Molecular dissection of the semaphorin 4D receptor Plexin-B1-stimulated R-Ras GTPase-activating protein activity and neurite remodeling in hippocampal neurons

Plexins serve as receptors for repulsive axonal guidance molecules semaphorins. The cytoplasmic domain of the semaphorin 4D (Sema4D) receptor, Plexin-B1 has two separated Ras GTPase-activating protein (GAP)-homologous domains, C1 and C2. Recently, we reported that the Rho family small GTPase Rnd1 associates with Plexin-B1, and the Plexin-B1-Rnd1 complex stimulates GTPase activity of R-Ras, inducing growth cone collapse in hippocampal neurons in response to Sema4D. However, the molecular mechanisms by which Plexin-B1 exhibits the GAP activity remain unclear. In this report, critical roles of Rnd1 and Sema4D in Plexin-B1-stimulated R-Ras GAP activity and neurite remodeling were examined. The N-terminal region of the cytoplasmic domain of Plexin-B1 containing the C1 domain interacts with the C-terminal region containing the C2 domain, and Rnd1 disrupts this interaction. On the other hand, Sema4D induces clustering of Rnd1-bound Plexin-B1, in parallel with inactivation of R-Ras in cells. Antibody clustering of the recombinant cytoplasmic domain of Plexin-B1 in the presence of Rnd1 triggers the R-Ras GAP activity. Deletion of the extracellular domain of Plexin-B1 causes ligand-independent clustering of the receptor, rendering the receptor constitutively active in the presence of Rnd1, and induces contraction of COS-7 cells and inhibition of neurite outgrowth in hippocampal neurons. These results indicate that Rnd1 opens the two R-Ras GAP domains of Plexin-B1, and Sema4D-induced receptor clustering stimulates R-Ras GAP activity and neurite remodeling in hippocampal neurons.