Binding of Rac1, Rnd1, and RhoD to a Novel Rho GTPase Interaction Motif Destabilizes Dimerization of the Plexin-B1 Effector Domain

Department of Physiology, Case Western Reserve University School of Medicine, Cleveland, Ohio 44106, USA.
Journal of Biological Chemistry (Impact Factor: 4.57). 01/2008; 282(51):37215-24. DOI: 10.1074/jbc.M703800200
Source: PubMed

ABSTRACT Plexins are the first known transmembrane receptors that interact directly with small GTPases. On binding to certain Rho family GTPases, the receptor regulates the remodeling of the actin cytoskeleton and alters cell movement in response to semaphorin guidance cues. In a joint solution NMR spectroscopy and x-ray crystallographic study, we characterize a 120-residue cytoplasmic independent folding domain of plexin-B1 that directly binds three Rho family GTPases, Rac1, Rnd1, and RhoD. The NMR data show that, surprisingly, the Cdc42/Rac interactive binding-like motif of plexin-B1 is not involved in this interaction. Instead, all three GTPases interact with the same region, beta-strands 3 and 4 and a short alpha-helical segment of the plexin domain. The 2.0 A resolution x-ray structure shows that these segments are brought together by the tertiary structure of the ubiquitin-like fold. In the crystal, the protein is dimerized with C2 symmetry through a four-stranded antiparallel beta-sheet that is formed outside the fold by a long loop between the monomers. This region is adjacent to the GTPase binding motifs identified by NMR. Destabilization of the dimer in solution by binding of any one of the three GTPases suggests a model for receptor regulation that involves bidirectional signaling. The model implies a multifunctional role for the GTPase-plexin interaction that includes conformational change and a localization of active receptors in the signaling mechanism.

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Available from: Matthias Buck, Jul 21, 2015
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    • "Crystal structures of the RBD [23] and of the cytoplasmic domain of plexinB1 in complex with Rac1 [32,33] have been determined. Bell et al. [33]., identified a second RhoGTPase binding site in addition to the RBD, adjacent to the Ras site, which stabilises a trimeric structure of plexinB1-Rac complexes. "
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    ABSTRACT: Semaphorins act as chemotactic cues for cell movement via their transmembrane receptors, plexins. Somatic missense mutations in the plexinB1 gene coupled with overexpression of the protein frequently occur in prostate tumours, indicating a role for plexinB1 in the pathogenesis of prostate cancer. Two specific mutations found in prostate cancer enhance RhoD binding and one other mutation results in loss of inhibition of Rac-dependent Pak1 phosphorylation and lamellipodia formation and in impairment of trafficking of plexinB1 to the membrane. None of the three characterised mutations affect PDZRhoGEF binding, RhoA activity, the interaction of plexinB1 with the oncogenes ErbB2 or c-Met or ErbB2 phosphorylation. The mutations have the net effect of increasing cell motility by blocking plexinB1-mediated inhibition of Rac while enhancing the interaction with RhoD, an anti-migratory factor. PlexinB1 mutations block plexinB1-mediated signalling pathways that inhibit cell motility.
    Molecular Cancer 03/2012; 11(1):11. DOI:10.1186/1476-4598-11-11 · 4.26 Impact Factor
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    • "The intracellular portion of the plexins contain a GTPase-activating protein (GAP)-like motif that downregulates activity of the G protein R-Ras, interrupted by a region capable of binding small Rho family GTPases [9], [10]. In the case of Plexin-B1 for example, this Rho GTPase binding domain (RBD) can associate with Rnd1, Rac1, and RhoD, the binding of which influences plexin functioning [10]. The small GTPases act as molecular switches that cycle between an active GTP-bound and inactive GDP-bound form to regulate microtubule dynamics, cell shape and cell mobility [11]. "
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    ABSTRACT: The semaphorins and their receptors, the plexins, are proteins related to c-Met and the scatter factors that have been implicated in an expanding signal transduction network involving co-receptors, RhoA and Ras activation and deactivation, and phosphorylation events. Our previous work has demonstrated that Semaphorin 4D (Sema4D) acts through its receptor, Plexin-B1, on endothelial cells to promote angiogenesis in a RhoA and Akt-dependent manner. Since NF-κB has been linked to promotion of angiogenesis and can be activated by Akt in some contexts, we wanted to examine NF-κB in Sema4D treated cells to determine if there was biological significance for the pro-angiogenic phenotype observed in endothelium. Using RNA interference techniques, gel shifts and NF-κB reporter assays, we demonstrated NF-κB translocation to the nucleus in Sema4D treated endothelial cells occurring downstream of Plexin-B1. This response was necessary for endothelial cell migration and capillary tube formation and protected endothelial cells against apoptosis as well, but had no effect on cell proliferation. We dissected Plexin-B1 signaling with chimeric receptor constructs and discovered that the ability to activate NF-κB was dependent upon Plexin-B1 acting through Rho and Akt, but did not involve its role as a Ras inhibitor. Indeed, inhibition of Rho by C3 toxin and Akt by LY294002 blocked Sema4D-mediated endothelial cell migration and tubulogenesis. We also observed that Sema4D treatment of endothelial cells induced production of the NF-κB downstream target IL-8, a response necessary for angiogenesis. Finally, we could show through co-immunofluorescence for p65 and CD31 that Sema4D produced by tumor xenografts in nude mice activated NF-κB in vessels of the tumor stroma. These findings provide evidence that Sema4D/Plexin-B1-mediated NF-κB activation and IL-8 production is critical in the generation a pro-angiogenic phenotype in endothelial cells and suggests a new therapeutic target for the anti-angiogenic treatment of some cancers.
    PLoS ONE 10/2011; 6(10):e25826. DOI:10.1371/journal.pone.0025826 · 3.23 Impact Factor
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    • "Starting from this structure, we tried several modeling strategies involving different extents of remodeling of the plexin loops. The most conservative strategy involved eliminating an extended loop that plexin uses for homodimerization [encompassing residues 1822–1834 according to the numbering in Protein Data Bank (PDB) entry 2REX4] followed by fixed-backbone docking to identify low-energy conformations. Another strategy eliminated the loop encompassing residues 1790–1800 on plexin. "
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    ABSTRACT: Modeling the conformational changes that occur on binding of macromolecules is an unsolved challenge. In previous rounds of the Critical Assessment of PRediction of Interactions (CAPRI), it was demonstrated that the Rosetta approach to macromolecular modeling could capture side chain conformational changes on binding with high accuracy. In rounds 13-19 we tested the ability of various backbone remodeling strategies to capture the main-chain conformational changes observed during binding events. These approaches span a wide range of backbone motions, from limited refinement of loops to relieve clashes in homologous docking, through extensive remodeling of loop segments, to large-scale remodeling of RNA. Although the results are encouraging, major improvements in sampling and energy evaluation are clearly required for consistent high accuracy modeling. Analysis of our failures in the CAPRI challenges suggest that conformational sampling at the termini of exposed beta strands is a particularly pressing area for improvement.
    Proteins Structure Function and Bioinformatics 11/2010; 78(15):3212-8. DOI:10.1002/prot.22784 · 2.63 Impact Factor
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