Abstract
Epidermal growth factor (EGF) and transforming growth factor-alpha (TGFalpha) are mitogenic hormones that exert their activity primarily by binding to the EGF receptor, also known as ErbB-1. We have recently characterized a set of EGF/TGFalpha chimeric molecules with similar high affinity for ErbB-1 as EGF and TGFalpha and shown that three of these chimeras induce mitogenic cell stimulation at already a 10-fold lower concentration than their wild-type counterparts (Lenferink, A. E., Kramer, R. H., van Vugt, M. J., Königswieser, M., DiFiore, P. P., van Zoelen, E. J., and van de Poll, M. L. (1997) Biochem. J. 327, 859-865). In the present study we show that these so-called superagonistic chimeras do not differ from EGF and TGFalpha in their ability to induce ErbB-1 tyrosine phosphorylation but are considerably more potent in activation of mitogen-activated protein kinase phosphorylation. Direct cell binding studies and analysis of ligand-receptor interaction by surface plasmon resonance measurements revealed that both the association rate constant (k(on)) and the dissociation rate constant (k(off)) of these superagonists is 3-5-fold higher in comparison with the wild-type ligands and nonsuperagonistic chimeras. These data indicate that the dynamic on and off rate constants for receptor binding may be more specific parameters for determining the mitogenic activity of peptide hormones than their constants for equilibrium receptor binding.
Highlights
From the ‡Department of Cell Biology, University of Nijmegen, Toernooiveld 1, 6525 ED Nijmegen, The Netherlands and the ʈBiotechnology Research Institute, National Research Council Canada, Montreal H4P 2R2, Canada
ErbB-1 Receptor Tyrosine Phosphorylation by Epidermal growth factor (EGF)/TGF␣ Chimeras—In a previous study we have shown that chimeras of hEGF and hTGF␣, obtained by exchanging domains bordered by their conserved cysteine residues, all have similar binding affinity for ErbB-1 [23]
To investigate if the superagonistic behavior of these chimeras correlates with an enhanced induction of intracellular second messengers, we first compared the ability of the superagonist E4T and the wild-type ligands EGF and TGF␣ to induce ErbB-1 receptor tyrosine phosphorylation in HER-14 cells in a dose-dependent manner
Summary
The following full text is a publisher's version. For additional information about this publication click this link. http://hdl.handle.net/2066/129332. In the present study we show on the basis of receptor tyrosine phosphorylation studies, mitogen-activated protein kinase (MAPK) phosphorylation experiments, in vitro ligand binding studies, and kinetic analysis of receptor-ligand interactions using surface plasmon resonance (SPR) measurements that, compared with EGF and TGF␣, these so-called superagonists have both enhanced association and dissociation rate constants for binding to the ErbB-1 receptor. These data indicate that the on and off kinetics of ligand binding may represent an important additional parameter in determining the biological response of growth factor action
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