Pathfinding axons change responses to guidance cues at intermediate targets. During midline crossing, spinal cord commissural axons acquire responsiveness to class 3 Semaphorins and Slits, which regulate their floor plate exit and restrict their post-crossing trajectory into a longitudinal pathway. We found that Sonic Hedgehog (Shh) could activate the repulsive response of pre-crossing axons to Semaphorins. Blocking Shh function with a monoclonal antibody to Shh, 5E1, in 'open-book' explants or by expressing a dominant-negative form of Patched-1, Ptch1(Delta loop2), or a Smoothened (Smo) shRNA construct in commissural neurons resulted in severe guidance defects, including stalling and knotting inside the floor plate, recrossing, randomized anterior-posterior projection and overshooting after crossing, reminiscent of Neuropilin-2 mutant embryos. Enhancing protein kinase A activity in pre-crossing axons diminished Shh-induced Semaphorin repulsion and caused profound midline stalling and overshooting/wandering of post-crossing axons. Therefore, a morphogen, Shh, can act as a switch of axon guidance responses.
Background: Intracellular domain (ICD) modifications regulate extracellular ectodomain cleavage by metalloproteases. How this inside-out signal is relayed is unknown. Results: Cleavage requires substrate homodimerization; ICD modifications likely induce a relative positional change of the dimerization partners, allowing cleavage. Conclusion: Substrate dimerization might be a general requirement for cleavage. Significance: Our results fill an important gap in understanding growth factor release by ectodomain cleavage.
Ectodomain cleavage (shedding) of transmembrane proteins by metalloproteases (MMP) generates numerous essential signaling molecules, but its regulation is not totally understood. CD44, a cleaved transmembrane glycoprotein, exerts both antiproliferative or tumor-promoting functions, but whether proteolysis is required for this is not certain. CD44-mediated contact inhibition and cellular proliferation are regulated by counteracting CD44 C-terminal interacting proteins, the tumor suppressor protein merlin (NF2) and ERM proteins (ezrin, radixin, moesin). We show here that activation or overexpression of constitutively active merlin or downregulation of ERMs inhibited 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced [as well as serum, hepatocyte growth factor (HGF), or plateletderived growth factor (PDGF)] CD44 cleavage by the metalloprotease ADAM10, whereas overexpressed ERM proteins promoted cleavage. Merlin-and ERM-modulated Ras or Rac activity was not required for this function. However, latrunculin (an actin-disrupting toxin) or an ezrin mutant which is unable to link CD44 to actin, inhibited CD44 cleavage, identifying a cytoskeletal C-terminal link as essential for induced CD44 cleavage. Cellular migration, an important tumor property, depended on CD44 and its cleavage and was inhibited by merlin. These data reveal a novel function of merlin and suggest that CD44 cleavage products play a tumor-promoting role. Neuregulin, an EGF ligand released by ADAM17 from its proform NRG1, is predominantly involved in regulating cellular differentiation. In contrast to CD44, release of neuregulin from its pro-form was not regulated by merlin or ERM proteins. Disruption of the actin cytoskeleton however, also inhibited NRG1 cleavage. This current study presents one of the first examples of substrate-selective cleavage regulation.
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