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. 2003 May 26;161(4):793–804. doi: 10.1083/jcb.200209019

Figure 6.

Figure 6.

VEGFR-2 association and dephosphorylation requires the β-catenin binding domain of VE-cadherin. VEC-null cells were transfected with VE-cadherin wild type or truncated mutants lacking β-catenin (Δ-βcat) or p120 (Δ-p120) binding domains. Intra, intracellular region; extra, extracellular region (C). (A) After stimulation with VEGF (80 ng/ml) for 5 and 30 min, cell extracts were immunoprecipitated (IP) with antibodies to VEGFR-2 (αVEGFR-2) and immunoblotted (IB) with antibodies to phosphotyrosine (αphosphoTyr), VEGFR-2 (αVEGFR-2), and VE-cadherin (αVEC). Wild-type (molecular mass, ∼120 kD) and Δ-p120 VE-cadherin (molecular mass, ∼100 kD) were coimmunoprecipitated with VEGFR-2 (A, lower panel). Receptor phosphorylation was significantly reduced in VEC-positive and Δ-p120, but not in Δ-βcat, in comparison with VEC-null cells. The quantification of receptor phosphorylation data from three experiments ± SD is shown in A on the right. The values represent the ratio between the phosphorylated and total amount of VEGFR-2 and are normalized to the ratio calculated in untreated VEC-positive cells. The peak of VEGFR-2 phosphorylation at 5 min is similar in VEC-null and Δ-βcat, but lower in Δ-p120. At longer stimulation (30 min), the level of phosphorylation of VEGFR-2 in Δ-p120 was comparable to VEC-positive cells. Incubation of VEC-positive cell extract with nonimmune (NI) rabbit immunoglobulin (matched with VEGFR-2 antibody used for IP) did not precipitate bands corresponding to either VEGFR-2 or VE-cadherin, last lane from the left (IP NI). (B) VE-cadherin mutants modulate endothelial growth induced by VEGF. VEC-null and Δ-βcat had comparable effects and were the most permissive mutations in terms of cell proliferation (>160% increase over VEC-positive cells). Mutations that affected binding of p120 (Δ-p120) allowed cell proliferation, but to a lower extent (60% increase over stimulation of VEC-positive cells). Proliferation was measured as BrdU incorporation as described in the legend to Fig. 1. Mean ± SD of three independent experiments, each in duplicate, is shown.