Article

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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    • "Binding of the specific ligand semaphorin-4D to the extracellular domain of Plexin- B1 induces a variety of signaling processes on the intracellular side, for which proteins of Rho family are known to be important components [1] [5]. Members of the Rho family have been shown to directly associate with the Rho binding domain of Plexin-B1 (B1RBD), such as Rnd1, Rac1 and RhoD [6] [7] [8] [9] [10]. Structural analysis of B1RBD in complex with Rho proteins has suggested that B1RBD binds to similar regions of Rnd1 and Rac1 [9] [11] [12]. "
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    ABSTRACT: Plexin-B1 regulates various cellular processes interacting directly with several Rho proteins. Molecular details of these interactions are, however, not well understood. In this study, we examined in vitro and in silico the interaction of the Rho binding domain (B1RBD) of human Plexin-B1 with eleven different Rho proteins. We show that B1RBD binds in a GTP-dependent manner to Rac1, Rac2, Rac3, Rnd1, Rnd2, Rnd3, and RhoD, but not to RhoA, Cdc42, RhoG, or Rif. Interestingly, Rnd1 competitively displaces the Rac1 from B1RBD but not vice versa. Structure-function analysis revealed a negatively charged loop region, called B1L(31), which may facilitate a selective B1RBD interaction with Rho proteins.
    Full-text · Article · Apr 2013 · Biochemical and Biophysical Research Communications
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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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    • "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.
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