title: In-vivo detection of binary PKA network interactions upon activation of endogenous GPCRs.
abstract: 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 (Rluc) 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 Rluc PCA for the quantification of PPI-based receptor-effector relationships in physiological and pathological model systems.
authors.affiliation: Institute of Biochemistry and Center for Molecular Biosciences, University of Innsbruck, Innrain 80/82, 6020 Innsbruck, Austria.
authors.forename: Ruth
authors.initials: R
authors.lastname: Röck
authors.affiliation: Institute of Biochemistry and Center for Molecular Biosciences, University of Innsbruck, Innrain 80/82, 6020 Innsbruck, Austria.
authors.forename: Verena
authors.initials: V
authors.lastname: Bachmann
authors.affiliation: Russell H. Morgan Department of Radiology and Radiological Science, Johns Hopkins Medical School, Baltimore, MD 21287, USA.
authors.forename: Hyo-Eun C
authors.initials: HE
authors.lastname: Bhang
authors.affiliation: Département de Biochimie, Université de Montréal, H3C 3J7 Montréal, Québec, Canada.
authors.forename: Mohan
authors.initials: M
authors.lastname: Malleshaiah
authors.affiliation: Institute of Biochemistry and Center for Molecular Biosciences, University of Innsbruck, Innrain 80/82, 6020 Innsbruck, Austria.
authors.forename: Philipp
authors.initials: P
authors.lastname: Raffeiner
authors.affiliation: Institute of Biochemistry and Center for Molecular Biosciences, University of Innsbruck, Innrain 80/82, 6020 Innsbruck, Austria.
authors.forename: Johanna E
authors.initials: JE
authors.lastname: Mayrhofer
authors.affiliation: Institute of Molecular Biology, University of Innsbruck, Technikerstrasse 25, 6020 Innsbruck, Austria.
authors.forename: Philipp M
authors.initials: PM
authors.lastname: Tschaikner
authors.affiliation: Institute of Biochemistry and Center for Molecular Biosciences, University of Innsbruck, Innrain 80/82, 6020 Innsbruck, Austria.
authors.forename: Klaus
authors.initials: K
authors.lastname: Bister
authors.affiliation: Institute of Molecular Biology, University of Innsbruck, Technikerstrasse 25, 6020 Innsbruck, Austria.
authors.forename: Pia
authors.initials: P
authors.lastname: Aanstad
authors.affiliation: Russell H. Morgan Department of Radiology and Radiological Science, Johns Hopkins Medical School, Baltimore, MD 21287, USA.
authors.forename: Martin G
authors.initials: MG
authors.lastname: Pomper
authors.affiliation: Département de Biochimie, Université de Montréal, H3C 3J7 Montréal, Québec, Canada.
authors.forename: Stephen W
authors.initials: SW
authors.lastname: Michnick
authors.affiliation: Institute of Biochemistry and Center for Molecular Biosciences, University of Innsbruck, Innrain 80/82, 6020 Innsbruck, Austria.
authors.forename: Eduard
authors.initials: E
authors.lastname: Stefan
mesh_terms: D000818:Animals; D015374:Biosensing Techniques; D045744:Cell Line, Tumor; D017868:Cyclic AMP-Dependent Protein Kinases / Q000378:metabolism*; D004625:Embryo, Nonmammalian / Q000378:metabolism; D017930:Genes, Reporter; D057809:HEK293 Cells; D006801:Humans; D051379:Mice; D012516:Osteosarcoma / Q000378:metabolism; D011485:Protein Binding; D025941:Protein Interaction Mapping; D043562:Receptors, G-Protein-Coupled / Q000378:metabolism*; D012441:Saccharomyces cerevisiae / Q000378:metabolism; D023041:Xenograft Model Antitumor Assays; D015027:Zebrafish / Q000196:embryology / Q000378:metabolism
chemical_list: D043562:Receptors, G-Protein-Coupled; D017868:Cyclic AMP-Dependent Protein Kinases