Superagonistic behaviour of epidermal growth factor/transforming growth factor-α chimaeras: correlation with receptor routing after ligand-induced internalization

Abstract

Human epidermal growth factor (EGF) and human transforming growth factor alpha (TGF-alpha) are structurally related polypeptide growth factors that exert their mitogenic activity through interaction with a common cell-surface receptor, the epidermal growth factor receptor (EGFR). The biological effect induced by these two ligands is quantitatively similar in most cases; in some test systems, however, TGF-alpha functions as a more potent form of EGF. In this study, we have compared EGF, TGF-alpha and ten previously described chimaeras of these two ligands in terms of their ability to generate a mitogenic response in cells carrying the human EGFR, and observed that three of the mutant growth factors (E3T, E4T and T3E4T) are mitogenic at concentrations 10-fold lower than that of either wild-type EGF or TGF-alpha. No difference in tyrosine kinase activity of the receptor towards an external substrate was observed after binding of the various mutants. It has been established before [Ebner and Derynck (1991) Cell Regulation 2, 599-612] that EGF and TGF-alpha differ in the processing of the receptor-ligand complex after internalization, as a result of their different pH sensitivities of receptor binding. Similar measurements on our chimaeric mutants revealed that the above superagonists show an enhanced pH dependence of binding in comparison with EGF. Furthermore, induction of receptor recycling by these superagonists is largely comparable with that induced by TGF-alpha. No superagonistic behaviour was observed on a cell-line containing an EGFR/erbB-2 chimaera which does not show ligand-induced internalization. These data show that EGF/TGFalpha chimaeras can be more active than the naturally occurring ligands, and that receptor recycling after ligand-induced internalization seems to be a prerequisite for this phenomenon.

Superagonistic activation of ErbB-1 by EGF-related growth factors with enhanced association and dissociation rate constants

“…These results regarding MAPK phosphorylation closely match the differences observed in the mitogenic activity of the various chimeric ligands (20). Taking into account the low ligand concentrations and short time intervals used in this study, it seems unlikely that the observed differences in MAPK phosphorylation result from altered routing of the receptor after ligand-induced internalization.…”

“…In the nomenclature used, a chimera such as E4T stands for a ligand with hEGF sequences N-terminal and hTGFα sequences C-terminal of the fourth cysteine (19). Three of those chimeras, designated E3T, E4T and T3E4T, have been shown to induce mitogenic activation of ErbB-1-containing cells at a ten-fold lower concentration than the wild-type growth factors EGF and TGFα, indicating their superiority in activating this receptor (20). These three superagonistic chimeras have also been shown to induce ErbB-1 recycling after ligand-induced internalization, similar to…”

“…TGFα, but unlike EGF and other non-superagonistic chimeras tested (20). Moreover, studies using cells with defined combinations of ErbB receptors have indicated that this superagonistic behaviour can be fully ascribed to activation of ErbB-1 receptors and is not the consequence of specificity in the formation of ErbB receptor heterodimers (10).…”

“…We have recently characterized a set of chimeric molecules composed of EGF and TGFα, which all have similar binding affinity to ErbB-1 (19). Nonetheless, growth stimulation studies showed that three of them, designated E3T, E4T and T3E4T, are able to induce a mitogenic response at already a ten-fold lower concentration than wild-type EGF and TGFα (20).…”

“…In the present study we show on the basis of receptor tyrosine phosphorylation studies, using radiolabeled ligand and mitogenic assays on these cells were performed as described (20).…”