The Epidermal Growth Factor Receptor Forms Location-Dependent Complexes in Resting Cells

Abstract

The epidermal growth factor receptor (EGFR) is a prototypical receptor tyrosine kinase involved in cell growth and proliferation and associated with various cancers. It is commonly assumed that after activation by binding of epidermal growth factor to the extracellular domain it dimerizes, followed by autophosphorylation of tyrosine residues at the intracellular domain. However, its oligomerization state before activation is controversial. In the absence of ligands, EGFR has been found in various, inconsistent amounts of monomeric, inactive dimeric, and oligomeric forms. In addition, evidence suggests that the active conformation is not a simple dimer but contains higher oligomers. As experiments in the past have been conducted at different conditions, we investigate here the influence of cell lines (HEK293, COS-7, and CHO-K1), temperature (room temperature and 37°C), and membrane localization on the quantitation of preformed dimers using SW-FCCS, DC-FCCS, quasi PIE-FCCS, and imaging FCCS. While measurement modality, temperature, and localization on upper or lower membranes have only a limited influence on the dimerization amount observed, the cell line and location to periphery versus center of the cell can change dimerization results significantly. The observed dimerization amount is strongly dependent on the expression level of endogenous EGFR in a cell line and shows a strong cell-to-cell variability even within the same cell line. In addition, using imaging FCCS, we find that dimers have a tendency to be found at the periphery of cells compared to central positions.

Figure 1

SW-FCCS experiments under different conditions. (A) SW-FCCS for resting cells at two different temperatures and basal and apical membranes. The plots (A–C) show the cross-correlation amounts q in CHO-K1, COS-7, and HEK293 cells, respectively, for different expression levels of mRFP-EGFR/EGFR-eGFP. Measurements with <200 or >200 receptors per μm² are indicated by solid or open circles, respectively. The upper and lower gray zones represent the range of the positive and negative controls with their width given by the mean ± SD. (D) SW-FCCS measurements of CHO-K1 cells expressing mRFP-EGFR/EGFR-eGFP. The bars represent q (mean ± SE) of control (solid bars), LAT-A (shaded bars), and mβCD-treated cells (open bars). LAT-A experiments and mβCD experiments conducted at room temperature. (E) Time series of q (mean ± SE) values for high-dose (100 ng/mL) and low-dose (10 ng/mL) EGF stimulation on mRFP-EGFR/EGFR-eGFP on CHO-K1 cells. (F) Value of q (mean ± SE) of mRFP-EGFR/EGFR-eGFP (apical membrane, RT) compared with mRFP-EGFR/EGFR (1706Q, V948R)-eGFP (apical membrane, RT), EGFR interaction in COS-7 cells at receptor densities <200/μm² and >200/μm² (basal membrane, RT) in CHO-K1 cells at <200/μm² and >200/μm² (basal membrane, RT), mRFP-EGFR/EGFR-eGFP/wt-EGFR (1.5:1:2) (basal and apical membrane, RT), and mRFP-EGFR/EGFR/wt-EGFR (1.5:1:1.3) basal and apical membrane, RT).

Figure 2

(A) ACF curves for EGFR-eGFP (dotted line); mRFP-EGFR (dashed line); and CCF curves (dash-dotted line) and their best fits (solid lines in corresponding colors) recorded by different FCCS modalities: SW-FCCS, quasi PIE-FCCS, DC-FCCS, and DC-ITIR-FCCS. The ACF curves from positive control mRFP-EGFR-eGFP are shown in the upper panels; the curves for mRFP-EGFR/EGFR-eGFP (the actual experiment) in the middle panels; and the negative control PMT-mRFP/EGFR-eGFP in the case of SW-FCCS and PMT-eGFP/PMT-mRFP for other FCCS modalities, in the lower panels. (B) The average cross-correlation amounts q from the different FCCS modalities are summarized in Table S1. To see this figure in color, go online.

Figure 3

Histogram of pooled q values and examples of q maps obtained by DC-ITIR-FCCS. The q values obtained in all pixels of all investigated cells were pooled together for the three series of measurements: the negative control (10,053 pixels from 10 cells), the positive control (6696 pixels from eight cells), and the actual experiment with EGFR-eGFP and mRFP-EGFR (16,779 pixels from 16 cells). The frequencies plotted in the histogram (A) are numbers of pixels in each bin divided by the total number of pixels in the respective series. Examples of q maps are shown for a cell from the negative control (B), positive control (C), and the actual experiment (D). The color-scale of q and a 5 μm scale bar are shown next to the maps. Pixels shown in white were excluded from fitting (either because of having lower than threshold intensity or because the parameters obtained by fitting of the ACFs were not within the set limits). To see this figure in color, go online.