Epidermal growth factor receptor binding is affected by structural determinants in the toxin domain of transforming growth factor-alpha-Pseudomonas exotoxin fusion proteins.
TGF-alpha-PE40 is a hybrid protein composed of transforming growth factor-alpha (TGF-alpha) fused to a 40,000-dalton segment of Pseudomonas exotoxin A (PE40). This hybrid protein possesses the receptor-binding activity of TGF-alpha and the cell-killing properties of PE40. These properties enable TGF-alpha-PE40 to bind to and kill tumor cells that possess epidermal growth factor (EGF) receptors. Unexpectedly, TGF-alpha-PE40 binds approximately 100-fold less effectively to EGF receptors than does native TGF-alpha (receptor-binding inhibition IC50 = 540 and 5.5 nM, respectively). To understand the factors governing receptor binding, deletions and site-specific substitutions were introduced into the PE40 domain of TGF-alpha-PE40. Removal of the N-terminal 59 or 130 amino acids from the PE40 domain of TGF-alpha-PE40 improved receptor binding (IC50 = 340 and 180 nM, respectively) but decreased cell-killing activity. Substitution of alanines for cysteines at positions 265 and 287 within the PE40 domain dramatically improved receptor binding (IC50 = 37 nM) but also decreased cell-killing activity. Similar substitutions of alanines for cysteines at positions 372 and 379 within the PE40 domain did not significantly affect receptor-binding or cell-killing activities. These studies indicate that the PE40 domain of TGF-alpha-PE40 interferes with EGF receptor binding. The cysteine residues at positions 265 and 287 of PE40 are responsible for a major part of this interference.
Department of Cancer Research, Merck Sharp and Dohme Research Laboratories, West Point, Pennsylvania 19486.
G M
GM
Edwards
D
D
DeFeo-Jones
J Y
JY
Tai
G A
GA
Vuocolo
D R
DR
Patrick
D C
DC
Heimbrook
A
A
Oliff
D036002:ADP Ribose Transferases; D001427:Bacterial Toxins; D002461:Cell Line, Transformed; D003001:Cloning, Molecular; D003545:Cysteine / Q000235:genetics; D004247:DNA / Q000235:genetics; D004591:Electrophoresis, Polyacrylamide Gel; D066246:ErbB Receptors / Q000235:genetics* / Q000378:metabolism; D005098:Exotoxins / Q000235:genetics* / Q000378:metabolism; D006801:Humans; D009154:Mutation; D010957:Plasmids; D011993:Recombinant Fusion Proteins / Q000235:genetics* / Q000378:metabolism; D011994:Recombinant Proteins / Q000235:genetics*; D015291:Transforming Growth Factors / Q000235:genetics* / Q000378:metabolism; D037521:Virulence Factors; D000097668:Pseudomonas aeruginosa Exotoxin A
D036002:ADP Ribose Transferases; D001427:Bacterial Toxins; D003545:Cysteine; D004247:DNA; D066246:ErbB Receptors; D005098:Exotoxins; D011993:Recombinant Fusion Proteins; D011994:Recombinant Proteins; D015291:Transforming Growth Factors; D037521:Virulence Factors; D000097668:Pseudomonas aeruginosa Exotoxin A