CELL-SPECIFIC TARGETING OF FUSION PROTEINS THROUGH HEPARIN BINDING - Fusion Protein Technologies for Biopharmaceuticals: Applications and Challenges - page 406

FIGURE 27.3 Neuregulin's heparin-binding domain retains heparin-binding ability and potenti-
ates an HER4 antagonist. (A) Schematic diagram of secreted NRG and four antagonist constructs.
The heparin-binding domain (HBD) was fused N-terminal or C-terminal to extracellular-binding
domain of HER4 receptor (H4) with or without a spacer domain (S). H4 alone was used as a control.
The heparin column flow through and successive elutions with increasing concentrations of NaCl
were measured by immunoblotting. The HBD-S-H4 protein had the highest affinity for heparin.
(B) Comparable amounts of each fusion protein were premixed with 50 pM NRG and applied to rat
L6 muscle cells to determine the effect of each protein on the phosphorylation of erbB receptors
(p185) normalized to erbB2 levels (bottom gel). Quantitation of phosphorylated erbB protein to total
erbB2 protein reveals that HBD-S-H4 is the most potent antagonist at blocking NRG signaling with
about 80% reduction of erbB activation. (C) L6 cells were treated with either purified HBD-S-H4 or
H4 as a control at 1 and 10 nM for 1 h. The cells were then washed to remove unbound fusion protein
and then challenged with 75 pM NRG. While H4 alone had no residual effects, HBD-S-H4 had
sustained effects completely blocking NRG-induced erbB receptor phosphorylation. Source: This
research was originally published in J. Biol. Chem. Reference 79.