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A High-content Imaging Workflow to Study Grb2 Signaling Complexes by Expression Cloning
Signal transduction by growth factor receptors is essential for cells to maintain proliferation and differentiation and requires tight control. Signal transduction is initiated by binding of an external ligand to a transmembrane receptor and activation of downstream signaling cascades. A key regulat...
Autores principales: | , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
MyJove Corporation
2012
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3499067/ https://www.ncbi.nlm.nih.gov/pubmed/23150065 http://dx.doi.org/10.3791/4382 |
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author | Freeman, Jamie Kriston-Vizi, Janos Seed, Brian Ketteler, Robin |
author_facet | Freeman, Jamie Kriston-Vizi, Janos Seed, Brian Ketteler, Robin |
author_sort | Freeman, Jamie |
collection | PubMed |
description | Signal transduction by growth factor receptors is essential for cells to maintain proliferation and differentiation and requires tight control. Signal transduction is initiated by binding of an external ligand to a transmembrane receptor and activation of downstream signaling cascades. A key regulator of mitogenic signaling is Grb2, a modular protein composed of an internal SH2 (Src Homology 2) domain flanked by two SH3 domains that lacks enzymatic activity. Grb2 is constitutively associated with the GTPase Son-Of-Sevenless (SOS) via its N-terminal SH3 domain. The SH2 domain of Grb2 binds to growth factor receptors at phosphorylated tyrosine residues thus coupling receptor activation to the SOS-Ras-MAP kinase signaling cascade. In addition, other roles for Grb2 as a positive or negative regulator of signaling and receptor endocytosis have been described. The modular composition of Grb2 suggests that it can dock to a variety of receptors and transduce signals along a multitude of different pathways(1-3). Described here is a simple microscopy assay that monitors recruitment of Grb2 to the plasma membrane. It is adapted from an assay that measures changes in sub-cellular localization of green-fluorescent protein (GFP)-tagged Grb2 in response to a stimulus(4-6). Plasma membrane receptors that bind Grb2 such as activated Epidermal Growth Factor Receptor (EGFR) recruit GFP-Grb2 to the plasma membrane upon cDNA expression and subsequently relocate to endosomal compartments in the cell. In order to identify in vivo protein complexes of Grb2, this technique can be used to perform a genome-wide high-content screen based on changes in Grb2 sub-cellular localization. The preparation of cDNA expression clones, transfection and image acquisition are described in detail below. Compared to other genomic methods used to identify protein interaction partners, such as yeast-two-hybrid, this technique allows the visualization of protein complexes in mammalian cells at the sub-cellular site of interaction by a simple microscopy-based assay. Hence both qualitative features, such as patterns of localization can be assessed, as well as the quantitative strength of the interaction. |
format | Online Article Text |
id | pubmed-3499067 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2012 |
publisher | MyJove Corporation |
record_format | MEDLINE/PubMed |
spelling | pubmed-34990672012-11-19 A High-content Imaging Workflow to Study Grb2 Signaling Complexes by Expression Cloning Freeman, Jamie Kriston-Vizi, Janos Seed, Brian Ketteler, Robin J Vis Exp Molecular Biology Signal transduction by growth factor receptors is essential for cells to maintain proliferation and differentiation and requires tight control. Signal transduction is initiated by binding of an external ligand to a transmembrane receptor and activation of downstream signaling cascades. A key regulator of mitogenic signaling is Grb2, a modular protein composed of an internal SH2 (Src Homology 2) domain flanked by two SH3 domains that lacks enzymatic activity. Grb2 is constitutively associated with the GTPase Son-Of-Sevenless (SOS) via its N-terminal SH3 domain. The SH2 domain of Grb2 binds to growth factor receptors at phosphorylated tyrosine residues thus coupling receptor activation to the SOS-Ras-MAP kinase signaling cascade. In addition, other roles for Grb2 as a positive or negative regulator of signaling and receptor endocytosis have been described. The modular composition of Grb2 suggests that it can dock to a variety of receptors and transduce signals along a multitude of different pathways(1-3). Described here is a simple microscopy assay that monitors recruitment of Grb2 to the plasma membrane. It is adapted from an assay that measures changes in sub-cellular localization of green-fluorescent protein (GFP)-tagged Grb2 in response to a stimulus(4-6). Plasma membrane receptors that bind Grb2 such as activated Epidermal Growth Factor Receptor (EGFR) recruit GFP-Grb2 to the plasma membrane upon cDNA expression and subsequently relocate to endosomal compartments in the cell. In order to identify in vivo protein complexes of Grb2, this technique can be used to perform a genome-wide high-content screen based on changes in Grb2 sub-cellular localization. The preparation of cDNA expression clones, transfection and image acquisition are described in detail below. Compared to other genomic methods used to identify protein interaction partners, such as yeast-two-hybrid, this technique allows the visualization of protein complexes in mammalian cells at the sub-cellular site of interaction by a simple microscopy-based assay. Hence both qualitative features, such as patterns of localization can be assessed, as well as the quantitative strength of the interaction. MyJove Corporation 2012-10-30 /pmc/articles/PMC3499067/ /pubmed/23150065 http://dx.doi.org/10.3791/4382 Text en Copyright © 2012, Journal of Visualized Experiments http://creativecommons.org/licenses/by-nc-nd/3.0/ This is an open-access article distributed under the terms of the Creative Commons Attribution-NonCommercial-NoDerivs 3.0 Unported License. To view a copy of this license, visithttp://creativecommons.org/licenses/by-nc-nd/3.0/ |
spellingShingle | Molecular Biology Freeman, Jamie Kriston-Vizi, Janos Seed, Brian Ketteler, Robin A High-content Imaging Workflow to Study Grb2 Signaling Complexes by Expression Cloning |
title | A High-content Imaging Workflow to Study Grb2 Signaling Complexes by Expression Cloning |
title_full | A High-content Imaging Workflow to Study Grb2 Signaling Complexes by Expression Cloning |
title_fullStr | A High-content Imaging Workflow to Study Grb2 Signaling Complexes by Expression Cloning |
title_full_unstemmed | A High-content Imaging Workflow to Study Grb2 Signaling Complexes by Expression Cloning |
title_short | A High-content Imaging Workflow to Study Grb2 Signaling Complexes by Expression Cloning |
title_sort | high-content imaging workflow to study grb2 signaling complexes by expression cloning |
topic | Molecular Biology |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3499067/ https://www.ncbi.nlm.nih.gov/pubmed/23150065 http://dx.doi.org/10.3791/4382 |
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