Betacellulin-Pseudomonas toxin fusion proteins bind but are not cytotoxic to cells expressing HER4; correlation of EGFR for cytotoxic activity

Abstract

Betacellulin (BTC) is a member of the EGF ligand family that directly binds to both EGFR and HER4 and induces the growth of certain epithelial cell types. Fusion proteins composed of the terminal 48 or 50 amino acids of mature betacellulin and a binding defective form of Pseudomonas exotoxin (BTC-TX₄₈ and BTC-TX₅₀, respectively), have been produced. BTC-TX₅₀ induced tyrosine phosphorylation of both EGFR and HER4, whereas BTC-TX₄₈ induced phosphorylation of HER4 but to a much lesser extent EGFR, indicating that the presence of two additional amino acid residues, Arg₆₂ and Lys₆₃, contribute to full kinase activity. BTC-TX₅₀ was up to 300-fold more active at inhibiting protein synthesis than BTC-TX₄₈ on cell lines expressing EGFR, most likely due to the >tenfold higher affinity of BTC-TX₅₀. MDA-MB-453 breast carcinoma cells which express HER4 but not EGFR, were not sensitive to either BTC-TX form. These data indicate that despite the ability of BTC-TX to bind and phosphorylate HER4, it was only cytotoxic to cells expressing EGFR. The inability of BTC-TX to kill cells was likely due to its failure to internalize through HER4.