Figure 6.
Sequestration of cytoplasmic p120ctn by exogenously expressed cadherin mutants causes internalization of cell surface VE-cadherin. The internalization of cell surface VE-cadherin was monitored in MEC expressing various IL-2R-VE-cadherin mutants. MEC were incubated with the BV6 antibody at 4°C and transferred to 37°C for 3 h in the presence of chloroquine. The cells were acid washed to remove surface bound VE-cadherin antibody, and fixed and processed for dual label immunofluorescence microscopy to detect both the internalized cadherin and the IL-2R-VE-cadherin mutants. Internalized VE-cadherin was detected in control MEC expressing empty adenoviral vector (A and B). VE-Cadherin internalization was dramatically increased in MEC expressing either the IL-2R-VE-cadcyto (C and D) or the IL-2R-VE-cadΔCBD mutant (E and F). However, the IL-2R-VE-cadJMDAAA mutant (G and H), which does not bind and compete for cytoplasmic p120ctn, failed to cause increased VE-cadherin internalization relative to cells expressing empty vector. (I) Quantitative representation of vesicular VE-cadherin detected in cells expressing the various cadherin mutants (results representative of greater than three independently conducted experiments. Error bars indicate the SD; n > 10 cells). (J) MEC were infected with empty virus, IL-2R-VE-cadcyto, IL-2R-VE-cadΔCBD, or the IL-2R-VE-cadJMD-AAA mutant and Western blot analysis was performed using an antibody against the extracellular domain of VE-cadherin to monitor endogenous VE-cadherin levels. Expression of the mutants was verified using the c-myc epitope tag (not depicted). Mutant cadherins that bind p120ctn cause down-regulation of endogenous VE-cadherin (top). In untreated cells, VE-cadherin was completely removed by trypsinization (middle). In MEC treated with chloroquine for 8 h, VE-cadherin is detected in trypsinized cells (bottom), indicating that this pool of VE-cadherin is intracellular. The results indicate that the IL-2R-VE-cadcyto and the IL-2R-VE-cadΔCBD mutant cause internalization of endogenous VE-cadherin, whereas the IL-2R-VE- cadJMD-AAA mutant does not trigger VE-cadherin internalization. Bar, 50 μm.