Figure 1.

Cultured neurons release minute quantities of soluble heparin-binding NRG. A, Western blot of immunoprecipitated erbB2 and erbB3 proteins from L6 myotubes treated with increasing amounts of NRG produces a sensitive and quantitative bioassay for NRG. The blot was probed with anti-phosphotyrosine (pY) antibodies and then stripped and reprobed with antibodies to erbB2 and erbB3 (erbB). Quantitation of this blot reveals the ratio of tyrosine-phosphorylated erbB protein to total erbB protein (pY/erbB) to be linear. The dashed line represents the linear regression of this data described by y = 0.4999x, R² = 0.9936. B, Specificity of this NRG assay was shown using reagents that block the erbB receptor phosphorylation induced by recombinant NRG (5 pm) as well as sensory neuron culture media. Conditioned culture media was pretreated with an NRG-blocking antibody (1:10), soluble heparin (10 mg/ml), or a soluble fusion protein of erbB4 and human IgG (1:10) and then assayed for NRG as in A. Each reagent inhibited NRG-induced erbB tyrosine phosphorylation from the neuron cultures in a pattern identical to recombinant NRG, indicating that sensory neurons secrete predominately the soluble Ig form of NRG.