Plexins are cell surface receptors that bind semaphorins and transduce signals for regulating neuronal axon guidance and other processes. Plexin signaling depends on their cytoplasmic GTPase activating protein (GAP) domain, which specifically inactivates the Ras homolog Rap through an ill-defined non-canonical catalytic mechanism. The plexin GAP is activated by semaphorin-induced dimerization, the structural basis for which remained unknown. Here we present the crystal structures of the active dimer of zebrafish PlexinC1 cytoplasmic region in the apo state and in complex with Rap. The structures show that the dimerization induces a large-scale conformational change in plexin, which opens the GAP active site to allow Rap binding. Plexin stabilizes the switch II region of Rap in an unprecedented conformation, bringing Gln63 in Rap into the active site for catalyzing GTP hydrolysis. The structures also explain the unique Rap-specificity of plexins. Mutational analyses support that these mechanisms underlie plexin activation and signaling.DOI: http://dx.doi.org/10.7554/eLife.01279.001

Signaling through plexin, the major cell surface receptor for semaphorin, plays critical roles in regulating processes such as neuronal axon guidance, angiogenesis and immune response. Plexin is normally kept inactive in the absence of semaphorin. Upon binding of semaphorin to the extracellular region, plexin is activated and transduces signal to the inside of the cell through its cytoplasmic region. The GTPase Activating Protein (GAP) domain in the plexin cytoplasmic region mediates the major intracellular signaling pathway. The substrate specificity and regulation mechanisms of the GAP domain have only been revealed recently. Many intracellular proteins serve as either upstream regulators or downstream transducers by directly interacting with plexin. The mechanisms of action for some of these proteins also start to emerge from recent studies. We review here these advances in the mechanistic understanding of plexin intracellular signaling from a structural perspective.

In most multicellular organisms, homeostasis is contingent upon maintaining epithelial integrity. When unanticipated insults breach epithelial barriers, dormant programmes of tissue repair are immediately activated. However, many of the mechanisms that repair damaged epithelia remain poorly characterized. Here we describe a role for Plexin A (PlexA), a protein with particularly well-characterized roles in axonal pathfinding, in the healing of damaged epithelia in Drosophila. Semaphorins, which are PlexA ligands, also regulate tissue repair. We show that Drosophila PlexA has GAP activity for the Rap1 GTPase, which is known to regulate the stability of adherens junctions. Our observations suggest that the inhibition of Rap1 activity by PlexA in damaged Drosophila epithelia allows epithelial remodelling, thus facilitating wound repair. We also demonstrate a role for Plexin A1, a zebrafish orthologue of Drosophila PlexA, in epithelial repair in zebrafish tail fins. Thus, plexins function in epithelial wound healing in diverse taxa.

PDZ domains are abundant protein interaction modules and typically recognize a short motif at the C terminus of their ligands, with a few residues in the motif endowing the binding specificity. The sequence-based rules, however, cannot fully account for the specificity between the vast number of PDZ domains and ligands in the cell. Plexins are transmembrane receptors that regulate processes such as axon guidance and angiogenesis. Two related guanine nucleotide exchange factors (GEFs), PDZ-RhoGEF and leukemiaassociated RhoGEF (LARG), use their PDZ domains to bind class B plexins and play critical roles in signaling. Here, we present the crystal structure of the full-length cytoplasmic region of PlexinB2 in complex with the PDZ domain of PDZ-RhoGEF. The structure reveals that, in addition to the canonical C-terminal motif/PDZ interaction, the 3D domain of PlexinB2 forms a secondary interface with the PDZ domain. Our biophysical and cell-based assays show that the secondary interface contributes to the specific interaction between plexin and PDZ-RhoGEF and to signaling by plexin in the cell. Formation of secondary interfaces may be a general mechanism for increasing affinity and specificity of modular domain-mediated interactions.PDZ | plexin | signaling | protein interaction module | specificity P lexins are cell surface receptors for semaphorins, extracellular cues that control essential processes such as neuronal axon guidance and vasculature development (1). Binding of semaphorin to the extracellular region of plexin induces formation of the active dimer of the cytoplasmic region, which transduces signal to downstream pathways (2-7). The plexin cytoplasmic region contains a juxtamembrane segment (JM-segment), a RhoGTPase binding domain (RBD), and a GTPase activating protein (GAP) domain (8-10). The GAP domain, activated by the dimerization, transduces signal through converting its substrate GTPase Rap from the GTP-bound active to the GDP-bound inactive state (2, 3). The RBD regulates plexin activity in response to binding of Rho family GTPases, such as Rac1 (reviewed in ref. 11).In addition to the common signaling pathways through the domains shared by all plexins, class B plexins (B1, B2, and B3) mediate a pathway through their unique C terminus. The conserved "VTDL" motif at the C terminus of these plexins binds to the N-terminal PDZ (PSD-95/Discs-large/ZO-1) domains of two related guanine nucleotide exchange factors (GEFs), PDZ-RhoGEF, and leukemia-associated RhoGEF (LARG) (12)(13)(14)(15)(16)(17). This interaction recruits PDZ-RhoGEF and LARG to the plasma membrane, where they promote the exchange of GDP for GTP on RhoA. GTP-bound RhoA binds its downstream effectors and contributes to plexin signaling (13)(14)(15)18). A recent study has shown that deletion of the C terminus of PlexinB2 causes defects in the development of the liver vasculature in mice, highlighting the critical role of the PDZ-RhoGEF/LARG-RhoA pathway in plexin function in vivo (19).More than 250 PDZ domains exist in the human proteome, con...

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Plexins are cell surface receptors that bind semaphorins and regulate essential processes such as axon guidance and angiogenesis. The cytoplasmic regions of plexins contain a functionally essential GTPase-activating protein (GAP) domain, which initiates downstream signaling by specifically inactivating the Rap GTPase. Here we describe the methods for expression and purification of the plexin cytoplasmic region in E. coli, and characterization of its GAP activity using a photometric assay. We also provide a protocol for measuring GAP activity of single-chain constructs with Rap covalently linked to the plexin cytoplasmic region.

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