Plasma Membrane Organization of Epidermal Growth Factor Receptor in Resting and Ligand-Bound States

Abstract

The spatial arrangement of the epidermal growth factor receptor (EGFR) on the cellular plasma membrane is one of the prime factors that control its downstream signaling pathways and related functions. However, the molecular organization, which spans the scale from nanometers to micrometer-size clusters, has not been resolved in detail, mainly due to a lack of techniques with the required spatiotemporal resolution. Therefore, we used imaging total internal reflection-fluorescence correlation spectroscopy to investigate EGFR dynamics on live CHO-K1 plasma membranes in resting and ligand-bound states. In combination with the fluorescence correlation spectroscopy diffusion law, this provides information on the subresolution organization of EGFR on cell membranes. We found that overall EGFR organization is sensitive to both cholesterol and the actin cytoskeleton. EGFR in the resting state is partly trapped in cholesterol-containing domains, whereas another fraction exhibits cholesterol independent trapping on the membrane. Disruption of the cytoskeleton leads to a broader range of EGFR diffusion coefficients and a reduction of hop diffusion. In the ligand-bound state we found a dose-dependent behavior. At 10 ng/mL EGF the EGFR is endocytosed and recycled to the membrane, whereas diffusion and organization do not change significantly. At 100 ng/mL EGF the EGFR forms clusters, which are subsequently internalized, whereas outside the clusters diffusivity increases and the organization of the receptor remains unchanged. After disruption of cholesterol-containing domains or actin cytoskeleton, EGF induces microscopic EGFR clusters on the membrane and endocytosis is inhibited.

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

“…This demonstrates the utility of DC-ITIR-FCCS (and imaging FCS modalities in general) for linking the cell-to-cell variability with the variability between regions within individual cells. The regions of high complex fractions were located predominantly at the cell periphery in agreement with previous reports (20,33). It remains to be investigated further what is the nature of those regions and whether they contain only elevated amounts of dimers or whether they contain higher EGFR oligomers.…”

“…These values are higher than the cross-correlation amounts measured in untreated cells, demonstrating that EGFR complex formation is to some extent inhibited by partitioning of the receptor into cholesterol-dependent domains. These results are in agreement with a previously published study (63) and are consistent with the findings that EGFR resides in cholesterol-dependent domains (33,64).…”

“…Our recent results show that EGFR partitions into cholesterol-dependent as well as cholesterol-independent domains and that its diffusion is affected by the actin cytoskeleton (33). Our data show that cholesterol depletion by mbCD leads to an increase in the EGFR complex fraction.…”

“…Techniques differ in the efficiency with which they can detect short-lived transient dimers as well as in the ability to distinguish true functional receptor dimers from groups of receptors in close proximity due to colocalization in membrane domains (25)(26)(27)(28). Partitioning of EGFR in cholesterol-rich microdomains has been reported in several studies (29)(30)(31)(32); we have shown recently that EGFR partitions into cholesterol-dependent as well as cholesterol-independent domains and interacts with the cytoskeleton (33).…”

“…Cholesterol was depleted from the plasma membranes by treatment with mbCD (at 3 mM final concentration). EGFR-eGFP/mRFP-EGFR dynamics were monitored after 25 min mbCD incubation to maximize cholesterol depletion effect at room temperature (33) (Fig. 1 D).…”