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, ␤-strands 3 and 4 and a short ␣-helical segment of the plexin domain. The 2.0 Å 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 ␤-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.Members of the plexin family of transmembrane receptors have important functions in guiding axon growth in the developing nervous system (1-3). Plexins also function in several developmental processes such as cardiovascular development and angiogenesis (4 -8), in the invasive growth of epithelial cells (9), and in the immune system (10). The extracellular part of plexin shares significant homology with that of the hepatocyte growth factor receptor as well as with semaphorins, the principal family of ligands for plexins. The cytoplasmic region of plexins is also well conserved (11). Two segments with homology to Ras GTPase-activating proteins (GAPs) 5 are linked by a region that has been identified as the location for the binding of several Rho family GTPases.Plexins are unique in that they are the first documented example of a transmembrane receptor that interacts directly with small GTPases. Most A-and B-family plexins bind activated Rac1 and Rnd1 (12-15), Rho family GTPases that are known regulators of cytoskeletal dynamics and cell adhesion (16). Plexin-A1 has also been shown to bind active RhoD (17), another Rho family GTPase involved in actin remodeling and endosomal dynamics and possibly in receptor down-regulation (18). The role of these plexin-Rho GTPase interactions has remained unclear, however, as have the characteristics of the binding region. Does the Rho GTPase binding segment provide a GTPase regulatory property, functioning akin to a guanine nucleotide dissociation inhibitor by sequestering certain Rho family GTPases, or does it act as an effector pro...

Plexin transmembrane receptors and their semaphorin ligands, as well as their co-receptors (Neuropilin, Integrin, VEGFR2, ErbB2, and Met kinase) are emerging as key regulatory proteins in a wide variety of developmental, regenerative, but also pathological processes. The diverse arenas of plexin function are surveyed, including roles in the nervous, cardiovascular, bone and skeletal, and immune systems. Such different settings require considerable specificity among the plexin and semaphorin family members which in turn are accompanied by a variety of cell signaling networks. Underlying the latter are the mechanistic details of the interactions and catalytic events at the molecular level. Very recently, dramatic progress has been made in solving the structures of plexins and of their complexes with associated proteins. This molecular level information is now suggesting detailed mechanisms for the function of both the extracellular as well as the intracellular plexin regions. Specifically, several groups have solved structures for extracellular domains for plexin-A2, -B1, and -C1, many in complex with semaphorin ligands. On the intracellular side, the role of small Rho GTPases has been of particular interest. These directly associate with plexin and stimulate a GTPase activating (GAP) function in the plexin catalytic domain to downregulate Ras GTPases. Structures for the Rho GTPase binding domains have been presented for several plexins, some with Rnd1 bound. The entire intracellular domain structure of plexin-A1, -A3, and -B1 have also been solved alone and in complex with Rac1. However, key aspects of the interplay between GTPases and plexins remain far from clear. The structural information is helping the plexin field to focus on key questions at the protein structural, cellular, as well as organism level that collaboratoria of investigations are likely to answer.

Members of the plexin family are unique transmembrane receptors in that they interact directly with Rho family small GTPases; moreover, they contain a GTPase-activating protein (GAP) domain for R-Ras, which is crucial for plexin-mediated regulation of cell motility. However, the functional role and structural basis of the interactions between the different intracellular domains of plexins remained unclear. Here we present the 2.4 Å crystal structure of the complete intracellular region of human plexin-B1. The structure is monomeric and reveals that the GAP domain is folded into one structure from two segments, separated by the Rho GTPase binding domain (RBD). The RBD is not dimerized, as observed previously. Instead, binding of a conserved loop region appears to compete with dimerization and anchors the RBD to the GAP domain. Cell-based assays on mutant proteins confirm the functional importance of this coupling loop. Molecular modeling based on structural homology to p120 GAP ⅐H-Ras suggests that Ras GTPases can bind to the plexin GAP region. Experimentally, we show that the monomeric intracellular plexin-B1 binds R-Ras but not H-Ras. These findings suggest that the monomeric form of the intracellular region is primed for GAP activity and extend a model for plexin activation.Plexins are single transmembrane receptors for guidance cues, called semaphorins, which regulate the motility and positional maintenance of certain cells. With this function, the receptors play critical roles in many developmental processes, including axon guidance, angiogenesis, and bone formation (1, 2). Moreover, plexins and their ligands are also involved in the regulation of the immune response, in cancer progression, and are thought to restrain tissue regeneration after injury (3, 4).Plexins are unusual receptors in that they interact directly with Rho and Ras family small GTPases (5-7). An intracellular region that has high homology to Ras GTPase-activating proteins (GAPs) 7 facilitates the hydrolysis of R-Ras-bound GTP. This deactivation of R-Ras leads to functional inhibition of integrins and to a loss of cell adhesion in response to semaphorins (5-8). Interestingly, no GAP activity of plexin-B1 was detected toward the R-Ras-homologous H-Ras (5), suggesting greater substrate specificity compared with the GAP protein p120 GAP (9). How the plexin receptor is activated and specifically how the GAP function is regulated have been questions of considerable interest (10 -12). A number of studies have pointed to a sequence segment that interrupts the GAP-homologous region and is capable of binding small Rho family GTPases. In the case of plexin-B1, this Rho GTPase binding domain (RBD) can associate with Rnd1, Rac1, and RhoD, which are thought to regulate plexin function. Specifically, in vitro studies in a number of laboratories have used the intracellular region of plexins expressed as two fragments, named C1 (containing the RBD and an N-terminal GAP-homologous segment) and C2 (C-terminal GAP segment). The studies suggest that such fragm...