Quantification and kinetic analysis of Grb2-EGFR interaction on micro-patterned surfaces for the characterization of EGFR-modulating substances.

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 syste...

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Autores principales:	Peter Lanzerstorfer, Daniela Borgmann, Gerhard Schütz, Stephan M Winkler, Otmar Höglinger, Julian Weghuber
Formato:	article
Lenguaje:	EN
Publicado:	Public Library of Science (PLoS) 2014
Materias:	Medicine R Science Q
Acceso en línea:	https://doaj.org/article/8f71c6e6e0b04c7c88432af3b64d39d4
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spelling	oai:doaj.org-article:8f71c6e6e0b04c7c88432af3b64d39d42021-11-18T08:26:45ZQuantification and kinetic analysis of Grb2-EGFR interaction on micro-patterned surfaces for the characterization of EGFR-modulating substances.1932-620310.1371/journal.pone.0092151https://doaj.org/article/8f71c6e6e0b04c7c88432af3b64d39d42014-01-01T00:00:00Zhttps://www.ncbi.nlm.nih.gov/pmc/articles/pmid/24658383/?tool=EBIhttps://doaj.org/toc/1932-6203The 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.Peter LanzerstorferDaniela BorgmannGerhard SchützStephan M WinklerOtmar HöglingerJulian WeghuberPublic Library of Science (PLoS)articleMedicineRScienceQENPLoS ONE, Vol 9, Iss 3, p e92151 (2014)
institution	DOAJ
collection	DOAJ
language	EN
topic	Medicine R Science Q
spellingShingle	Medicine R Science Q Peter Lanzerstorfer Daniela Borgmann Gerhard Schütz Stephan M Winkler Otmar Höglinger Julian Weghuber Quantification and kinetic analysis of Grb2-EGFR interaction on micro-patterned surfaces for the characterization of EGFR-modulating substances.
description	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.
format	article
author	Peter Lanzerstorfer Daniela Borgmann Gerhard Schütz Stephan M Winkler Otmar Höglinger Julian Weghuber
author_facet	Peter Lanzerstorfer Daniela Borgmann Gerhard Schütz Stephan M Winkler Otmar Höglinger Julian Weghuber
author_sort	Peter Lanzerstorfer
title	Quantification and kinetic analysis of Grb2-EGFR interaction on micro-patterned surfaces for the characterization of EGFR-modulating substances.
title_short	Quantification and kinetic analysis of Grb2-EGFR interaction on micro-patterned surfaces for the characterization of EGFR-modulating substances.
title_full	Quantification and kinetic analysis of Grb2-EGFR interaction on micro-patterned surfaces for the characterization of EGFR-modulating substances.
title_fullStr	Quantification and kinetic analysis of Grb2-EGFR interaction on micro-patterned surfaces for the characterization of EGFR-modulating substances.
title_full_unstemmed	Quantification and kinetic analysis of Grb2-EGFR interaction on micro-patterned surfaces for the characterization of EGFR-modulating substances.
title_sort	quantification and kinetic analysis of grb2-egfr interaction on micro-patterned surfaces for the characterization of egfr-modulating substances.
publisher	Public Library of Science (PLoS)
publishDate	2014
url	https://doaj.org/article/8f71c6e6e0b04c7c88432af3b64d39d4
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Quantification and kinetic analysis of Grb2-EGFR interaction on micro-patterned surfaces for the characterization of EGFR-modulating substances.

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