Plexins Are GTPase-Activating Proteins for Rap and Are Activated by Induced Dimerization

Abstract

Plexins are cell surface receptors that bind to semaphorins and transduce signals that regulate neuronal development, immune responses, and other processes. Signaling through plexins has been proposed to rely on specific GTPase activating protein (GAP) activity for R-Ras and M-Ras. Activation of this GAP activity appears to require simultaneous binding of semaphorin to the plexin extracellular domain and of a RhoGTPase to the cytoplasmic region. However, GAP activity of plexins has eluded detection in several recent studies. We show that the purified cytoplasmic region of plexin uses a non-canonical catalytic mechanism to act as a GAP for Rap, but not for R-Ras or M-Ras. The RapGAP activity of plexins was normally autoinhibited and could be activated by induced dimerization. Our biochemical and crystallographic analyses demonstrate that binding of the RhoGTPases did not directly contribute to activation of plexin RapGAP activity. Semaphorin stimulated the RapGAP activity of full-length plexin in cells, which was required for plexin-mediated neuronal growth cone collapse. These findings together define a pathway for plexin signaling and provide new insights into the mechanism for semaphorin-induced activation of plexin.

PKA-induced dimerization of the RhoGAP DLC1 promotes its inhibition of tumorigenesis and metastasis

“…The formation of srGAP2 homodimers promotes neuronal cell membrane protrusion and cell spreading. Semaphorin stimulates the RapGAP activity of plexin receptors in cells 33 , and the RapGAP activity of the purified plexin cytoplasmic region is stimulated by dimerization. Furthermore, the GAP activities of both srGAP2 and plexins are autoinhibited and activated by induced dimerization, implicating a GAP activation mechanism that is regulated by intermolecular interactions.…”

Genetic dissection of plexin signaling in vivo

“…A common feature of all plexins is their intracellular GAP domain, which catalyzes the inactivation of R-Ras, M-Ras, and Rap1 upon binding of semaphorin ligands (11,14,15). Surprisingly, although plexins have been shown to regulate several independent signaling pathways in vitro, mutations in the GAP domain were sufficient to fully phenocopy the null mutants in vivo with respect to development of the nervous, the vascular, and the skeletal system.…”

“…The functional significance of the enzymatic activity of the plexin GAP domain has not been resolved hitherto in studies performed in vitro, owing to controversial observations: whereas several reports have shown that GAP domain-mediated inactivation of R-Ras and M-Ras regulates cellular behavior, including migration, axonal growth cone collapse, and dendrite morphology (11,14,38), others suggest that binding and sequestration of active R-Ras, rather than R-Ras inactivation, is required for the biological effects of plexins (15,51). We found that mutations in all three critical arginines or the two N-terminal arginines of the Plexin-B2 GAP domain block both R-Ras GAP activity as well as R-Ras binding, whereas mutation of the C-terminal arginine results in the loss of GAP activity toward R-Ras while retaining the ability to bind R-Ras, thereby allowing a differentiation between these two scenarios.…”

“…These in vitro approaches have often yielded conflicting results with respect to the functional relevance of individual plexin-mediated signaling pathways. For example, the axonal growth cone collapse of primary neurons has been suggested to be caused by plexinmediated deactivation of R-Ras (11,12), activation of RhoA (20,22), and deactivation of Rap1 (15). Which plexin signaling pathways are functionally relevant in vivo is largely unknown.…”

“…The intracellular domain of all plexins shares homology with GTPase-activating proteins (GAPs) and confers the deactivation of R-Ras, M-Ras, and Rap1 (11)(12)(13)(14)(15)(16)(17). The GAP activity toward R-Ras and M-Ras, but not toward Rap1, requires binding of Rnd GTPases to the plexin receptor (11,12,15,16). Plexins of the B-subfamily differ from all other plexins in that they carry a C-terminal PDZ domain interaction motif that mediates a stable interaction with the Rho guanine nucleotide exchange factor (RhoGEF) proteins PDZ-RhoGEF and LARG (18)(19)(20).…”