Large-Scale Screening Assay to Measure Epidermal Growth Factor Internalization

Abstract

Recently, we showed that the internalization of the epidermal growth factor (EGF) receptor is inhibited by hydrogen peroxide (H(2)O(2)) in human fibroblasts. In order to test the effect of various stress conditions on receptor internalization and to test a variety of antioxidants in their capacity to prevent or reduce the H(2)O(2)-induced inhibition of internalization, a screening assay was developed to measure the internalization in 96-well plates. In this assay, cells are exposed to biotin-conjugated EGF and the amount of internalized EGF is detected with horseradish peroxidase-conjugated streptavidin. We show that the results obtained by this new assay are comparable with those from internalization studies performed with radioactive labeled EGF. Therefore, the cellular internalization assay as presented here is a reliable method to measure EGF receptor internalization. Moreover, because elaborate processing of the cells is not required, the assay is a relatively fast and inexpensive method to study ligand-induced internalization in 96-well plates and thereby is suitable for large-scale screening of compounds or conditions interfering with this internalization.

Proteomic Analysis of the Epidermal Growth Factor Receptor (EGFR) Interactome and Post-translational Modifications Associated with Receptor Endocytosis in Response to EGF and Stress

“…EGFR internalization rates were determined by using the method of de Wit et al (33,34) with slight modification. Briefly, cells were plated at 20,000/well in 96-well plates, grown for 24 h, and then shifted to 1 g/ml tetracycline and 50 g/ml PEI-containing media for 24 h. To measure surface EGFR, cultures were washed and treated with 50 ng/ml biotin-labeled EGF (Invitrogen Molecular Probes) in PBS containing Ca 2ϩ /Mg 2ϩ /glucose for 1 h at 4°C, then washed with PBS and fixed in PBS containing 3% paraformaldehyde, 0.25% glutaraldehyde, and 0.25% Triton X-100.…”

“…Next, biotinylated EGF was used to quantify EGFR localization after EGF treatment (33,34). To measure surface EGFR, biotinylated EGF was incubated with WT, Y998A and 4A EGFR cells at 4°C for 1 h. HRP-conjugated streptavidin was used to quantify biotinylated EGF.…”

Hydrogen peroxide reversibly inhibits epidermal growth factor (EGF) receptor internalization and coincident ubiquitination of the EGF receptor and Eps15

“…at 0°C. After being washed twice with cold PBS, total amounts of protein after different treatments were determined with protein assay reagent BCA as described by the manufacturer (Pierce, Rockford, Ill.), while the amount of internalized EGFbiotin in cells in other wells was determined as described previously (20).…”

“…Recently, we have found that H 2 O 2 inhibits the internalization of the EGF receptor in fibroblasts in a concentration-dependent manner (19,20). The internalization and subsequent degradation of activated EGF receptors is one of the negative-feedback mechanisms to attenuate EGF-induced signaling.…”

Amplified Ras-MAPK signal states correlate with accelerated EGFR internalization, cytostasis and delayed HER2 tumor onset in Fer-deficient model systems

“…The procedure has been described previously and was adopted for biotin-EGF-based microtitre assays. [50][51][52] Briefly, cells were serum starved for 12-16 h and pulsed with 100 ng/ml biotin-EGF for 0-10 min at 37°C in serum-free medium in the presence or absence of 100-fold molar excess unlabeled EGF. EGFR internalization was assessed by tracking surface and internalized levels of biotin-EGF over time using immunocytochemistry.…”