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...

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...

The innate immune response must be mobilized promptly yet judiciously via TLRs to protect the lungs against pathogens. Stimulation of murine peritoneal macrophage (PMφ) TLR4 or TLR3 by pathogen-associated molecular patterns (PAMPs) typically induces type I IFN-β, leading to autocrine activation of the transcription factor STAT1. Because it is unknown whether STAT1 plays a similar role in the lungs, we studied the response of resident alveolar macrophages (AMφ) or control PMφ from normal C57BL/6 mice to stimulation by PAMPs derived from viruses (polyriboinosinic:polyribocytidylic acid, specific for TLR3) or bacteria (Pam3Cys, specific for TLR2, and repurified LPS, specific for TLR4). AMφ did not activate STAT1 by tyrosine phosphorylation on Y701 following stimulation of any of these three TLRs, but readily did so in response to exogenous IFN-β. This unique AMφ response was not due to altered TLR expression, or defective immediate-early gene response, as measured by expression of TNF-α and three β chemokines. Instead, AMφ differed from PMφ in not producing bioactive IFN-β, as confirmed by ELISA and by the failure of supernatants from TLR-stimulated AMφ to induce STAT1 phosphorylation in PMφ. Consequently, AMφ did not produce the microbicidal effector molecule NO following TLR4 or TLR3 stimulation unless exogenous IFN-β was also added. Thus, murine AMφ respond to bacterial or viral PAMPs by producing inflammatory cytokines and chemokines, but because they lack the feed-forward amplification typically mediated by autocrine IFN-β secretion and STAT1 activation, require exogenous IFN to mount a second phase of host defense.