Sema3A-induced growth-cone collapse is mediated by Rac1 amino acids 17-32

Background: Neurons project their axons along specific pathways in order to establish appropriate connections with their target cells. The rate and di rection of axonal growth is determined by interactions between the highly m otile growth cone and environmental cues that can act in either an attracti ve or a repulsive manner. Locomotion is ultimately dependent upon the reorg anisation of the actin cytoskeleton and an established role for the Rho fam ily of small GTPases in regulating this process in non-neuronal cells ident ifies them as candidate signalling molecules in growth cones, An inactive f orm of Rad has recently been shown to inhibit the 'growth-cone collapse' re sponse induced by chick Sema3A, a protein that has recently been establishe d as an important guidance cue. The molecular basis for this inhibition rem ains unclear. Results: We have made a series of overlapping peptides from the amino-termi nal region of Rad and rendered them cell permeable by synthesis in tandem w ith an established internalisation vector. We report here that a peptide en compassing Rad amino acids 17-32 binds directly to the established Rad-inte racting molecules PAK, WASP, 3BP-1 and p85 beta(PI3K), but not to p67(Phox) . Furthermore, the peptide can compete with activated Rad for target bindin g, and inhibits Sema3A-induced growth-cone collapse. We also synthesised ce ll-permeable peptides that correspond to the Cdc42/Rac1-binding (CRIB) moti fs present in PAK and N-WASP. Our results show that a CRIB-containing pepti de from PAK, but not that from N-WASP, inhibits growth-cone collapse and th at the inhibitory activity correlates with binding to Rad and not to Cdc42. Conclusions: Our results suggest that Sema3A-induced growth-cone collapse i s mediated by Rad amino acids 17-32, and demonstrate the feasibility of des igning new cell-permeable inhibitors of small GTPases.