The Laminin-Induced Phosphorylation of PKCδ Regulates AQP4 Distribution and Water Permeability in Rat Astrocytes

In astrocytes, the water-permeable channel aquaporin-4 (AQP4) is concentrated at the endfeet that abut the blood vessels of the brain. The asymmetric distribution of this channel is dependent on the function of dystroglycan (DG), a co-expressed laminin receptor, and its associated protein complex. We have demonstrated that the addition of laminin to astrocytes in culture causes the clustering of AQP4, DG, and lipid rafts. The last, in particular, have been associated with the initiation of cell signaling. As laminin binding to DG in muscle cells can induce the tyrosine phosphorylation of syntrophin and laminin requires tyrosine kinases for acetylcholine receptor clustering in myotubes, we asked if signal transduction might also be involved in AQP4 clustering in astrocytes. We analyzed the timecourse of AQP4, DG, and monosialotetrahexosylganglioside (GM1) clustering in primary cultures of rat astrocytes following the addition of laminin, and determined that the clustering of DG precedes that of AQP4 and GM1. We also showed that laminin induces the formation of phosphotyrosine-rich clusters and that the tyrosine kinase inhibitor, genistein, disrupts the laminin-induced clustering of both beta-DG and AQP4. Using the Kinexus antibody microarray chip, we then identified protein-serine kinase C delta (PKC delta) as one of the main proteins exhibiting high levels of tyrosine phosphorylation upon laminin treatment. Selective inhibitors of PKC and siRNA against PKC delta disrupted beta-DG and AQP4 clustering, and also caused water transport to increase in astrocytes treated with laminin. Our results demonstrate that the effects of laminin on AQP4 localization and function are relayed, at least in part, through PKC signaling.

Automated summary

In astrocytes, the clustering of AQP4, DG, and GM1 is induced by laminin, with DG clustering preceding AQP4 and GM1. This process involves PKC signaling, demonstrating the role of PKC in the localization and function of AQP4 in response to laminin treatment.