Regulation of cell-cell junction by ephrin-A3: Possible implications for astrocytes function

RUBIO DE LA TORRE E; SILVEYRA MX; PASQUALE EB

San Diego

Encuentro; 43rd Annual Meeting of the Society for Neuroscience; 2013

Society for Neuroscience

Ephrin-A3, a GPI-linked ligand for the EphA receptor tyrosine kinases, is concentrated in perisynaptic astrocyte processes of the mouse hippocampus and mediates a form of neuron-glia communication by interacting with the EphA4 receptor expressed in excitatory synapses. By promoting EphA4 activation, ephrin-A3 regulates dendritic spine structural remodeling. Upon binding to EphA4, ephrin-A3 also triggers ?reverse? signals in the astrocytes that reduce glutamate uptake,enabling hippocampal synaptic plasticity. However, the mechanisms underlying ephrin-A3 reverse signaling are poorly understood. To determine if ephrin-A3 reverse signals in the adult hippocampus may also depend on interaction with other EphA receptors, we performed pulldown experiments with ephrin-A3 Fc followed by mass spectrometry analysis of bound Eph receptors. Based on he number of spectral counts, we found that EphA4 is the main Eph receptor that interacts with ephrin-A3 but we also detected substantial binding of EphA5 and EphA7 and, to a lesser extent, EphA3 and EphA6. Since in vitro ephrin-A3 binds to EphA4 with lower affinity than to other Eph receptors, our data suggest that the preferential interaction of ephrin-A3 with EphA4 is driven by the high abundance of EphA4. To characterize the effects of ephrin-A3 signaling in cells, we generated stable HEK293 cells expressing ephrin-A3 and chicken antibodies specifically recognizing ephrin-A3 in immunoblotting and immunocytochemistry experiments. It is known that other ephrin-As are present in lipid rafts, where their signaling increases protein tyrosine phosphorylation. In our cell system, ephrin-A3 was also detected in lipid raft fractions, but after EphA2 Fc treatment to stimulate ephrin-A3 reverse signaling we observed a redistribution of tyrosine phosphorylated proteins to cell-cell junctions without changes in overall tyrosine phosphorylation levels. EphA2 Fc also triggered ephrin-A3 relocalization to cell junctions together with accumulation of the cell adhesion molecule N-cadherin and the associated cytoplasmic proteins β-catenin and ZO-1. In addition, EphA2 Fc stimulation increased phosphotyrosine and vinculin immunolabeling at focal adhesions and induced the extension of filopodial-like cellular projections containing ephrin-A3. Thus, ephrin-A3 reverse signals induce changes in cell morphology and intercellular junctions suggesting that astrocytic ephrin-A3 may play a role not only in synaptic plasticity, but also in the regulation of astrocyte morphology and cell-cell interactions to affect astrocyte communication with other astrocytes or different cell types, such as endothelial cells.