Peptides designed based on TD binding sites inhibit CME. (A) Design of TAT-tagged peptides. Previously reported key residues of the four binding sites in clathrin terminal domain are listed on the left column. Peptides encoded the TAT sequence (black) followed by 10 amino acids derived from TD (red, key residues indicated in bold). Cbox1, Wbox1, a2L, and site4 are peptides derived from the linear TD sequences incorporating the key residues. Cbox2 and Wbox2 were designed to incorporate discontinuous key residues located on adjacent blades, bringing them together in a 10-aa peptide engineered to encompass the entire binding motif. (B) Amino acid sequences of six designed peptides are shown as colored sticks on the TD (PDB accession no. 1BPO). (C) Comparison of the inhibitory activities of the six peptides on TfnR uptake in ARPE/HPV cells. Cells were preincubated with peptides (55 µM) for 60 min at 37°C before uptake assay. Error bars are SD. N.S., not significant. (D) Effect of preincubation time (10–60 min at 37°C, as indicated) on the inhibitory activity of 20 µM Wbox2. (E) Reversibility of Wbox2 inhibition. TfnR uptake efficiency was measured in ARPE/HPV cells immediately after a 1-h preincubation at 37°C with 20 µM WBox2 or after removing peptides and incubating cells at 37°C for 3 and 6 additional hours. (F) Relative TfnR uptake efficiency with increasing concentrations of TAT or the indicated TD-derived peptides, as well as Wbox2_mutant, after 60-min preincubation at 37°C. In C–F, values were normalized to control = 1. Data ± SD are from n = 4 replicates. (G) Effect of Wbox2 peptides on ¹²⁵I-LDL uptake in human fibroblasts. Data ± SD are from n = 3 replicates. (H and I) Effect of Wbox2 peptides (20 µM) on fluid phase uptake of HRP (H) or CIE of CD44 and CD59 (I). Error bars are SD from n = 4 replicates. ***, P ≤ 0.001.