Interaction between Rnd GTPases and plexin subfamilies. A, COS-7 cells were transfected with HA-tagged Rnd1, Rnd2, or Rnd3, and cell lysates were incubated with GST or GST-fused Myc-tagged cytoplasmic domain of Plexin-A1 (Plexin-A1-cyt, amino acids 1296–1894), Plexin-B1 (Plexin-B1-cyt, amino acids 1543–2136), Plexin-C1 (Plexin-C1-cyt, amino acids 972–1575), and Plexin-D1 (Plexin-D1-cyt, amino acids 1293–1926). Lysate inputs (lower panel) and bound proteins (upper panels) were analyzed by immunoblotting with anti-HA antibody. B, lysates from COS-7 cells transfected with the indicated plasmids were immunoprecipitated with anti-HA antibody. Lysate inputs and bound proteins were analyzed by immunoblotting with anti-HA or anti-Myc antibody.

Plexin-C1 inactivates R-Ras activity regardless of the presence of Rnd GTPase. A, schematic representation of Plexin-C1 constructs used in this study. The R-Ras GAP domains are indicated by gray columns. B, COS-7 cells were transfected with Myc-tagged chimeric Plexin-B1/C1 and HA-tagged R-Ras in the absence or presence of GFP-tagged Rnd1. The cells were stimulated with (+) or without (–) 0.5 nm Sema4D (+) for 5 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 as described in the legend to Fig. 2, C and E. The value from HA-R-Ras-transfected cells was defined as 1. 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.

Plexin-D1 and Plexin-C1 inhibit the ECM-mediated cell migration through R-Ras GAP activity. A, COS-7 cells expressing the indicated plasmids were tested in a transwell assay using the chambers coated with FN (10 μg/ml) in the presence of 0.5 nm Sema3E or control Fc. Migrated cells were visualized by the fluorescence of GFP. Bars, 100 μm. B, relative cell migration was determined by the number of the migrated cells normalized to the total number of the transfected cells. Expression levels of the constructs were verified by immunoblot analysis. Results are the means ± S.E. of three independent experiments. The value from Myc-Plexin-D1-transfected cells that were stimulated with control Fc was defined as 1. The statistical difference was determined by two-sided Mann-Whitney's U test. *, p < 0.05. C, COS-7 cells transfected with the indicated plasmids were tested in transwell assay using the chambers coated with FN (10μg/ml). Bars, 100μm. D, relative cell migration was determined. Results are the means ± S.E. of five independent experiments. The value from GFP-R-Ras-WT-transfected cells was defined as 1. The statistical difference was determined by two-sided onesample t test or two-sided Student's t test. *, p < 0.01.

Plexin-C1 and Plexin-D1 display M-Ras GAP activity. A and C, COS-7 cells were transfected with Myr-Plexin-C1-cyt (wild-type or RA mutant) and either HA-TC21 (A) or HA-M-Ras (C). The cell lysates were incubated with GST-RBD, and bound Ras GTPases and total lysates were analyzed by immunoblotting. E and G, COS-7 cells were transfected with Myr-Plexin-D1-cyt (wild-type or RA mutant), GFP-Rnd2, and either HA-TC21 (E) or HA-M-Ras (G). The cell lysates were incubated with GST-RBD, and bound Ras GTPases and total lysates were analyzed by immunoblotting. B, D, F, and H, relative Ras activity was determined as described in the legend to Fig. 2, C and E. The value from Ras-transfected or Ras and Rnd2-transfected cells was defined as 1. The statistical significance was determined by two-sided one-sample t test or two-sided Student's t test. *, p < 0.01; **, p < 0.001.

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.

Interactions of N-terminal and C-terminal regions within the cytoplasmic domains of Plexin-D1 and Plexin-C1. A, schematic representation of the Plexin-D1 and Plexin-C1 constructs used in this study. The R-Ras GAP domains are indicated by gray columns. B, lysate from COS-7 cells transfected with HA-tagged Rnd2 was used in a pull-down assay with GST-fused C1 domain-containing N-terminal region (N-cyt) and C2 domain-containing C-terminal region (C-cyt) of Plexin-C1 and Plexin-D1. Bound and total HA-Rnd2 were analyzed by immunoblotting with anti-HA antibody. C, COS-7 cells were transfected with Myc-tagged Plexin-C1-N-cyt or Plexin-D1-N-cyt alone or in combination with HA-tagged Rnd2. The cell lysates were used in a pull-down assay with GST, GST-Plexin-C1-C-cyt, or GST-Plexin-D1-C-cyt. Bound proteins and total cell lysates were analyzed by immunoblotting with anti-HA and anti-Myc antibodies.

Rnd2 is required for Sema3E-mediated inhibition of axon outgrowth. A, lysate from ventrolateral cortex of P2 rats was immunoprecipitated with anti-Rnd2 antibody or with anti-Myc antibody as a control. The lysate input and immunoprecipitates were analyzed by immunoblotting with anti-Plexin-D1 or anti-Rnd2 antibody. For detection of immunoprecipitates of Plexin-D1, 3/4 volume of the total immunoprecipitates was applied. B, neurons from ventrolateral cortex of E19 rats were nucleofected with GFP together with Rnd1 shRNA or Rnd2 shRNA (shRNA-221 or shRNA-665) and cultured for 3 days. The cell lysates were immunoblotted with anti-Rnd2 or anti-α-tubulin antibody. C, COS-7 cells were transfected with Myc-rPlexin-D1-cyt together with Rnd1 shRNA or Plexin-D1 shRNA (shRNA-4683 or shRNA-4958) and cultured for 3 days. The cell lysates were immunoblotted with anti-Myc or anti-α-tubulin antibody. D, neurons of ventrolateral cortex were nucleofected with GFP together with Rnd1 shRNA, Rnd2 shRNA (shRNA-221 or shRNA-665), or Plexin-D1 shRNA (shRNA-4683 or shRNA-4958) and cultured for 3 days in the presence or absence of 1 nm Sema3E. Scale bar, 50 μm. F, neurons of ventrolateral cortex were nucleofected with GFP or GFP-R-RasQL and cultured for 2 days in the presence or absence of 1 nm Sema3E. Scale bar, 50 μm. E and G, quantification of axon length. Results are the means ± S.E. of 100 neurons from three independent experiments. The statistical difference was determined by two-sided Mann-Whitney's U test. ***, p < 0.0005.