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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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Using cadherin chimeras to map VEcad-specific functional domains. (A) Endothelial cells orient into the direction of flow. Shown is an example of shear-induced alignment in HUVECs after exposure to 12 dynes/cm2 laminar shear stress for 16 h in a parallel plate flow chamber. Slides were fixed and stained for VEcad, phalloidin–Alexa Fluor 647, and Hoechst. Bar, 30 µm. (B) VEcad- (VEcad−/−) endothelial cells were reconstituted with human VEcad or Ncad, then exposed to 12 dynes/cm2 laminar shear stress for 16 h. Cell alignment in the direction of flow (±23°) was quantified. Values are means ± SEM (error bars), n ≥ 3. (C) Domain organization of VEcad and Ncad. Each cadherin has an extracellular domain containing five cadherin repeats (CA1–5), a single-pass TMD, and an intracellular domain (ICD) including p120 and β-catenin binding sites. A Flag tag was also added to each construct. (D) VEcad−/− cells reconstituted with WT or chimeric VEcad/Ncad were assayed for alignment as in B. (E) VEcad−/− cells were reconstituted with mouse, WT VEcad, or VEcad containing the human Ncad-TMD (VEcadN-TMD). Alignment in flow was analyzed as in A. Values are means ± SEM (error bars), n ≥ 3. *, P < 0.05 significance to VEcad−/− by one-way analysis of variance (ANOVA). The broken lines in each graph indicate random alignment.
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fig1: Using cadherin chimeras to map VEcad-specific functional domains. (A) Endothelial cells orient into the direction of flow. Shown is an example of shear-induced alignment in HUVECs after exposure to 12 dynes/cm2 laminar shear stress for 16 h in a parallel plate flow chamber. Slides were fixed and stained for VEcad, phalloidin–Alexa Fluor 647, and Hoechst. Bar, 30 µm. (B) VEcad- (VEcad−/−) endothelial cells were reconstituted with human VEcad or Ncad, then exposed to 12 dynes/cm2 laminar shear stress for 16 h. Cell alignment in the direction of flow (±23°) was quantified. Values are means ± SEM (error bars), n ≥ 3. (C) Domain organization of VEcad and Ncad. Each cadherin has an extracellular domain containing five cadherin repeats (CA1–5), a single-pass TMD, and an intracellular domain (ICD) including p120 and β-catenin binding sites. A Flag tag was also added to each construct. (D) VEcad−/− cells reconstituted with WT or chimeric VEcad/Ncad were assayed for alignment as in B. (E) VEcad−/− cells were reconstituted with mouse, WT VEcad, or VEcad containing the human Ncad-TMD (VEcadN-TMD). Alignment in flow was analyzed as in A. Values are means ± SEM (error bars), n ≥ 3. *, P < 0.05 significance to VEcad−/− by one-way analysis of variance (ANOVA). The broken lines in each graph indicate random alignment.

Mentions: To confirm that VEcad is unique in its involvement in shear stress signaling and to validate the experimental system, VEcad- endothelial cells (VEcad−/−; Carmeliet et al., 1999) were infected with lentivirus coding for with either VEcad or Ncad. Cells showing equal expression were selected (Fig. S1 A) and used for subsequent studies. Both cadherins localized well to cell–cell borders and bound β-catenin similarly (Fig. S1 B). To assess flow responsiveness, we examined alignment after 18 h of flow at 12 dynes/cm2 (Fig. 1 A). Only VEcad rescued the alignment defect of VEcad−/− cells (Fig. 1 B). Next, we used this assay to examine chimeras with different regions of VEcad and Ncad (see Fig. 1 C for domain organization). All of the chimeric cadherins also localized well to cell–cell contacts and bound β-catenin (Fig. S1, B and C; and data not depicted). The first round of experiments showed that a segment comprising the extracellular domain (ECD) and TMD of VEcad conferred flow sensitivity, whereas the cytoplasmic domain did not (Fig. 1 D). Further dissection of the extracellular/transmembrane regions showed that the transmembrane sequence of VEcad fully rescued alignment when inserted into Ncad (NcadVE-TMD; Fig. 1 D). Conversely, substitution of the Ncad TMD into VEcad (VEcadN-TMD) completely abrogated alignment (Fig. 1 E).


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)

Using cadherin chimeras to map VEcad-specific functional domains. (A) Endothelial cells orient into the direction of flow. Shown is an example of shear-induced alignment in HUVECs after exposure to 12 dynes/cm2 laminar shear stress for 16 h in a parallel plate flow chamber. Slides were fixed and stained for VEcad, phalloidin–Alexa Fluor 647, and Hoechst. Bar, 30 µm. (B) VEcad- (VEcad−/−) endothelial cells were reconstituted with human VEcad or Ncad, then exposed to 12 dynes/cm2 laminar shear stress for 16 h. Cell alignment in the direction of flow (±23°) was quantified. Values are means ± SEM (error bars), n ≥ 3. (C) Domain organization of VEcad and Ncad. Each cadherin has an extracellular domain containing five cadherin repeats (CA1–5), a single-pass TMD, and an intracellular domain (ICD) including p120 and β-catenin binding sites. A Flag tag was also added to each construct. (D) VEcad−/− cells reconstituted with WT or chimeric VEcad/Ncad were assayed for alignment as in B. (E) VEcad−/− cells were reconstituted with mouse, WT VEcad, or VEcad containing the human Ncad-TMD (VEcadN-TMD). Alignment in flow was analyzed as in A. Values are means ± SEM (error bars), n ≥ 3. *, P < 0.05 significance to VEcad−/− by one-way analysis of variance (ANOVA). The broken lines in each graph indicate random alignment.
© Copyright Policy - openaccess
Related In: Results  -  Collection

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

fig1: Using cadherin chimeras to map VEcad-specific functional domains. (A) Endothelial cells orient into the direction of flow. Shown is an example of shear-induced alignment in HUVECs after exposure to 12 dynes/cm2 laminar shear stress for 16 h in a parallel plate flow chamber. Slides were fixed and stained for VEcad, phalloidin–Alexa Fluor 647, and Hoechst. Bar, 30 µm. (B) VEcad- (VEcad−/−) endothelial cells were reconstituted with human VEcad or Ncad, then exposed to 12 dynes/cm2 laminar shear stress for 16 h. Cell alignment in the direction of flow (±23°) was quantified. Values are means ± SEM (error bars), n ≥ 3. (C) Domain organization of VEcad and Ncad. Each cadherin has an extracellular domain containing five cadherin repeats (CA1–5), a single-pass TMD, and an intracellular domain (ICD) including p120 and β-catenin binding sites. A Flag tag was also added to each construct. (D) VEcad−/− cells reconstituted with WT or chimeric VEcad/Ncad were assayed for alignment as in B. (E) VEcad−/− cells were reconstituted with mouse, WT VEcad, or VEcad containing the human Ncad-TMD (VEcadN-TMD). Alignment in flow was analyzed as in A. Values are means ± SEM (error bars), n ≥ 3. *, P < 0.05 significance to VEcad−/− by one-way analysis of variance (ANOVA). The broken lines in each graph indicate random alignment.
Mentions: To confirm that VEcad is unique in its involvement in shear stress signaling and to validate the experimental system, VEcad- endothelial cells (VEcad−/−; Carmeliet et al., 1999) were infected with lentivirus coding for with either VEcad or Ncad. Cells showing equal expression were selected (Fig. S1 A) and used for subsequent studies. Both cadherins localized well to cell–cell borders and bound β-catenin similarly (Fig. S1 B). To assess flow responsiveness, we examined alignment after 18 h of flow at 12 dynes/cm2 (Fig. 1 A). Only VEcad rescued the alignment defect of VEcad−/− cells (Fig. 1 B). Next, we used this assay to examine chimeras with different regions of VEcad and Ncad (see Fig. 1 C for domain organization). All of the chimeric cadherins also localized well to cell–cell contacts and bound β-catenin (Fig. S1, B and C; and data not depicted). The first round of experiments showed that a segment comprising the extracellular domain (ECD) and TMD of VEcad conferred flow sensitivity, whereas the cytoplasmic domain did not (Fig. 1 D). Further dissection of the extracellular/transmembrane regions showed that the transmembrane sequence of VEcad fully rescued alignment when inserted into Ncad (NcadVE-TMD; Fig. 1 D). Conversely, substitution of the Ncad TMD into VEcad (VEcadN-TMD) completely abrogated alignment (Fig. 1 E).

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