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

Röck R, Bachmann V, Bhang HE, Malleshaiah M, Raffeiner P, Mayrhofer JE, Tschaikner PM, Bister K, Aanstad P, Pomper MG, Michnick SW, Stefan E - Sci Rep (2015)

Bottom Line: 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.This extends the application spectrum of Rluc PCA for the quantification of PPI-based receptor-effector relationships in physiological and pathological model systems.

View Article: PubMed Central - PubMed

Affiliation: Institute of Biochemistry and Center for Molecular Biosciences, University of Innsbruck, Innrain 80/82, 6020 Innsbruck, Austria.

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.

No MeSH data available.


Related in: MedlinePlus

Impact of receptor activities on PPIs of PKA in human osteosarcoma cells.(A) Ligand-mediated activations of GPCR pathways linked to cAMP production lead to activation of PKA holoenzymes. cAMP binds to R subunits and triggers dissociation of active PKAc subunits which phosphorylate substrates in cytoplasm and nucleus. PKI inactivates nuclear PKAc (C) complexes. (B) U2OS cells transiently expressing indicated Rluc PCA pairs were exposed to different doses of isoproterenol (15 min). PPIs were determined using the Rluc PCA as read out (normalized to the untreated control for each PPI reporter; n = 3 independent experiments; SEM).
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f3: Impact of receptor activities on PPIs of PKA in human osteosarcoma cells.(A) Ligand-mediated activations of GPCR pathways linked to cAMP production lead to activation of PKA holoenzymes. cAMP binds to R subunits and triggers dissociation of active PKAc subunits which phosphorylate substrates in cytoplasm and nucleus. PKI inactivates nuclear PKAc (C) complexes. (B) U2OS cells transiently expressing indicated Rluc PCA pairs were exposed to different doses of isoproterenol (15 min). PPIs were determined using the Rluc PCA as read out (normalized to the untreated control for each PPI reporter; n = 3 independent experiments; SEM).

Mentions: The prototypical GPCR coupled to cAMP production is the beta adrenergic receptor family (βAR). Activation of different βAR subtypes are related to proliferation, cardiac function, and memory and learning3738394041. We chose the human osteosarcoma cell line U2OS (which exclusively expresses β2ARs) for the first perturbation experiments of cellular cAMP-levels33. We tested how β2AR-mediated cAMP-production affects binary PPIs of the PKA network (Fig. 3A). We activated endogenous β2AR in U2OS cells with the non-selective beta adrenergic agonist isoproterenol. Following overexpression of indicated Rluc PCA pairs we observed that nM concentrations of isoproterenol are sufficient to induce type I and type II PKA activation, indicated by a decrease of PPI/bioluminescence. Activation of β2ARs for 15 min has no impact on the RII dimer and on PKAc:PKI interactions (Fig. 3B).


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

Röck R, Bachmann V, Bhang HE, Malleshaiah M, Raffeiner P, Mayrhofer JE, Tschaikner PM, Bister K, Aanstad P, Pomper MG, Michnick SW, Stefan E - Sci Rep (2015)

Impact of receptor activities on PPIs of PKA in human osteosarcoma cells.(A) Ligand-mediated activations of GPCR pathways linked to cAMP production lead to activation of PKA holoenzymes. cAMP binds to R subunits and triggers dissociation of active PKAc subunits which phosphorylate substrates in cytoplasm and nucleus. PKI inactivates nuclear PKAc (C) complexes. (B) U2OS cells transiently expressing indicated Rluc PCA pairs were exposed to different doses of isoproterenol (15 min). PPIs were determined using the Rluc PCA as read out (normalized to the untreated control for each PPI reporter; n = 3 independent experiments; SEM).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4477410&req=5

f3: Impact of receptor activities on PPIs of PKA in human osteosarcoma cells.(A) Ligand-mediated activations of GPCR pathways linked to cAMP production lead to activation of PKA holoenzymes. cAMP binds to R subunits and triggers dissociation of active PKAc subunits which phosphorylate substrates in cytoplasm and nucleus. PKI inactivates nuclear PKAc (C) complexes. (B) U2OS cells transiently expressing indicated Rluc PCA pairs were exposed to different doses of isoproterenol (15 min). PPIs were determined using the Rluc PCA as read out (normalized to the untreated control for each PPI reporter; n = 3 independent experiments; SEM).
Mentions: The prototypical GPCR coupled to cAMP production is the beta adrenergic receptor family (βAR). Activation of different βAR subtypes are related to proliferation, cardiac function, and memory and learning3738394041. We chose the human osteosarcoma cell line U2OS (which exclusively expresses β2ARs) for the first perturbation experiments of cellular cAMP-levels33. We tested how β2AR-mediated cAMP-production affects binary PPIs of the PKA network (Fig. 3A). We activated endogenous β2AR in U2OS cells with the non-selective beta adrenergic agonist isoproterenol. Following overexpression of indicated Rluc PCA pairs we observed that nM concentrations of isoproterenol are sufficient to induce type I and type II PKA activation, indicated by a decrease of PPI/bioluminescence. Activation of β2ARs for 15 min has no impact on the RII dimer and on PKAc:PKI interactions (Fig. 3B).

Bottom Line: 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.This extends the application spectrum of Rluc PCA for the quantification of PPI-based receptor-effector relationships in physiological and pathological model systems.

View Article: PubMed Central - PubMed

Affiliation: Institute of Biochemistry and Center for Molecular Biosciences, University of Innsbruck, Innrain 80/82, 6020 Innsbruck, Austria.

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.

No MeSH data available.


Related in: MedlinePlus