ABSTRACT
We have identified the adenine nucleotide translocator (ANT) isoforms ANT1 and ANT2 that are present in the plasma membrane of mouse cerebellar neurons as novel binding partners of the cell adhesion molecule L1. The direct interaction between ANT and L1 is mediated by sites within the fibronectin type III domains of L1 and the first and third extracellular loops of the ANT proteins. We also show that L1 interacts with the ANT binding partner matrix metalloprotease 14 (MMP14) and that the ANT proteins bind directly to the L1 interaction partner glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Moreover, we provide evidence that the functional interplay between L1, ANT proteins, MMP14, and GAPDH at the plasma membrane mediates L1-induced neurite outgrowth of cerebellar neurons. Disruption of this interplay by ANT inhibitors, ANT-derived synthetic peptides, and/or function-blocking MMP14 and ANT antibodies leads to alterations in L1-dependent neurite outgrowth. Stimulation of L1-mediated signaling in cerebellar neurons triggers transient ATP secretion via ANT proteins and leads to transient src family-dependent tyrosine phosphorylation of L1, ANT1, ANT2, and MMP14. Thus, our results indicate that plasma membrane-localized ANT1 and ANT2 regulate L1-mediated neurite outgrowth in conjunction with MMP14.
Subject(s)
Adenine Nucleotide Translocator 1/metabolism , Adenine Nucleotide Translocator 2/metabolism , Cerebellum/metabolism , Matrix Metalloproteinase 14/metabolism , Membrane Proteins/metabolism , Neural Cell Adhesion Molecule L1/physiology , Neurites/physiology , Animals , Cells, Cultured , Cerebellum/cytology , Female , Male , Membrane Proteins/physiology , Mice , Mice, Inbred C57BL , Neurons/metabolism , Protein Binding/physiologyABSTRACT
Polysialic acid (PSA) is a large and highly negatively charged glycan that plays crucial roles in nervous system development and function in the adult. It has been suggested to facilitate cell migration, neurite outgrowth, and synaptic plasticity because its hydration volume could enhance flexibility of cell interactions. Evidence for receptors of PSA has so far been elusive. We now identified histone H1 as binding partner of PSA via a single-chain variable fragment antibody using an anti-idiotypic approach. Histone H1 directly binds to PSA as shown by ELISA. Surface biotinylation of cultured cerebellar neurons indicated an extracellular localization of histone H1. Immunostaining of live cerebellar neurons and Schwann cells confirmed that an extracellular pool of histone H1 colocalizes with PSA at the cell surface. Histone H1 was also detected in detergent-insoluble synaptosomal membrane subfractions and postsynaptic densities. When applied in vitro, histone H1 stimulated neuritogenesis, process formation and proliferation of Schwann cells, and migration of neural precursor cells via a PSA-dependent mechanism, further indicating that histone H1 is active extracellularly. These in vitro observations suggested an important functional role for the interaction between histone H1 and PSA not only for nervous system development but also for regeneration in the adult. Indeed, histone H1 improved functional recovery, axon regrowth, and precision of reinnervation of the motor branch in adult mice with femoral nerve injury. Our findings encourage investigations on the therapeutic potential of histone H1 in humans.
Subject(s)
Cell Differentiation/physiology , Extracellular Space/physiology , Histones/physiology , Nerve Regeneration/physiology , Sialic Acids/physiology , Animals , Cell Movement/physiology , Cell Proliferation , Cells, Cultured , Disease Models, Animal , Extracellular Space/metabolism , Femoral Nerve/metabolism , Femoral Nerve/pathology , Femoral Nerve/physiology , Histones/metabolism , Mice , Mice, Inbred C57BL , Motor Neurons/metabolism , Myelin Sheath/metabolism , Myelin Sheath/pathology , Myelin Sheath/physiology , Neurites/physiology , Protein Binding/physiology , Schwann Cells/cytology , Schwann Cells/metabolism , Sialic Acids/metabolismABSTRACT
We have identified glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as a binding partner for the cell adhesion molecule L1. GAPDH binds to sites within the extracellular domain of L1, namely the immunoglobulin-like domains I-VI and the fibronectin type III homologous repeats 4-5. Extracellular GAPDH was detected at the cell surface of neuronal cells by surface biotinylation and immunocytochemistry. Addition of GAPDH antibodies to cultured cerebellar neurons inhibited L1-dependent neurite outgrowth in the presence of ATP, while the application of exogenous GAPDH promoted L1-dependent neurite outgrowth. Pre-treatment of substrate-coated L1-Fc with ATP and GAPDH, which phosphorylates L1, subsequently led to an enhanced neurite outgrowth. Furthermore, aggregation of L1-Fc carrying beads was enhanced in the presence of both GAPDH and ATP. L1-dependent neurite outgrowth and aggregation of L1 were diminished in the presence of alkaline phosphatase or a protein kinase inhibitor. Our results show that GAPDH-dependent phosphorylation of L1 is a novel mechanism in regulating L1-mediated neurite outgrowth.
