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 β-DG and AQP4. Using the Kinexus antibody microarray chip, we then identified protein-serine kinase C delta (PKCδ) as one of the main proteins exhibiting high levels of tyrosine phosphorylation upon laminin treatment. Selective inhibitors of PKC and siRNA against PKCδ disrupted β-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.

在星形胶质细胞中，透水通道水通道蛋白 4 (AQP4) 集中在与大脑血管相邻的末端。该通道的不对称分布取决于肌营养不良聚糖 (DG)、共表达的层粘连蛋白受体及其相关蛋白复合物的功能。我们已经证明，向培养中的星形胶质细胞添加层粘连蛋白会导致 AQP4、DG 和脂筏聚集。最后，特别是与细胞信号传导的启动有关。由于层粘连蛋白与肌肉细胞中的 DG 结合可诱导肌营养不良蛋白的酪氨酸磷酸化，而层粘连蛋白需要酪氨酸激酶用于肌管中的乙酰胆碱受体聚集，我们询问信号转导是否也可能参与星形胶质细胞中的 AQP4 聚集。我们分析了 AQP4、DG、添加层粘连蛋白后大鼠星形胶质细胞原代培养物中单唾液酸四己糖神经节苷脂 (GM1) 的聚集，并确定 DG 的聚集先于 AQP4 和 GM1 的聚集。我们还表明，层粘连蛋白诱导富含磷酸酪氨酸的簇的形成，并且酪氨酸激酶抑制剂染料木黄酮破坏了层粘连蛋白诱导的 β-DG 和 AQP4 的聚集。使用 Kinexus 抗体微阵列芯片，我们随后将蛋白丝氨酸激酶 C δ (PKCδ) 鉴定为在层粘连蛋白处理后表现出高水平酪氨酸磷酸化的主要蛋白质之一。PKC 和针对 PKCδ 的 siRNA 的选择性抑制剂破坏了 β-DG 和 AQP4 的聚集，并且还导致用层粘连蛋白处理的星形胶质细胞中的水运输增加。