(A) Mutations in the Rac effector domain abolish plexin-B1 interaction. Various myc-Rac mutants were transfected into HEK293 cells, and cell lysates were subjected to in vitro pulldown by MBP-plexin-B1. Expression of different Rac mutants in cell lysates is shown (lower). The mutations Y32F (lane 5), T35S (lane7), and F37L (lane 9) in the Rac effector domain completely abolished interaction with plexin-B1. In contrast, the mutations P34G (lane 6) and Y40C (lane 9) had no significant effect on the interaction with plexin-B1. (B) Cdc42 and RhoA do not interact with plexin-B1. Various mutants of myc-RhoA, -Cdc42, and -Rac were transfected into HEK293 cells. Cell lysates were used in either MBP-plexin-B1 or GST-PAK-RBD pulldown. Neither RhoL63 (lane 2) nor Cdc42L61 (lane 4) bind to plexin-B1. As a control, GST-PAK-RBD binds both RacL61 (lane 9) and Cdc42L61 (lane 11).

(A) CD100 stimulates the interaction between plexin-B1 and RacL61. VSV epitope-tagged plexin-B1 or plexin-A1 (0.5 μg) was cotransfected with myc-Rac (0.2 μg) and/or semaphorin (0.5 μg) in HEK293 cells as indicated. Cells were lysed and immunoprecipitated with anti-myc antibody. The immunoprecipitated samples were probed with anti-VSV antibody. Expression of Rac (Bottom, detected by anti-myc) and plexin (second panel from bottom, detected by anti-VSV) are shown. VSV-Plexin-B1 coimmunoprecipitated by myc-Rac is shown in the top two panels (for short and long exposure). Cotransfection of CD100 and Sema3A is indicated. Lanes 8 and 9 also were cotransfected with neuropilin-1 (0.5 μg). (B) A proposed model for Rac-GTP in CD100-stimulated plexin-B1 signaling. The ability of Rac-GTP to bind the plexinB1 receptor is enhanced on stimulation with CD100. Active Rac binds the receptor CRIB motif (denoted as a filled circle) and is predicted to affect actin reorganization.

(A) Interaction between activated Rac and plexin-B1. Active mutants of HA-RasV12 (lanes 1–4), myc-RacL61 (lanes 5–8), and HA-RapV12 (lanes 9–12) were transfected into HEK293 cells. Cells lysates (75 μl) were incubated with 5 μg of MBP-plexin prebound to amylose resin. The resin was washed and eluted in buffer containing 10 mM maltose. The eluted samples were analyzed by SDS/PAGE and detected by Western blot (WB) with the antibodies as indicated. MBP-plexin-A3, -B1, or -C1 are indicated on top of each lane. Lanes labeled Lys denote total cell lysate (7.5 μl). (B) Plexin-B1 interacts with constitutively active RacL61 but not with dominant negative RacN17. In vitro binding of purified GST-Rac/N17/L61 was incubated with MBP-plexin-A3, -B1, and -C1 prebound to amylose resin. MBP-plexins and associated proteins were isolated and stained by Coomassie (upper) or subject to WB with α-Rac antibody (lower). GST-RacL61 was quantitatively recovered by MBP-plexin-B1 pulldown (lane 6). GST-wild-type Rac (WT), RacN17 (N17), and RacL61 (L61) are indicated on top of corresponding lanes. (C) Plexin-B1 interacts with Rac-GTP but not Rac-GDP. Wild-type Rac was preloaded with GTPγS (a nonhydrolyzable GTP analogue, lane 3) or GDP (lane 4) before in vitro binding with MBP-plexin-B1. The interaction with MBP-plexin-B1 was analyzed as in B, with the exception that α-GST antibody was used. RacL61 (lane 1) and RacN17 (lane 2) were included as positive and negative controls, respectively.

(A) Mapping the Rac binding domain in plexin-B1. The intracellular domain of plexin-B1 (residues 1512–2135) contains two conserved regions as depicted by the hatched bars. The filled circle denotes the CRIB motif found in plexin-B1, which is not conserved in other members of the plexin family. The results of the in vitro interaction are summarized in the right column. (B) Interaction between RacL61 and deletion mutants of plexin-B1. Various MBP-plexin-B1 deletion proteins were expressed and purified from E. coli and used for in vitro interaction with GST-RacL61. The samples were stained with Coomassie (lower) or detected by α-GST WB (upper). (C) Sequence alignment of the CRIB domain of plexin-B1. The CRIB consensus defined by Burbelo et al. is shown (upper) (31). Residues conserved among ACK, WASP, PAK, and plexin-B1 are shown (lower). The invariable P, H, and D are boxed whereas * denotes partially conserved residues. (D) Mutation in the CRIB motif disrupts interaction with plexin-B1. MBP-plexin-B1 fusion proteins, containing mutations in the CRIB domain as indicated, were expressed and purified from E. coli and used for in vitro interaction with GST-RacL61. The samples were analyzed by α-GST WB.