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.