Activation of insulin-epidermal growth factor (EGF) receptor chimerae regulates EGF receptor binding affinity.

Abstract

. Cell surface tyrosine kinase receptors are subject to a rapid activation by their ligand, which is followed by secondary regulatory processes . The IHE2 cell line is a unique model system to study the regulation of EGF binding to EGF receptors after activation of the EGF receptor kinase. IHE2 cells express both a chimeric insulin-EGF receptor kinase (IER) and a kinase-deficient EGF receptor (HER K721A) . We have previously reported that IER is an insulin-responsive EGF receptor tyrosine kinase that activates one or several serine/threonine kinases, which in turn phosphorylate(s) the unoccupied HER K721A . In this article we show that insulin through IER activation induces a decrease in 'ZSI-EGF binding to IHE2 cells . Scatchard analysis indicates that, as for TPA, the effect of insulin can be accounted for by a loss of the high affinity bind-HE EGF receptor is a 170-kD transmembrane glycoprotein. The extracellular region contains the growth factor binding domain, whereas the intracellular re gion carries the tyrosine kinase catalytic domain (33,38) . These two functions of the EGF receptor, i .e., EGF binding and tyrosine kinase activity, play cardinal roles in the induction of the EGF biological effects and are subject to fine regulatory mechanisms. Receptor tyrosine kinase activity is positively regulated by EGF binding, which leads to tyrosine phosphorylation of the receptor (15,20,21,28) and of cellular substrates (38) . Recent data have indicated that tyrosine phosphorylation of the carboxy-terminal tail of the EGF receptor promotes the interaction with signaling molecules such as phospholipase C-.y and GTP-ase activating protein (GAP) ' (29) . In addition to these positive regulations, both the binding function and the kinase activity of the EGF receptor are subject to negative controls, which are mainly mediated by activation of protein kinase C. Thus, activators of protein kinase C, such as tetradecanoyl phorbol acetate (TPA) or PDGF, decrease the intrinsic tyrosine kinase ac- ing of EGF to HER K721A. Since this receptor transmodulation persists in protein kinase C downregulated IHE2 cells, it is likely to be due to a mechanism independent of protein kinase C activation. Using an in vitro system of 121 I-EGF binding to transmodulated IHE2 membranes, we illustrate that the inhibition of EGF binding induced by IER activation is related to the phosphorylation state of HER K721A. Further, studies with phosphatase 2A, or at a temperature (4°C) where only IER is functional, strongly suggest that the loss of high affinity EGF binding is related to the serine/ threonine phosphorylation of HER K721A after IER activation . Our results provide evidence for a "homologous desensitization" of EGF receptor binding after activation of the EGF receptor kinase of the IER receptor. tivity of the EGF receptor (7,16,17,23) . The major EGF receptor site phosphorylated by protein kinase C appears to be threonine 654 (12,22) . Studies using site-directed mutagenesis have shown that phosphorylation of the EGF recepto...

Uncoupling between Epidermal Growth Factor Receptor and Downstream Signals Defines Resistance to the Antiproliferative Effect of Gefitinib in Bladder Cancer Cells

“…We were able to document heterodimer formation between EGFR and PDGFRh in UM-UC13 (data not shown); however, the presence of heterodimers in the other cell lines and their biological roles remain to be further established. This effect, termed ''transmodulation, '' was described a decade ago and seems to serve in decreasing the binding affinity of EGF to EGFR (45)(46)(47).…”

Signalling through the insulin receptor and the insulin-like growth factor-I receptor

“…situated in trans [28]. Note that intermolecular phosphorylation appears to be the rule, since it has been found for all the tyrosine kinase receptors examined so far [7,29,30]. Concerning the biological implications of such an intermolecular transphosphorylation/transactivation model involving insulin receptors we have suggested that it could underlie the phenomenon of spare receptors seen in insulin action [27].…”

Cross talk among tyrosine kinase receptors in PC12 cells: desensitization of mitogenic epidermal growth factor receptors by the neurotrophic factors, nerve growth factor and basic fibroblast growth factor.

“…The second one, induced by EGF, platelet-derived growth factor (PDGF), acidic fibroblast growth factor, basic fibroblast growth factor (bFGF), interleukin 1 (IL-1), and tumor necrosis factor a (TFNa), appears to be independent of PKC activation (Olashaw et al, 1986;Bird and Saklatvala, 1989;Hicks et al, 1989;Tartare et al, 1992). Moreover, it has been demonstrated that EGF receptor serine/ threonine phosphorylation plays a crucial role in EGF binding inhibition induced by EGF (Tartare et al, 1992), PDGF (Olashaw et al, 1986;Davis and Czech, 1987;Countaway et al, 1989), IL-1, and TNFa (Bird and Saklatvala, 1990), but the kinase(s) involved is un-known.…”