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Measurement of Epidermal Growth Factor Receptor-Derived Signals Within Plasma Membrane Clathrin Structures

  • Stefanie Lucarelli
  • Ralph Christian Delos Santos
  • Costin N. Antonescu
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 1652)

Abstract

The epidermal growth factor (EGF) receptor (EGFR) is an important regulator of cell growth, proliferation, survival, migration, and metabolism. EGF binding to EGFR triggers the activation of the receptor’s intrinsic kinase activity, in turn eliciting the recruitment of many secondary signaling proteins and activation of downstream signals, such as the activation of phosphatidylinositol-3-kinase (PI3K) and Akt, a process requiring the phosphorylation of Gab1. While the identity of many signals that can be activated by EGFR has been revealed, how the spatiotemporal organization of EGFR signaling within cells controls receptor outcome remains poorly understood. Upon EGF binding at the plasma membrane, EGFR is internalized by clathrin-mediated endocytosis following recruitment to clathrin-coated pits (CCPs). Further, plasma membrane CCPs, but not EGFR internalization, are required for EGF-stimulated Akt phosphorylation. Signaling intermediates such as phosphorylated Gab1, which lead to Akt phosphorylation, are enriched within CCPs upon EGF stimulation. These findings indicate that some plasma membrane CCPs also serve as signaling microdomains required for certain facets of EGFR signaling and are enriched in key EGFR signaling intermediates. Understanding how the spatiotemporal organization of EGFR signals within CCP microdomains controls receptor signaling outcome requires imaging methods that can systematically resolve and analyze the properties of CCPs, EGFR and key signaling intermediates. Here, we describe methods using total internal reflection fluorescence microscopy imaging and analysis to systematically study the enrichment of EGFR and key EGFR-derived signals within CCPs.

Key words

Epidermal growth factor receptor Total internal reflection fluorescence (TIRF) microscopy Clathrin Signaling microdomain Akt Gab1 Spatiotemporal signal organization Image analysis 

Notes

Acknowledgments

This was supported by an Operating Grant from the Canadian Institutes of Health Research (CIHR) to C.N.A (grant no. 125854).

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Copyright information

© Springer Science+Business Media LLC 2017

Authors and Affiliations

  • Stefanie Lucarelli
    • 1
    • 2
  • Ralph Christian Delos Santos
    • 1
    • 2
  • Costin N. Antonescu
    • 1
    • 2
    • 3
  1. 1.Department of Chemistry and BiologyRyerson UniversityTorontoCanada
  2. 2.Graduate Program in Molecular ScienceRyerson UniversityTorontoCanada
  3. 3.Keenan Research Centre for Biomedical Science of St. Michael’s HospitalTorontoCanada

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