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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 flow signaling. (A) Requirement for VEcad. HUVECS were infected with scrambled or anti-VEcad shRNA-containing lentiviruses then subjected to 12 dynes/cm2 laminar shear for 1 min. Activation of SFKs (SFKpY416, ∼55 kD) and VEGFR2 (VEGFR2pY1175, ∼250 and 220 kD) were assayed by immunoblotting, with actin as a loading control. (B) VEcad TMD requirement for VEGFR activation. VEcad−/− cells reconstituted with VEcadWT, NcadVE-TMD, NcadWT, and VEcadN-TMD were subjected to laminar shear stress for 1 min, then VEGFR2 activation was assayed as in A. (C) VEcad requirement for PI3K signaling. Cells were subjected to laminar shear stress, then p85 immunoprecipitated and immunoblotted with anti-p85pY458 antibody. Values beneath each panel indicate phosphorylation relative to cells without flow, quantified by densitometry with total p85 serving as a loading control. For all panels, values are means ± SEM, n = 3. IB, immunoblotting.
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fig2: VEcad TMD in flow signaling. (A) Requirement for VEcad. HUVECS were infected with scrambled or anti-VEcad shRNA-containing lentiviruses then subjected to 12 dynes/cm2 laminar shear for 1 min. Activation of SFKs (SFKpY416, ∼55 kD) and VEGFR2 (VEGFR2pY1175, ∼250 and 220 kD) were assayed by immunoblotting, with actin as a loading control. (B) VEcad TMD requirement for VEGFR activation. VEcad−/− cells reconstituted with VEcadWT, NcadVE-TMD, NcadWT, and VEcadN-TMD were subjected to laminar shear stress for 1 min, then VEGFR2 activation was assayed as in A. (C) VEcad requirement for PI3K signaling. Cells were subjected to laminar shear stress, then p85 immunoprecipitated and immunoblotted with anti-p85pY458 antibody. Values beneath each panel indicate phosphorylation relative to cells without flow, quantified by densitometry with total p85 serving as a loading control. For all panels, values are means ± SEM, n = 3. IB, immunoblotting.

Mentions: We next examined additional flow responses through the junctional complex. VEcad was previously reported to be downstream of shear-induced SFK activation but upstream of VEGFR2 activation (Tzima et al., 2005). We first confirmed this using VEcad knockdown in human umbilical cord endothelial cells (HUVECs). Cells expressing control or anti-VEcad shRNA were treated with laminar shear stress for 1 min. Shear stress activated SFK in both cells but VEGFR2 was only activated in control cells (Fig. 2 A). We then tested VEGFR2 transactivation in our chimeric cadherin-expressing cells. Onset of laminar flow transactivated VEGFR2 in cells expressing wild-type (WT) VEcad or NcadVE-TMD, but not Ncad or VEcadN-TMD (Fig. 2 B). Phosphorylation of the PI 3-kinase p85 subunit by onset of flow showed similar characteristics (Fig. 2 C). Together, these data show that the VEcad TMD is the critical VE-specific region required for responses to fluid shear stress through the junctional complex.


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 flow signaling. (A) Requirement for VEcad. HUVECS were infected with scrambled or anti-VEcad shRNA-containing lentiviruses then subjected to 12 dynes/cm2 laminar shear for 1 min. Activation of SFKs (SFKpY416, ∼55 kD) and VEGFR2 (VEGFR2pY1175, ∼250 and 220 kD) were assayed by immunoblotting, with actin as a loading control. (B) VEcad TMD requirement for VEGFR activation. VEcad−/− cells reconstituted with VEcadWT, NcadVE-TMD, NcadWT, and VEcadN-TMD were subjected to laminar shear stress for 1 min, then VEGFR2 activation was assayed as in A. (C) VEcad requirement for PI3K signaling. Cells were subjected to laminar shear stress, then p85 immunoprecipitated and immunoblotted with anti-p85pY458 antibody. Values beneath each panel indicate phosphorylation relative to cells without flow, quantified by densitometry with total p85 serving as a loading control. For all panels, values are means ± SEM, n = 3. IB, immunoblotting.
© Copyright Policy - openaccess
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4384728&req=5

fig2: VEcad TMD in flow signaling. (A) Requirement for VEcad. HUVECS were infected with scrambled or anti-VEcad shRNA-containing lentiviruses then subjected to 12 dynes/cm2 laminar shear for 1 min. Activation of SFKs (SFKpY416, ∼55 kD) and VEGFR2 (VEGFR2pY1175, ∼250 and 220 kD) were assayed by immunoblotting, with actin as a loading control. (B) VEcad TMD requirement for VEGFR activation. VEcad−/− cells reconstituted with VEcadWT, NcadVE-TMD, NcadWT, and VEcadN-TMD were subjected to laminar shear stress for 1 min, then VEGFR2 activation was assayed as in A. (C) VEcad requirement for PI3K signaling. Cells were subjected to laminar shear stress, then p85 immunoprecipitated and immunoblotted with anti-p85pY458 antibody. Values beneath each panel indicate phosphorylation relative to cells without flow, quantified by densitometry with total p85 serving as a loading control. For all panels, values are means ± SEM, n = 3. IB, immunoblotting.
Mentions: We next examined additional flow responses through the junctional complex. VEcad was previously reported to be downstream of shear-induced SFK activation but upstream of VEGFR2 activation (Tzima et al., 2005). We first confirmed this using VEcad knockdown in human umbilical cord endothelial cells (HUVECs). Cells expressing control or anti-VEcad shRNA were treated with laminar shear stress for 1 min. Shear stress activated SFK in both cells but VEGFR2 was only activated in control cells (Fig. 2 A). We then tested VEGFR2 transactivation in our chimeric cadherin-expressing cells. Onset of laminar flow transactivated VEGFR2 in cells expressing wild-type (WT) VEcad or NcadVE-TMD, but not Ncad or VEcadN-TMD (Fig. 2 B). Phosphorylation of the PI 3-kinase p85 subunit by onset of flow showed similar characteristics (Fig. 2 C). Together, these data show that the VEcad TMD is the critical VE-specific region required for responses to fluid shear stress through the junctional complex.

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