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Published on: October 30, 2012
Quantification and kinetic analysis of Grb2-EGFR interaction on micro-patterned surfaces for the characterization of
Peter Lanzerstorfer1, Daniela Borgmann2, Gerhard Schütz3
1University of Applied Sciences Upper Austria, Wels, Austria.
Abstract:
The identification of the epidermal growth factor receptor (EGFR) as an oncogene has led to the development of several anticancer therapeutics directed against this receptor tyrosine kinase. However, drug resistance and low efficacy remain a severe challenge, and have led to a demand for novel systems for an efficient identification and characterization of new substances. Here we report on a technique which combines micro-patterned surfaces and total internal reflection fluorescence (TIRF) microscopy (μ-patterning assay) for the quantitative analysis of EGFR activity. It does not simply measure the phosphorylation of the receptor, but instead quantifies the interaction of the key signal transmitting protein Grb2 (growth factor receptor-bound protein 2) with the EGFR in a live cell context. It was possible to demonstrate an EGF dependent recruitment of Grb2 to the EGFR, which was significantly inhibited in the presence of clinically tested EGFR inhibitors, including small tyrosine kinase inhibitors and monoclonal antibodies targeting the EGF binding site. Importantly, in addition to its potential use as a screening tool, our experimental setup offers the possibility to provide insight into the molecular mechanisms of bait-prey interaction. Recruitment of the EGFR together with Grb2 to clathrin coated pits (CCPs) was found to be a key feature in our assay. Application of bleaching experiments enabled calculation of the Grb2 exchange rate, which significantly changed upon stimulation or the presence of EGFR activity inhibiting drugs.
Insights
A new assay quantifies epidermal growth factor receptor (EGFR) activity by measuring interactions with Grb2 protein in live cells. This method aids in developing new anticancer drugs by assessing EGFR inhibitor efficacy and understanding drug resistance mechanisms.
Area of Science:
- Oncology
- Cell Biology
- Biophysics
Background:
- Epidermal growth factor receptor (EGFR) is an oncogene targeted by anticancer drugs.
- Drug resistance and low efficacy necessitate novel methods for identifying and characterizing new therapeutic substances.
- Existing methods for assessing EGFR activity, such as phosphorylation assays, have limitations.
Purpose of the Study:
- To develop a novel assay for quantitative analysis of EGFR activity in live cells.
- To characterize the interaction between EGFR and the signal-transmitting protein Grb2.
- To evaluate the efficacy of EGFR inhibitors and gain insights into molecular mechanisms of drug resistance.
Main Methods:
- Development of a micro-patterning assay combining micro-patterned surfaces and total internal reflection fluorescence (TIRF) microscopy.
- Quantitative analysis of Grb2 protein recruitment to EGFR in live cells.
- Application of bleaching experiments to calculate Grb2 exchange rates.
Main Results:
- Demonstrated EGF-dependent recruitment of Grb2 to EGFR.
- Showed significant inhibition of Grb2 recruitment by clinically tested EGFR inhibitors (TKIs and monoclonal antibodies).
- Identified recruitment of EGFR and Grb2 to clathrin-coated pits (CCPs) as a key feature and observed changes in Grb2 exchange rates upon stimulation or drug treatment.
Conclusions:
- The μ-patterning assay provides a quantitative method for analyzing EGFR activity by measuring EGFR-Grb2 interactions.
- This assay can serve as a screening tool for new anticancer therapeutics targeting EGFR.
- The assay offers insights into the molecular mechanisms underlying EGFR signaling, drug resistance, and bait-prey interactions.
