFR3 were initially activated similarly, but VEGFR3 exhibited a more sustained response (Fig. 4 A). The activation kinetics of each receptor were confirmed with additional phospho-VEGFR antibodies, anti-VEGFR2pY1175 and anti-VEGFR3pY1230, which recognize sites commonly phosphorylated in response to ligand (Fig. 4 A). Thus, VEGFR3, like VEGFR2, shows activation by flow in the absence of ligand.

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