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Published on: August 2, 2018
Nanoscale imaging of epidermal growth factor receptor clustering: effects of inhibitors
Abedelnasser Abulrob1, Zhengfang Lu, Ewa Baumann
1Institute for Biological Sciences, National Research Council of Canada, Ottawa, Ontario K1A 0R5, Canada. Abedelnasser.Abulrob@nrc.gc.ca
Abstract:
The development of some solid tumors is associated with overexpression of the epidermal growth factor receptor (EGFR) and often correlates with poor prognosis. Near field scanning optical microscopy, a technique with subdiffraction-limited optical resolution, was used to examine the influence of two inhibitors (the chimeric 225 antibody and tyrosine phosphorylation inhibitor AG1478) on the nanoscale clustering of EGFR in HeLa cells. The EGFR is organized in small clusters, average diameter of 150 nm, on the plasma membrane for both control and EGF-treated cells. The numbers of receptors in individual clusters vary from as few as one or two proteins to greater than 100. Both inhibitors yield an increased cluster density and an increase in the fraction of clusters with smaller diameters and fewer receptors. Exposure to AG1478 also decreases the fraction of EGFR that colocalizes with both rafts and caveolae. EGF stimulation results in a significant loss of the full-length EGFR from the plasma membrane with the concomitant appearance of low molecular mass proteolytic products. By contrast, AG1478 reduces the level of EGFR degradation. Changes in receptor clustering provide one mechanism for regulating EGFR signaling and are relevant to the design of strategies for therapeutic interventions based on modulating EGFR signaling.
Insights
Investigating epidermal growth factor receptor (EGFR) clustering with inhibitors revealed that these treatments increase EGFR cluster density and reduce receptor numbers per cluster. This suggests a mechanism for regulating EGFR signaling in cancer therapy.
Area of Science:
- Cell biology
- Molecular oncology
- Biophysics
Background:
- Epidermal growth factor receptor (EGFR) overexpression is linked to solid tumor development and poor prognosis.
- EGFR signaling pathways are crucial in cancer progression and represent therapeutic targets.
Purpose of the Study:
- To investigate the nanoscale clustering of EGFR in HeLa cells using near-field scanning optical microscopy.
- To examine the effects of two EGFR inhibitors, the 225 antibody and AG1478, on EGFR nanoscale organization and signaling.
Main Methods:
- Utilized near-field scanning optical microscopy (NSOM) for subdiffraction-limited imaging of EGFR clusters.
- Applied two inhibitors: a chimeric 225 antibody and the tyrosine phosphorylation inhibitor AG1478.
- Analyzed EGFR cluster density, size, receptor content, and colocalization with membrane rafts and caveolae.
Main Results:
- EGFR forms nanoscale clusters (average 150 nm diameter) on the plasma membrane, varying in size and receptor number.
- Both inhibitors increased EGFR cluster density and favored smaller clusters with fewer receptors.
- AG1478 reduced EGFR colocalization with rafts and caveolae and decreased EGFR degradation compared to EGF stimulation.
Conclusions:
- Modulating EGFR nanoscale clustering by inhibitors alters receptor organization and signaling.
- Changes in EGFR clustering represent a mechanism for regulating EGFR-mediated signaling.
- Findings are relevant for designing therapeutic strategies targeting EGFR signaling in cancer.
