In-vivo detection of binary PKA network interactions upon activation of endogenous GPCRs

Membrane receptor-sensed input signals affect and modulate intracellular protein-protein interactions (PPIs). Consequent changes occur to the compositions of protein complexes, protein localization and intermolecular binding affinities. Alterations of compartmentalized PPIs emanating from certain de...

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Bibliographic Details
Published in:Scientific reports 2015-06, Vol.5 (1), p.11133-11133, Article 11133
Main Authors: Röck, Ruth, Bachmann, Verena, Bhang, Hyo-eun C, Malleshaiah, Mohan, Raffeiner, Philipp, Mayrhofer, Johanna E, Tschaikner, Philipp M, Bister, Klaus, Aanstad, Pia, Pomper, Martin G, Michnick, Stephen W, Stefan, Eduard
Format: Article
Language:English
Subjects:
631/1647/338/469
631/80/86/2363
631/92/275
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82
82/111
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Animals
Biosensing Techniques
Cancer
Cell Line, Tumor
Complementation
Cyclic AMP-Dependent Protein Kinases - metabolism
Embryo, Nonmammalian - metabolism
Embryos
G protein-coupled receptors
Genes, Reporter
HEK293 Cells
Humanities and Social Sciences
Humans
Kinases
Localization
Mice
multidisciplinary
Osteosarcoma - metabolism
Protein Binding
Protein interaction
Protein Interaction Mapping
Protein kinase A
Proteins
Receptors, G-Protein-Coupled - metabolism
Saccharomyces cerevisiae - metabolism
Science
Xenograft Model Antitumor Assays
Xenografts
Yeasts
Zebrafish - embryology
Zebrafish - metabolism
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Summary:Membrane receptor-sensed input signals affect and modulate intracellular protein-protein interactions (PPIs). Consequent changes occur to the compositions of protein complexes, protein localization and intermolecular binding affinities. Alterations of compartmentalized PPIs emanating from certain deregulated kinases are implicated in the manifestation of diseases such as cancer. Here we describe the application of a genetically encoded Protein-fragment Complementation Assay (PCA) based on the Renilla Luciferase ( R luc) enzyme to compare binary PPIs of the spatially and temporally controlled protein kinase A (PKA) network in diverse eukaryotic model systems. The simplicity and sensitivity of this cell-based reporter allows for real-time recordings of mutually exclusive PPIs of PKA upon activation of selected endogenous G protein-coupled receptors (GPCRs) in cancer cells, xenografts of mice, budding yeast and zebrafish embryos. This extends the application spectrum of R luc PCA for the quantification of PPI-based receptor-effector relationships in physiological and pathological model systems.
ISSN:2045-2322
2045-2322
DOI:10.1038/srep11133