The Extracellular Domain of the Epidermal Growth Factor Receptor

Abstract

The binding of epidermal growth factor (EGF) or an EGF-like growth factor to the EGF receptor is the initial event which leads to receptor activation, and consequently the induction of cell growth. In order to study this binding interaction in detail, we produced the extracellular domain of the EGF receptor (EGFR) using the baculovirus expression system. Affinity-labeling and Western-blot analyses revealed that the baculovirus-infected insect cells secrete active EGFR extracellular domain relatively efficiently, however a significant amount of inactive EGFR extracellular domain is retained within the cells. The apparent dissociation constant (&) of the secreted EGFR extracellular domain for EGF and transforming growth factor a (TGF-a), as determined using an immobilized receptor binding assay, was approximately 200 nM. Interestingly, this Kd value is 30-40-fold lower than that of the full-length EGFR derived from detergent-solubilized A431 cell membranes. The stoichiometry of binding of the EGFR extracellular domain to EGF and TGF-a was examined by band-shift analysis on non-denaturing PAGE and was estimated to be 1 : 1. We have also shown, using sedimentation equilibrium analysis, that ligand binding induces significant dimerization of the EGFR extracellular domain. Finally, we carried out site-specific mutagenesis on the EGFR extracellular domain in order to define the ligand-binding region. We identified amino acid residues which are close to the binding site since they are common to the epitopes of several ligand-competitive monoclonal antibodies. However, these residues do not contribute directly to ligand binding since the affinity of the mutated EGFR extracellular domain for EGF and TGF-a was unaffected.Binding of epidermal growth factor (EGF) to the extracellular domain of its 170-kDa receptor (EGFR) leads to activation of the receptor cytoplasmic tyrosine kinase, substrate phosphorylation and ultimately mitogenesis (reviewed by Carpenter and Cohen, 1990;Ullrich and Schlessinger, 1990). Fundamental questions concerning the initial EGFEGFR interaction which leads to receptor activation can be addressed using a truncated form of the EGFR which consists of only the extracellular ligand-binding domain of the receptor. We have utilized the baculovirus expression system to produce large quantities of the extracellular domain and this has enabled us to study the affinity and stoichiometry of ligand binding, ligand-induced dimerization, as well as to attempt to identify receptor residues which are critical for ligand binding.In relation to the issue of affinity, the dissociation constant (Kd) of the soluble EGFR extracellular domain for EGF

HER2/Neu: mechanisms of dimerization/oligomerization

“…Two sEGFR populations have been detected in solution: one with a high a nity (2 ± 20 nM) and the other with a low a nity (400 ± 550 nM) for EGF (Domagala et al, 2000). The results of these studies are complicated by the fact that some groups have used soluble receptor fragments, while others have used full-length receptor present on the surface of intact cells; the a nity of EGF for a soluble extracellular EGFR fragment has been shown to be markedly lower than for the full-length receptor (Brown et al, 1994). Furthermore, interpretation of binding studies with erbB receptors must take into account that the receptors are in a monomer/oligomer equilibrium which is not governed solely by ligand concentration.…”

“…A general scheme based on current understanding of EGF induced oligomerization is shown in Figure 2. Multiple studies have concluded that EGF binds EGFR with a 1 : 1 stoichiometry (Brown et al, 1994;Domagala et al, 2000;Lemmon et al, 1997;Odaka et al, 1997;Weber et al, 1984). Subdomains I and especially III of the extracellular portion of EGFR have been identi®ed as important in ligand binding (Lax et al, 1998(Lax et al, , 1989Lee et al, 1995;Summer®eld et al, 1996), as has subdomain IV, one of two cysteine rich domains (Saxon and Lee, 1999).…”

Dual effect of erbB-2 depletion on the regulation of DNA repair and cell cycle mechanisms in non-small cell lung cancer cells

“…The immunoprecipitates were then washed four times in cold PBS containing 0.1% Tween-20, resuspended in SDS sample bu er, boiled for 5 min and pelleted by centrifugation at 2000 g for 1 min. The eluate was loaded onto a 7.5% SDS-polyacrylamide gel, transferred to nitrocellulose (Costar, Cambridge, MA, USA), blocked overnight in PBS containing 3% dried milk, then incubated with polyclonal antibody against EGFR (Brown et al, 1994), erbB-2 (C-18, Santa Cruz Biotechnology), erbB-3 (C-17, Santa Cruz Biotechnology), or erbB-4 (C-18, Santa Cruz Bio-technology), or monoclonal anti-phosphotyrosine (clone 4G10, Upstate Biotechnology Inc., NY, USA). Blots were developed using the alkaline phosphatase substrate detection kit from Vector Laboratories (Burlingame, CA, USA).…”

Decorin Binds to a Narrow Region of the Epidermal Growth Factor (EGF) Receptor, Partially Overlapping but Distinct from the EGF-binding Epitope

“…It has been shown that the amino acid residues in the EGFR that are essential for binding the LA22 monoclonal antibody (Ala 351 -Asp 364 ) are not critical for the binding of EGF or TGF-␣ because the epitope was eliminated, whereas ligand binding was unaffected (77). Thus, because the monoclonal antibody is bulky it is likely that the EGF binding site is located in the near vicinity and that the competition with EGF occurs by steric hindrance.…”