The PDZ binding motif of NRP-1 is required for recycling of VEGFR-2/NRP-1 complexes to the plasma membrane through the Rab11 compartment. PAECs expressing VEGFR-2 were transiently transfected with either full-length NRP-1 (A,C,E) or NRP-1-S921X (B-D-F). Cells were analyzed before (A-B), 30 minutes (C-D), and 180 minutes after stimulation with VEGF-A₁₆₅a (E-F). (G) Quantification of panels A through F (*P < .01; paired Student t test). (H-I) PAECs expressing VEGFR-2 were transiently transfected with NRP-1-S921X and fluorescently tagged Rab GTPase constructs. Cells were stimulated for 10 minutes with VEGF-A₁₆₅a. No colocalization of NRP-1-S921X with Rab11 was found. Arrowheads indicate vesicles that are positive for NRP-1 and VEGFR-2, but negative for Rab11. Scale bar: 20 μm (insets: 5 μm). Quantification revealed that NRP-1-S921X is not able to enter the Rab11 compartment. Images of Rab11 transfected cells are shown in panel (I), images of all other Rab GTPases stimulated for various time points are shown in supplemental Figure 5. (J) Degradation of VEGFR-2 is slower in the presence of wild-type NRP-1. VEGFR-2 was nearly completely degraded in the presence of NRP-1-S921X after 180 minutes whereas > 30% of VEGFR-2 remained in the presence of NRP-1 (mean ± SD, n = 3; *P < .05; paired Student t test).