Agrin signaling in cortical neurons is mediated by a tyrosine kinase-dependent increase in intracellular Ca2+ that engages both CaMKII and MAPK signal pathways

Agrin has been implicated in multiple aspects of central nervous system (CNS) neuron differentiation and function including neurite formation, synaptogenesis, and synaptic transmission. However, little is known about the signaling mechanisms whereby agrin exerts its effects. We have recently identified a neuronal receptor for agrin, whose activation induces expression of c-fos, and provided evidence that agrin binding to this receptor is associated with a rise in intracellular Ca2+, a ubiquitous second messenger capable of mediating a wide range of effects. To gain further insight into agrin's role in brain, we used Ca2+ imaging to explore agrin signal transduction in cultured cortical neurons. Bath application of either z+ or z- agrin isoforms resulted in marked changes in intracellular Ca2+ concentration specifically in neurons. Propagation of the Ca2+ response was a two-step process characterized by an initial increase in intracellular Ca2+ mediated by ryanodine receptor (RyR) release from intracellular stores, supplemented by influx through voltage-gated calcium channels (VGCCs). Agrin-induced increases in intracellular Ca2+ were blocked by genistein and herbimycin, suggesting that the agrin receptor is a tyrosine kinase. Ca2+ release from intracellular stores activates both calcium/calmodulin-dependent kinase II (CaMKII) and mitogen activated protein kinase (MAPK). Activation of CaMKII is required for propagation of the Ca2+ wave itself, whereas both MAPK and CaMKII play a role in mediating long latency responses such as induction of c-fos. These results suggest that an agrin-dependent tyrosine kinase could play a critical role in modulating levels of intracellular Ca2+ and activity of MAPK and CaMKII in CNS neurons. (C) 2004 Wiley Periodicals, hic.