Mechanism of desensitization of the epidermal growth factor receptor protein-tyrosine kinase.

J L Countaway,A C Nairn,R J Davis

doi:10.1016/s0021-9258(18)48406-2

J L Countaway, A C Nairn + Show 1 more

Open Access

https://doi.org/10.1016/s0021-9258(18)48406-2

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Abstract

The intrinsic protein-tyrosine kinase activity of the epidermal growth factor (EGF) receptor is required for signal transduction. Increased protein-tyrosine kinase activity is observed following the binding of EGF to the receptor. However, signaling is rapidly desensitized during EGF treatment. We report that EGF receptors isolated from desensitized cells exhibit a lower protein-tyrosine kinase activity than EGF receptors isolated from control cells. The mechanism of desensitization of kinase activity can be accounted for, in part, by the EGF-stimulated phosphorylation of the receptor at Ser1046/7, a substrate for the multifunctional calmodulin-dependent protein kinase II in vitro. Mutation of Ser1046/7 by replacement with Ala residues blocks desensitization of the EGF receptor protein-tyrosine kinase activity. Furthermore, this mutation causes a marked inhibition of the EGF-stimulated endocytosis and down-regulation of cell surface receptors. Thus, the phosphorylation site Ser1046/7 is required for EGF receptor desensitization in EGF-treated cells. This regulatory phosphorylation site is located at the carboxyl terminus of the EGF receptor within the subdomain that binds src homology 2 regions of signaling molecules.

Highlights

From the YHoward Hughes Medical Institute, $Program in Molecular Medicine,$Department of Biochemistry& Molecular Biology, University of Massachusetts Medical School, Worcester, Massachusetts 01605 and the §Laboratoryof Molecular &
The binding of EGF to the extracellular domain of the receptor causes a stimulation of the cytoplasmic domain protein-tyrosine kinase activity (Ushiro the tyrosine autophosphorylation of EGF receptors isolated from EGF-treated cells is reduced as compared with receptors isolated from control cells (Chinkers and Garbers, 1986; Lai et al, 1989; McCune et al, 1990)
We show thatthe mechanism of desensitization of theEGF receptor protein-tyrosine kinase activity is mediated by the phosphorylation of the receptor at a negative regulatory site (Ser1046/7 ) located at the carboxyl terminus of the receptor

Summary

RESULTS

Tyrosine phosphorylation of the EGF receptor was examined hy Western blot analysis using the anti-phosphotvrosine nnti-. EGF receptor tyrosine phosphorylation was examined using a Western blot procedure employingthe anti-phosphotyrosine antibody PY20. Addition of EGF caused a marked increase in thelevel of EGF receptor tyrosine phosphorylation afte5r min of EGF treatment (Fig. 1, lanes 1 and 2). Level of phosphorylation was foundafter 30 min of EGF CHO cells expressing wild-type EGF receptors were Iabelcd t)y incutreatment (Fig. 1, lane 3 ). The medium was replaced tyrosine phosphorylation observed after prolonged treatment and the cells were treated without ( 0 )and with 1 nM E(;F ( A )or 100 of cells with EGF could be caused by two different mecha- nM EGF (0f)or defined times. EGF receptor expression was observedafter 5-15 min of EGF treatment (Fig. 2).

EGF Causes Desensitization of the ECF Receptor

Phosphorylation of synthetic peptide substratesby CAM kinase II

Findings

EGF Receptor