Immunoenzymetric Assay of Epidermal Growth Factor Receptor

Grimaux, M.; Laine-Bidron, C.; Magdelénat, H.

doi:10.1159/000217618

Cited by 14 publications

(16 citation statements)

References 5 publications

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“…The binding constant (K d ϭ 102.1 Ϯ 13.4 nM) for the recombinant ECD (Fig. 2) was similar to previously published values (24,26,31). Neither GM3 nor GM1 had a significant effect on binding of EGF to the ECD (Fig.…”

Section: Expression and Purification Of The Recombinant Ecd-insupporting

confidence: 89%

Interaction of the Extracellular Domain of the Epidermal Growth Factor Receptor with Gangliosides

Miljan¹,

Meuillet²,

Mania-Farnell³

et al. 2002

Journal of Biological Chemistry

161

113

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Ganglioside GM3 inhibits epidermal growth factor (EGF)-dependent cell proliferation in a variety of cell lines. Both in vitro and in vivo, this glycosphingolipid inhibits the kinase activity of the EGF receptor (EGFR). Furthermore, membrane preparations containing EGFR can bind to GM3-coated surfaces. These data suggest that GM3 may interact directly with the EGFR. In this study, the interaction of gangliosides with the extracellular domain (ECD) of the EGFR was investigated. The purified human recombinant ECD from insect cells bound directly to ganglioside GM3. The ganglioside interaction site appears to be distinct from the EGF-binding site. In agreement with previous reports on the effects of specific gangliosides on EGFR kinase activity, the ECD preferentially interacted with GM3. The order of relative binding of other gangliosides investigated was as follows: GM3 > > GM2, GD3, GM4 > GM1, GD1a, GD1b, GT1b, GD2, GQ1b > lactosylceramide. These data suggest that NeuAc-lactose is essential for binding and that any sugar substitution reduces binding. In agreement with the specificity of soluble ECD binding to gangliosides, GM3 specifically inhibited EGFR autophosphorylation. Identification of a ganglioside interaction site on the ECD of the EGFR is consistent with the hypothesis that endogenous GM3 may function as a direct modulator of EGFR activity.

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Section: Expression and Purification Of The Recombinant Ecd-insupporting

confidence: 89%

Interaction of the Extracellular Domain of the Epidermal Growth Factor Receptor with Gangliosides

Miljan¹,

Meuillet²,

Mania-Farnell³

et al. 2002

Journal of Biological Chemistry

161

113

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“…An ELISA assay (Grimaux et al, 1989) was employed to determine the quantity of receptor produced by recombinant baculovirus-infected insect cells and by cultured A431 cells. A431 cells, due to gene rearrangement, naturally secrete the extracellular domain of the EGFR Weber et al, 1984;Lin et al, 1984) and we used this human source for comparison with the baculovirus-expressed extracellular domain.…”

Section: Expression Of the Egfr Extracellular Domain In Recombinant Bmentioning

confidence: 99%

“…(unpurified) (W) was assayed using an EGF-binding assay (Grimaux et al, 1989; Materials and Methods). The data was analyzed using the LIGAND program of Munson and Rodbard (1980).…”

Section: Fig 2 Scatchard Analysis Of Equilibrium Binding Of Egf To mentioning

confidence: 99%

“…Based on the results of studies utilizing humankhicken EGF receptor chimeras (Lax et al, 1989), we predicted that this change from the human to the chicken sequence within the 14-amino-acid epitope of the ligand-competitive EGFR monoclonal antibody LA22 (mAb EGFR extracellular domain (W ; estimated by Westem-blot analysis) was incubated with varying concentrations of T -E G F in the absence or presence of a 50-fold molar excess of unlabeled EGF to demonstrate specificity of binding. The binding assay was carried out as previously described in Grimaux et al (1989) and in the Materials and Methods. The data was analyzed using the LIGAND program of Munson and Rodbard (1980).…”

Section: Dimerization As Assessed By Crosslinking and Analytical Ultrmentioning

confidence: 99%

“…Although it has been stated previously that this value is similar to that of the detergent-solubilized full-length receptor (Yarden and Schlessinger, 1987), the affinity of the EGFR extracellular domain and the full-length EGFR have never been directly compared in the same assay. In the present report, we use an immobilized receptor-binding assay (Grimaux et al, 1989) to systematically compare the affinity of the full-length EGFR with that of the truncated EGFR extracellular domain. We find that the affinity of the truncated EGFR extracellular domain for both EGF and transforming growth factor-a (TGF-a) is 30-40-fold lower than that of the detergent-solubilized full-length EGFR.…”

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confidence: 99%

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The Extracellular Domain of the Epidermal Growth Factor Receptor

Brown

Debanne

Grothé

et al. 1994

European Journal of Biochemistry

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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

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Serum-free production of recombinant proteins and adenoviral vectors by 293SF-3F6 cells

Côté

Garnier

Massie

et al. 1998

Biotechnol. Bioeng.

127

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This article describes the step‐wise approach undertaken to select a serum‐free medium (SFM) for the efficient production of a recombinant adenoviral vectors expressing β‐galactosidase (Ad5 CMV‐LacZ), in the complementing human embryonic kidney 293S cells. In the first step, a 293S‐derived transfectoma, secreting a soluble epidermal growth factor receptor sEGFr (D2‐22), was used to estimate the potential of selected serum‐free formulations to support the production of a recombinant protein as compared to serum‐containing medium. Assays showed that only one among six commercial serum‐free formulations could support both sEGFr production and cell growth in static or suspension culture. In commercially available calcium‐containing serum‐free formulations, the cell aggregates reached up to 3 mm in diameter. In the second step, 293S cells were gradually adapted to a low‐calcium version of the selected medium (LC‐SFM). Cells were cloned, then screened according to their ability to grow at a rate and an extent comparable to parental cells in serum‐containing medium (standard) as single cells or small aggregates. The 293SF‐3F6 clone, first adapted to and then cloned in the selected serum‐free medium, was selected for further experiments. Bioreactor run performed with the 293SF‐3F6 clone showed similar growth curve as in the shake‐flask controls. In the final step, the recombinant viral vector productivity of the 293S cells and the 293SF‐3F6 clone was tested. The 293SF‐3F6 cells infected by Ad5 CMV‐LacZ in 3 L‐scale bioreactor maintained the specific productivities of both β‐galactosidase and adenoviral vector equivalent to the shake‐flask controls in suspension culture. Results from this study clearly demonstrate that the 293SF‐3F6 cell line thus selected may be used either for establishing stable transfected cell line or for the production of adenoviral vectors required for gene therapy studies. © 1998 John Wiley & Sons, Inc. Biotechnol Bioeng 59: 567–575, 1998.

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Immunoenzymetric Assay of Epidermal Growth Factor Receptor

Cited by 14 publications

References 5 publications

Interaction of the Extracellular Domain of the Epidermal Growth Factor Receptor with Gangliosides

Interaction of the Extracellular Domain of the Epidermal Growth Factor Receptor with Gangliosides

The Extracellular Domain of the Epidermal Growth Factor Receptor

Serum-free production of recombinant proteins and adenoviral vectors by 293SF-3F6 cells

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