Plexin-D1 inactivates R-Ras activity in the presence of Rnd2. A, schematic representation of Plexin-D1 constructs used in this study. Letters indicate specific amino acid residues within domains (A, Ala; F, Phe; L, Leu; R; Arg), and numbers indicate amino acid positions within the sequences. TM, transmembrane; Myr, myristoylated. B, COS-7 cells plated onto FN-coated dishes were transfected with Myc-tagged Plexin-D1, GFP-tagged Rnd proteins, and HA-tagged R-Ras. The cells were stimulated with 0.5 nm recombinant Sema3E (+) or control Fc (–) for 7 min. The cell lysates were incubated with GST-RBD, and bound R-Ras and total cell lysates were analyzed by immunoblotting. D, COS-7 cells were transfected with the indicated plasmids. The cell lysates were incubated with GST-RBD, and bound R-Ras and total cell lysates were analyzed by immunoblotting. C and E, relative R-Ras activity was determined by the amount of R-Ras bound to GST-RBD normalized to the amount of R-Ras in cell lysates analyzed by NIH Image software. The value from Myc-Plexin-D1 or Myr-Myc-Plexin-D1-cyt with HA-R-Ras-transfected cells was defined as 1. Results are the means ± S.E. of more than three independent experiments. The statistical significance was determined by two-sided one-sample t test or two-sided Student's t test. *, p < 0.05; **, p < 0.001.