The core p120-binding region of VE-cadherin functions as an endocytic signal. (A) Portions of the VE-cadherin (VE-cad) cytoplasmic tail (gray) were fused to the extracellular and transmembrane domains of the interleukin-2 receptor α chain (IL-2R; black) to create chimeric proteins. The core p120-binding region is indicated by asterisks. CBD, catenin-binding domain; JMD, juxtamembrane domain; cyto, entire VE-cadherin cytoplasmic tail; Δ657 and Δ644, VE-cadherin cytoplasmic tail truncated at residues 657 and 644. (B) IL-2R–VE-cadherin chimeras were expressed in COS-7 cells and isolated by immunoprecipitation of IL-2R. β-Catenin (β) coprecipitates only with the chimera containing the catenin-binding domain, and p120 coprecipitates only with the chimeras containing the entire juxtamembrane domain. (C) Fluorescence-based internalization assay of IL-2R–VE-cadherin chimeras expressed in COS-7 cells. (top row) Internalized chimera was identified by antibody labeling of surface IL-2R followed by a 10-min incubation to allow internalization and a low pH wash to remove antibody remaining at the cell surface. (bottom row) Cells were then fixed and stained for total IL-2R. (D) Quantification of the ratio of internalized to total chimera. Means ± SEM (n = 8–16 cells per group); *, P < 0.05; **, P < 0.01; ***, P < 0.001 compared with IL-2R and Δ644. (E) The VE-cadherin core p120-binding region (core; asterisk) was fused to IL-2R with a short linker peptide (linker; white oval) used to maintain spacing from the plasma membrane. (F and G) Fluorescence-based internalization assay with a 10-min internalization (intern.) period. Means ± SEM (n = 8–11 cells per group); **, P < 0.01 compared with IL-2R; ^◊◊, P < 0.01 compared with linker. IP, immunoprecipitation. Bars, 20 µm.