Binding of ATP to nerve growth factor: Characterization and relevance for bioactivity

Growth factors and their mechanisms of action have been studied extensively. However, it remained widely unrecognized that binding of ATP to growth factors is a prerequisite for their bioactivity. Here we demonstrated the binding of ATP to nerve growth factor (NGF) as well as its relevance for neuroprotection. By using mass spectrometry-based methodology we identified one or two molecules of ATP as being bound to NGF. To test neuroprotective activity of NGF we used primary cultures of rat cortical neurons damaged by staurosporine. ATP was indispensable for the neuroprotective effect of NGF. When the ATP concentration in the culture medium was reduced below approximately 2 nM by adding alkaline phosphatase (AP) or ATPase the neuroprotective activity of NGF was abolished. Site-directed mutagenesis within the heparin-binding domain (HBD) of NGF abolished ATP-binding and the neuroprotective effect. Thus, NGF has to bind ATP to be capable of protecting neurons.