Ethanol inhibits L1 cell adhesion molecule tyrosine phosphorylation and dephosphorylation and activation of pp60^src

Abstract

Fetal alcohol syndrome is a leading cause of mental retardation. The neuropathology found in patients with fetal alcohol syndrome overlaps with those with mutations in the gene for cell adhesion molecule (L1). We have previously shown that L1‐mediated neurite outgrowth and L1 activation of extracellular receptor kinases 1/2 are inhibited at low concentrations of ethanol. One possible mechanism for this effect is through disruption of a tyrosine‐based sorting signal, Y(1176)RSLE, on the cytoplasmic domain of L1. Our goal was to determine if ethanol inhibited the sorting signal or its phosphorylation state. Using cerebellar granule neurons and dorsal root ganglion neurons, we found that ethanol had no effect on L1 distribution to the growth cone or its ability to be expressed on the cell surface as determined by confocal microscopy. In cerebellar granule neurons, clustering of L1 resulted in increased dephosphorylation of Y(1176), increased L1 tyrosine phosphorylation, and an increase in the activation of pp60src as measured by immunoblot. All changes were inhibited by 25 mM ethanol. Using PP2 to inhibit pp60src activation resulted in inhibition of increases in L1 tyrosine and extracellular receptor kinases 1/2 phosphorylation, and Y(1176) dephosphorylation. We conclude that ethanol disrupts L1 trafficking/signaling following its expression on the surface of the growth cone, and prior to its activation of pp60src.

The Interaction between Cell Adhesion Molecule L1, Matrix Metalloproteinase 14, and Adenine Nucleotide Translocator at the Plasma Membrane Regulates L1-Mediated Neurite Outgrowth of Murine Cerebellar Neurons

“…L1 stimulates src-dependent tyrosine phosphorylation of ANT and MMP14 and triggers ATP release via ANT Since L1 stimulation activates nonreceptor tyrosine kinase src (Ignelzi et al, 1994;Schmid et al, 2000;Loers et al, 2005;Yeaney et al, 2009) stimulation of cerebellar neurons would lead to tyrosine phosphorylation of ANT and MMP14. After incubating cerebellar neurons without or with L1-Fc for different time periods, cells were lysed, and the tyrosine-phosphorylated proteins were immunoprecipitated and subjected to Western blot analysis using L1, ANT, and MMP14 antibodies.…”

Two Alcohol Binding Residues Interact across a Domain Interface of the L1 Neural Cell Adhesion Molecule and Regulate Cell Adhesion

“…Concentrations of ethanol attained after one drink inhibit the adhesion of L1 expressed in fibroblasts, neural cell lines, and cerebellar granule neurons (CGN) (6,7). Furthermore, ethanol inhibits L1-mediated neurite outgrowth in CGNs with similar potency to its inhibition of L1 adhesion (11,12). Finally, drugs that block ethanol inhibition of L1 adhesion also prevent ethanol teratogenesis in mouse embryos (13)(14)(15)(16)(17)(18).…”

Choline Partially Prevents the Impact of Ethanol on the Lipid Raft Dependent Functions of L1 Cell Adhesion Molecule

“…Cells were grown overnight, then placed in medium without serum for 2 h. After 2 h, ethanol was added for 1 h, then L1 was clustered by the addition of a cross-linked antibody as previously described (Yeaney et al, 2009; Tang et al, 2006). To form cross-linked ASCS4 antibody complexes, purified ASCS4 was mixed with goat anti-mouse IgG (H+L)(1:2.5 g/g) for 1 h at 4°C.…”

“…The complexes were added to cultures for a final concentration of 30 μg/mL of media. Mouse IgG 30 μg/mL final concentration was added as control (Schmid et al, 2000; Yeaney et al, 2009; Tang et al, 2006). 10 min after addition of the complexes, the tissue culture dishes were placed on ice, the media was removed, and the CGN were washed with ice-cold Hank’s Buffered Saline Solution (HBSS).…”

“…L1 mediates neurite outgrowth through a downstream signaling cascade consisting of activation of pp60 src and ERK1/2, dephosphorylation of tyrosine 1176 in the cytoplasmic domain of L1, and phosphorylation of other tyrosines in the cytoplasmic domain of L1 (Schmid et al, 2000; Schaefer et al, 1999; Schaefer et al, 2002; Tuvia et al, 1997; Yeaney et al, 2009). This cascade is inhibited by ethanol (Yeaney et al, 2009; Bearer et al, 1999; Littner et al, 2013; Tang et al, 2006; Watanabe et al, 2004). The neurite outgrowth of cerebellar granule neurons (CGN) grown on a substrate of the extracellular domain of L1 is effectively suppressed by ethanol at pharmacologic concentrations (Bearer et al, 1999).…”