Replacement of N-terminal portions of TGF-alpha with corresponding heregulin sequences affects ligand-induced receptor signaling and intoxication of tumor cells by chimeric growth-factor toxins.
ErbB receptor tyrosine kinases play an important role in developmental processes and tumor formation. Their activity is regulated by a family of structurally related ligands that bind to distinct ErbB receptor subsets, with transforming growth factor (TGF)-alpha preferentially interacting with epidermal growth-factor receptor (EGFR) and heregulin (HRG)-beta1 recognizing ErbB3 and ErbB4. To investigate the contribution of N-terminal ligand sequences to binding specificity, we have constructed 2 chimeric growth factors termed H181T8 and H194T20, which contain N-terminal HRG-beta1 sequences linked to complementary fragments of TGF-alpha. For bacterial expression and analysis of cell binding, the chimeric ligands were genetically fused to truncated Pseudomonas exotoxin A (ETA). H181T8-ETA and H194T20-ETA toxins both were cytotoxic for human tumor cell lines overexpressing EGFR but did not significantly affect the growth of cells that express ErbB receptors other than EGFR. Binding of H181T8, which contains HRG-beta1 residues 177-181, induced rapid autophosphorylation of EGFR, but in contrast to a previously described chimeric ligand based on EGF was unable to activate other ErbB receptors. H194T20, which contains HRG-beta1 residues 177-194, despite specific binding to EGFR was unable to induce autophosphorylation of any of the ErbB family members. However, H194T20 enhanced and modified the activity of parental TGF-alpha and HRG-beta1 when these ligands were simultaneously present. Our results show that modification of the N-terminal TGF-alpha sequence can have a significant effect on the signaling properties of the ligand and suggest that different EGF-like ligands can synergize in the activation of ErbB receptors.
Schmidt
Mathias
M
Chemotherapeutisches Forschungsinstitut Georg-Speyer-Haus, Paul-Ehrlich-Strasse 42-44, D-60596 Frankfurt am Main, Germany.
Wels
Winfried
W
D036002:ADP Ribose Transferases; D000595:Amino Acid Sequence; D001427:Bacterial Toxins; D001667:Binding, Competitive; D002470:Cell Survival; D016895:Culture Media, Serum-Free / Q000378:metabolism; D019281:Dimerization; D004305:Dose-Response Relationship, Drug; D004789:Enzyme Activation; D066246:ErbB Receptors / Q000378:metabolism; D005098:Exotoxins / Q000494:pharmacology; D008024:Ligands; D008969:Molecular Sequence Data; D009154:Mutation; D020890:Neuregulin-1 / Q000737:chemistry* / Q000235:genetics*; D010766:Phosphorylation; D019000:Phosphotyrosine / Q000378:metabolism; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D011994:Recombinant Proteins / Q000378:metabolism; D015398:Signal Transduction; D013997:Time Factors; D016211:Transforming Growth Factor alpha / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D014407:Tumor Cells, Cultured; D014443:Tyrosine / Q000378:metabolism; D037521:Virulence Factors; D000097668:Pseudomonas aeruginosa Exotoxin A
D036002:ADP Ribose Transferases; D001427:Bacterial Toxins; D016895:Culture Media, Serum-Free; D066246:ErbB Receptors; D005098:Exotoxins; D008024:Ligands; D020890:Neuregulin-1; D019000:Phosphotyrosine; D011993:Recombinant Fusion Proteins; D011994:Recombinant Proteins; D016211:Transforming Growth Factor alpha; D014443:Tyrosine; D037521:Virulence Factors; D000097668:Pseudomonas aeruginosa Exotoxin A