Figure 5. p190RhoGAP mediates Rnd3 inhibitory function towards RhoA and competes with Plexin B2 for Rnd3 binding.

(a) In vitro FRET analysis of RhoA activity in dissociated cortical cells in culture, 2 days after the electroporation of the constructs indicated. Upper panels show the CFP signal from the FRET probe; the RFP signal (in insets) marks electroporated cells (scale bar in insets, 10 μm). Lower panels show FRET efficiency. Scale bar, 10 μm. (b) Mean±s.e.m.; (n>11 cells for each condition, from three independent experiments; t-test: *P<0.05 and **P<0.01 compared with control). (c) Mouse embryonic cortices electroporated in utero with control shRNA or p190RhoGAP shRNA at E14.5 and analysed 3 days later. Scale bar, 200 μm. The distribution of GFP⁺ cells in the different cortical compartments revealed defects in the migration of p190RhoGAP-depleted neurons. Mean±s.e.m. from six sections prepared from three different experiments; t-test; **P<0.01 and ****P<0.0001. (d) Plexin B2 interacts with the same site as p190RhoGAP on Rnd3 protein. COS7 cells were co-transfected with HA-PlexinB2 (cytoplasmic domain) and wild-type and mutated (T55V) FLAG-Rnd3 constructs. The lysates were immunoprecipitated (IP) with anti-FLAG antibody and immunoblotted with anti-HA or anti-FLAG antibodies. The mutation of a single residue, Threonine 55 in the effector binding domain, of Rnd3 disrupted the interaction with Plexin B2, similar to p190RhoGAP4. (e) Competition between Plexin B2 and p190RhoGAP for Rnd3 binding. Expression vectors encoding FLAG-Rnd3 and Myc-p190RhoGAP-B (middle domain) were co-transfected with increasing amounts of VSV-Plexin B2 into COS7 cells. Cell lysates were IP with anti-FLAG antibody, then immunoblotted with the indicated antibodies. For full blots see Supplementary Fig. 6.