Figure 4.

Binding of NRG3^EGF.Fc to ErbB4. (A) Binding of NRG3^EGF.Fc to K562^erbB cells as analyzed by FACS. (Panels 1-4) Parental K562 cells (1) or K562 cells expressing either ErbB2 (K562^erbB2 cells, 2), ErbB3 (K562^erbB3 cells, 3), or ErbB4 (K562^erbB4 cells, 4) were examined for the expression of corresponding receptors. Cells were incubated with anti-ErbB2, anti-ErbB3, or anti-ErbB4 antibodies as indicated before the phycoerythrin-conjugated secondary antibody was added. The binding of antibodies was analyzed by FACS. LOG PE, relative fluorescent intensity; Counts, cell numbers. (Panels 5-8) NRG3^EGF.Fc binds to ErbB4 expressing cells. Parental K562 cells (5), K562^erbB2 cells (6), K562^erbB3 cells (7), and K562^erbB4 cells (8) were incubated with or without NRG3^EGF.Fc (containing gD tag) for 1 hr, followed by anti-gD-tag primary antibody and phycoerythrin-conjugated secondary antibody. The binding of antibodies was evaluated by FACS analysis. (B) Competitive inhibition of ¹²⁵I-NRG3^EGF.Fc binding to immobilized ErbB4.Fc by NRG3^EGF.Fc or NRG1^EGF. Iodination of NRG3^EGF.Fc and the binding assay were as described in Materials and Methods. ErbB4.Fc was immobilized on 96-well plates and incubated with various concentrations of unlabeled NRG3^EGF.Fc or NRG1^EGF and a constant amount of ¹²⁵I-labeled NRG3^EGF.Fc for 1.5 hr at room temperature. Unbound ligand was removed and the plate was extensively washed. The fraction of radioactivity bound over total ¹²⁵I-NRG3^EGF.Fc input is plotted against the concentration of competitor. Data of a representative experiment from four independent assays are shown. Error bars indicate standard deviation of quadruplicate samples of this experiment.