title: Sema3A-induced growth-cone collapse is mediated by Rac1 amino acids 17-32.
abstract: BACKGROUND
  Neurons project their axons along specific pathways in order to establish appropriate connections with their target cells. The rate and direction of axonal growth is determined by interactions between the highly motile growth cone and environmental cues that can act in either an attractive or a repulsive manner. Locomotion is ultimately dependent upon the reorganisation of the actin cytoskeleton and an established role for the Rho family of small GTPases in regulating this process in non-neuronal cells identifies them as candidate signalling molecules in growth cones. An inactive form of Rac1 has recently been shown to inhibit the 'growth-cone collapse' response induced by chick Sema3A, a protein that has recently been established as an important guidance cue. The molecular basis for this inhibition remains unclear.
  
  
  RESULTS
  We have made a series of overlapping peptides from the amino-terminal region of Rac1 and rendered them cell permeable by synthesis in tandem with an established internalisation vector. We report here that a peptide encompassing Rac1 amino acids 17-32 binds directly to the established Rac1-interacting molecules PAK, WASP, 3BP-1 and p85beta(P13K), but not to p67(Phox). Furthermore, the peptide can compete with activated Rac1 for target binding, and inhibits Sema3A-induced growth-cone collapse. We also synthesised cell-permeable peptides that correspond to the Cdc42/Rac1-binding (CRIB) motifs present in PAK and N-WASP. Our results show that a CRIB-containing peptide from PAK, but not that from N-WASP, inhibits growth-cone collapse and that the inhibitory activity correlates with binding to Rac1 and not to Cdc42.
  
  
  CONCLUSIONS
  Our results suggest that Sema3A-induced growth-cone collapse is mediated by Rac1 amino acids 17-32, and demonstrate the feasibility of designing new cell-permeable inhibitors of small GTPases.
authors.affiliation: Molecular Neurobiology Group GKT School of Medicine King's College London London, SE1 9RT, Computational Genome Analysis Laboratory ICRF 44 Lincoln's Inn Fields, London, WC2A 3PX, UK.
authors.forename: I
authors.initials: I
authors.lastname: Västrik
authors.forename: B J
authors.initials: BJ
authors.lastname: Eickholt
authors.forename: F S
authors.initials: FS
authors.lastname: Walsh
authors.forename: A
authors.initials: A
authors.lastname: Ridley
authors.forename: P
authors.initials: P
authors.lastname: Doherty
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D019556:COS Cells; D002352:Carrier Proteins / Q000378:metabolism; D002642:Chick Embryo; D002522:Chlorocebus aethiops; D020690:GTPase-Activating Proteins; D006023:Glycoproteins / Q000502:physiology*; D020439:Growth Cones / Q000187:drug effects / Q000378:metabolism / Q000648:ultrastructure*; D008958:Models, Molecular; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000138:chemical synthesis / Q000494:pharmacology*; D019869:Phosphatidylinositol 3-Kinases / Q000378:metabolism; D010750:Phosphoproteins / Q000378:metabolism; D011487:Protein Conformation; D017434:Protein Structure, Tertiary; D011506:Proteins / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000494:pharmacology; D040121:Semaphorin-3A; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D051316:Wiskott-Aldrich Syndrome Protein; D020764:cdc42 GTP-Binding Protein / Q000502:physiology; D020830:rac1 GTP-Binding Protein / Q000138:chemical synthesis / Q000737:chemistry / Q000494:pharmacology* / Q000502:physiology*
chemical_list: D002352:Carrier Proteins; D020690:GTPase-Activating Proteins; D006023:Glycoproteins; D010446:Peptide Fragments; D019869:Phosphatidylinositol 3-Kinases; D010750:Phosphoproteins; D011506:Proteins; D011993:Recombinant Fusion Proteins; D040121:Semaphorin-3A; D051316:Wiskott-Aldrich Syndrome Protein; D020764:cdc42 GTP-Binding Protein; D020830:rac1 GTP-Binding Protein; C403751:Rac1 GTP-binding protein (17-32); C094762:Sh3bp1 protein, mouse; C079789:neutrophil cytosol factor 67K