Systems Biological Analysis of Epidermal Growth Factor Receptor Internalization Dynamics for Altered Receptor Levels

Abstract

Epidermal growth factor (EGF) receptor (EGFR) overexpression is a hallmark of many cancers. EGFR endocytosis is a critical step in signal attenuation, raising the question of how receptor expression levels affect the internalization process. Here we combined quantitative experimental and mathematical modeling approaches to investigate the role of the EGFR expression level on the rate of receptor internalization. Using tetramethylrhodamine-labeled EGF, we established assays for quantifying EGF-triggered EGFR internalization by both high resolution confocal microscopy and flow cytometry. We determined that the flow cytometry approach was more sensitive for examining large populations of cells. Mathematical modeling was used to investigate the relationship between EGF internalization kinetics, EGFR expression, and internalization machinery. We predicted that the standard parameter used to assess internalization kinetics, the temporal evolution r(t) of the ratio of internalized versus surface-located ligand⅐receptor complexes, does not describe a straight line, as proposed previously. Instead, a convex or concave curve occurs depending on whether initial receptor numbers or internalization adaptors are limiting the uptake reaction, respectively. To test model predictions, we measured EGF-EGFR binding and internalization in cells expressing different levels of green fluorescent protein-EGFR. As expected, surface binding of rhodaminelabeled EGF increased with green fluorescent protein-EGFR expression level. Unexpectedly, internalization of ligand⅐ receptor complexes increased linearly with increasing receptor expression level, suggesting that receptors and not internalization adaptors were limiting the uptake in our experimental model. Finally, determining the ratio of internalized versus surface-located ligand⅐receptor complexes for this cell line confirmed that it follows a convex curve, supporting our model predictions.

Identification and super-resolution imaging of ligand-activated receptor dimers in live cells

“…4). In our imaging conditions, this bleaching time being shorter than the lifetime of the activated receptors at the membrane before endocytosis20, the super-resolution images exclusively display EGFRs that are present at the membrane. Capturing receptors in their early states following ligand binding would not be possible with photo-activation based super-resolution methods since fluorophore photoactivation and ligand binding processes are not time-correlated.…”

“…2). Functionality of fluorescent ligand was controlled by observing that EGFR internalization occurs within minutes following binding of fluorescent EGF20 (Supplementary Fig. 3).…”

“…Binding of EGF to receptors indeed induces receptor dimerization and tyrosine autophosphorylation that activates intracellular signaling cascades leading to phenotypic changes like increased proliferation and migration. Importantly, receptor endocytosis occurs in the minute time scale following EGF binding20 such that activated EGFRs dimers are only transiently present at the plasma membrane making their study challenging at high resolution in living cells.…”

miR-17-5p Regulates Endocytic Trafficking through Targeting TBC1D2/Armus

“…Dil-LDL cellular uptake assay was performed as described elsewhere [32]. Prior to EGF-Alexa555 uptake assay, cells were starved for 12 h. EGF-Alexa555 internalization assay was performed as described elsewhere [33]. Prior to transferrin-Alexa568 uptake assay, cells were maintained in the starvation medium supplemented with 1 mg/ml BSA for 1 h and transferrin-Alexa568 internalization assay was performed as described previously [34].…”

Quantitative kinetics analysis of BMP2 uptake into cells and its modulation by BMP antagonists

“…Ligand internalization was measured in single cells using high resolution confocal microscopy and flow cytometry (FACS). Automated image processing was used to quantify microscopic images on the level of single endosomes (Schmidt-Glenewinkel et al, 2009). Thereby, various endosome parameters could be defined, which were used to evaluate the temporal evolution of endosomes during BMP2 endocytosis.…”